## THE FIBROBLAST GROWTH FACTOR SIGNALING PATHWAY IN METABOLIC REGULATION, DEVELOPMENT, DISEASE AND REPAIR AFTER INJURY

EDITED BY : Zhouguang Wang, Xiaokun Li, Li Lin, Saverio Bellusci and Jin-San Zhang PUBLISHED IN : Frontiers in Pharmacology

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ISSN 1664-8714 ISBN 978-2-88966-128-2 DOI 10.3389/978-2-88966-128-2

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## THE FIBROBLAST GROWTH FACTOR SIGNALING PATHWAY IN METABOLIC REGULATION, DEVELOPMENT, DISEASE AND REPAIR AFTER INJURY

Topic Editors:

Zhouguang Wang, Albert Einstein College of Medicine, United States Xiaokun Li, Wenzhou Medical University, China Li Lin, Wenzhou Medical University, China Saverio Bellusci, University of Giessen, Germany Jin-San Zhang, Mayo Clinic, United States

Citation: Wang, Z., Li, X., Lin, L., Bellusci, S., Zhang, J.-S., eds. (2020). The Fibroblast Growth Factor Signaling Pathway in Metabolic Regulation, Development, Disease and Repair After Injury. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-128-2

# Table of Contents


Huan Lin, Chuanren Zhou, Yushu Hou, Qi Li, Guanting Qiao, Yang Wang, Zhifeng Huang and Jianlou Niu


Lei Zheng, Chuan-Li Shen, Jian-Min Li, Yu-Lei Ma, Ning Yan, Xin-Qiao Tian and Ying-Zheng Zhao


Sipin Zhu, Mengji Chen, Min Chen, Jiahui Ye, Yibo Ying, Qiuji Wu, Haicheng Dou, Liyunian Bai, Fangmin Mao, Wenfei Ni and Kehe Yu *104 The Protective Effect of Basic Fibroblast Growth Factor on Diabetic Nephropathy Through Remodeling Metabolic Phenotype and Suppressing Oxidative Stress in Mice* Tingting Wei, Qi Shu, Jie Ning, Shuaijie Wang, Chen Li, Liangcai Zhao, Hong Zheng and Hongchang Gao *116 Progress in Research on the Role of FGF in the Formation and Treatment of Corneal Neovascularization* Mengji Chen, Licheng Bao, Mengying Zhao, Jiarong Cao and Haihua Zheng *122 FGF21 Attenuated LPS-Induced Depressive-Like Behavior* via *Inhibiting the Inflammatory Pathway* Xue Wang, Liyun Zhu, Jian Hu, Ruili Guo, Shasha Ye, Fei Liu, Dongxue Wang, Yeli Zhao, Aiping Hu, Xiaojie Wang, Kaiming Guo and Li Lin *137 Temporospatial Expression of Fgfr1 and 2 During Lung Development, Homeostasis, and Regeneration* Tingting Yuan, Kylie Klinkhammer, Handeng Lyu, Shan Gao, Jie Yuan, Seantel Hopkins, Jin-San Zhang and Stijn P. De Langhe *147 Non-Mitogenic Fibroblast Growth Factor 1 Enhanced Angiogenesis Following Ischemic Stroke by Regulating the Sphingosine-1-Phosphate 1 Pathway* Yuchi Zou, Jian Hu, Wenting Huang, Shasha Ye, Fanyi Han, Jingting Du, Mingjie Shao, Ruili Guo, Jingjing Lin, Yeli Zhao, Ye Xiong and Xue Wang *159 Fibroblast Growth Factor 1 Ameliorates Diabetes-Induced Liver Injury by Reducing Cellular Stress and Restoring Autophagy* Zeping Xu, Yanqing Wu, Fan Wang, Xiaofeng Li, Ping Wang, Yuying Li, Junnan Wu, Yiyang Li, Ting Jiang, Xindian Pan, Xie Zhang, Longteng Xie, Jian Xiao and Yanlong Liu *170 The Role of Fibroblast Growth Factor 10 Signaling in Duodenal Atresia* Matthew L. M. Jones, Gulcan Sarila, Pierre Chapuis, John M. Hutson, Sebastian K. King and Warwick J. Teague *178 SIRT1 Mediates Effects of FGF21 to Ameliorate Cisplatin-Induced Acute Kidney Injury* Qiongzhen Chen, Junfeng Ma, Xiaoning Yang, Qinyao Li, Zhuofeng Lin and Fanghua Gong *191 Fibroblast Growth Factors in the Management of Acute Kidney Injury Following Ischemia-Reperfusion* Lian-Cheng Deng, Tahereh Alinejad, Saverio Bellusci and Jin-San Zhang

# Editorial: The Fibroblast Growth Factor Signaling Pathway in Metabolic Regulation, Development, Disease, and Repair After Injury

Zhouguang Wang1,2\*, Li Lin1\*, Jin-San Zhang1,3, Xingxing Zhong2 , Saverio Bellusci <sup>4</sup> and Xiaokun Li 1\*

<sup>1</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, United States, <sup>3</sup> Division of Oncology Research, Mayo Clinic, Rochester, MN, United States, <sup>4</sup> Cardiopulmonary Institute, Member of the German Lung Center, Justus Liebig University, Giessen, Germany

Keywords: FGF (fibroblast growth factor), regeneration, development, metabolic regulation, ischemareperfusion injury

Editorial on the Research Topic

### Edited and reviewed by:

Alastair Stewart, The University of Melbourne, Australia

#### \*Correspondence:

Zhouguang Wang wzhouguang@gmail.com Li Lin linliwz@163.com Xiaokun Li lixk1964@163.com

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 23 July 2020 Accepted: 24 August 2020 Published: 08 September 2020

#### Citation:

Wang Z, Lin L, Zhang J-S, Zhong X, Bellusci S and Li X (2020) Editorial: The Fibroblast Growth Factor Signaling Pathway in Metabolic Regulation, Development, Disease, and Repair After Injury. Front. Pharmacol. 11:586654. doi: 10.3389/fphar.2020.586654 The Fibroblast Growth Factor Signaling Pathway in Metabolic Regulation, Development, Disease and Repair After Injury

Fibroblast growth factors, or FGFs, are a family of structurally related proteins with diverse functions during embryonic development, tissue repair, cancer, and metabolic homeostasis. In humans, 22 members of the FGFs family have been identified, all of which are structurally related signaling molecules. They have been alternately referred to as "pluripotent" growth factors and as "promiscuous" growth factors due to their multiple actions on a wide range of cell types. Four receptor subtypes of FGFs can be activated by more than 20 different FGF ligands. Thus, the functions of FGFs in developmental processes can include mesoderm induction, anterior-posterior patterning, limb development, neural induction, and neural development.

This Research Topic gathers original research and review papers on the different roles of FGFs/ FGFRs in early development, organogenesis, musculoskeletal biology, nervous system, metabolism, tumorigenesis, intracellular signaling, and emerging research areas. This collection of papers sheds light on the drug development of FGF, with a focus on the new development for FGFs treatment or mechanisms of action, ranging from basic research to clinical translational studies.

The 17 accepted articles consist of 14 Original Research articles and 3 Reviews or Mini-Reviews, which demonstrated roles of FGFs in various diseases including neural diseases, diabetes-related diseases, renal injury, lung diseases, digestive diseases, and vascular diseases.

Four research articles elucidated that FGFs are involved in alleviating neural diseases via different mechanisms. By using 6-OHDA–induced Parkinson's disease (PD) mice model, Zhong et al. found that acid FGF promoted autophagy by inhibiting ER stress-induced TRB3 overexpression during PD development and subsequently ameliorated 6-OHDA–induced neuronal apoptosis (Zhong et al.). Zhu S. et al. reported the anti-apoptotic effect of another FGF member FGF22 in mediating neural disease (Zhu S. et al.). FGF22 treatment was associated with reduced pro-apoptosis proteins and increased recovery of the spinal cord injury in mouse animals. Intriguingly, the number of neurons and expression of an axon regeneration related protein (growth-associated protein 43) were also increased

**5**

after FGF22 administration. The beneficial effects of FGF22 in ER stress-induced spinal cord injury could be partially due to neuron regeneration (Zhu S. et al.). This hypothesis is consistent with what has been claimed by Dong et al. that FGF10 treatment promoted axonal regeneration and functional recovery in sciatic nerve injury rat (Dong et al.). FGF10 was also revealed to prevent Schwann cells from oxidative stress-induced apoptosis, which was probably related to the activation of phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling (Dong et al.). In addition to the roles of antiapoptosis and promoting regeneration, the anti-inflammatory function of FGFs were also involved in repairing neural diseases. Wang et al. treated LPS-induced depression mouse model with recombinant human FGF2 and found that depressive-like behavior was significantly relieved (Wang et al.). The decreased microglial expression of proinflammatory cytokines suggested the involvement of FGF2 in NF-kB suppression (Wang et al.).

Four of these research articles investigated roles of FGFs in diabetes-related diseases, providing new insights into treatment of diabetes and related complications. In Xu et al.'s research, db/db mice revealed improved blood glucose level and diabetes-induced liver steatosis, fibrosis and apoptosis after intraperitoneally injected with FGF1 (Xu et al.). Mechanistic investigations suggested that these effects were the results of attenuated oxidative stress and ER stress (Xu et al.). The anti-oxidative stress function of another FGF member, basic Fibroblast Growth Factor, was shown to play vital roles in ameliorating diabetic nephropathy (Wei et al.). Studies have shown that FGF1 has a wide range of physiological functions, the application in vivo is limited because of the lack of an efficient and safe delivery system. Nanoliposomes and ultrasound targeted microbubble destruction techniques provided hopes into solving this problem. Zheng et al. evaluated the preventive effect of FGF1 loaded nanoliposomes (FGF1-nlip) combined with ultrasoundtargeted microbubble destruction (UTMD) on diabetic cardiomyopathy using ultrasound examination and found that echocardiographic indexes were significantly higher than those in FGF1 and FGF1-nlip treatment groups (Zheng et al.). Islet transplantation is considered a potential therapy for diabetes. However, the extracellular matrix (EXM) proteins essential for islets survival are impaired in the isolation process before islet transplantation. Zhu Q. et al. elucidated the beneficial effects of recombinant human collagen with FGF2 application in islet transplantation. The system provided insights into islet transplantation with a simulated EXM microenvironment for the revascularization and attachment of islets to the transplantation region (Zhu Q. et al.).

On another hand, Tan et al. investigated the molecular pathways underlying the protective effect of FGF10 on renal ischemia-reperfusion (I/R) injury using Sprague-Dawley rat model (Tan et al.). It was shown that FGF10 attenuated I/Rinduced renal epithelial apoptosis by suppressing excessive ER stress in renal I/R injury, and the function was partially mediated by the activation of the MEK–ERK1/2 signaling pathway. Accumulating evidence suggests that FGFs play important roles in renal I/R injury. As summarized in Deng et al.'s review (Deng et al.), FGFs mediate repair process of I/R injury-caused acute kidney injury and could offer a potential therapeutic option in the future. Chen Q. et al. studied the physiological role of FGF21 in cisplatin-treated AKI and it was revealed that recombinant FGF21 significantly improved renal function in cisplatin-induced damage via SITT1 signaling pathway (Chen Q. et al.). Non-mitogenic FGF1 was found to enhance angiogenesis following ischemic stroke by regulating the sphingosine-1 phosphate 1 pathway (Zou et al.). Two reviews summarized the progress of FGF in corneal neovascularization (Chen M. et al.) and fibroblast FGF10 in duodenal atresia (Jones et al.).

In addition to neural diseases, diabetes-related diseases and renal damage, functions of FGFs were also investigated in other diseases. It was claimed by Lin et al. that administration of engineered FGF1 mutant-FGF1DHBS protected liver in alpha naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis mice by reducing hepatic bile acid accumulation (Lin et al.). Yuan et al. found that the temporospatial expression of FGFR1 and FGFR2 varied during lung development, homeostasis, and regeneration, indicating the involvement of FGF signaling pathways in lung development and diseases (Yuan et al.). To overcome the treatment resistance toward problem of the tyrosine kinase inhibitor (TKI) in non-small-cell lung cancer (NSCLC), Chen G. et al. evaluated the efficacy of the compound 15c, a novel dual inhibitor of EGFRL858R/T790M and FGFR1, which revealed that the compound 15c efficiently overcame the EGFR-TKI resistance of NSCLC (Chen G. et al.).

In summary, both the research articles and reviews in this Research Topic are an excellent source of information about the current knowledge in the fibroblast growth factor signaling pathway in metabolic regulation, development, disease, and repair after injury field.

### AUTHOR CONTRIBUTIONS

ZW and XZ wrote this article. LL, J-SZ, SB and XL have made a direct and intellectual contribution to the work. All authors have approved the article for publication.

### FUNDING

This work was supported by grants from Advanced Postdoctoral Programs of Zhejiang (zj2019030 to Z.W.), China Postdoctoral Science Foundation (2019M662015 to Z.W.). National Natural Science Foundation of China (No.81771284, 81971180 to L.L.), Research Unit of Research and Clinical Translation of Cell Growth Factors and Diseases, Chinese Academy of Medical Science (No.2019RU010 to X.L.).

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Wang, Lin, Zhang, Zhong, Bellusci and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# FGF10 Enhances Peripheral Nerve Regeneration *via* the Preactivation of the PI3K/Akt Signaling-Mediated Antioxidant Response

*Lvpeng Dong1,2†, Rui Li2,3†, Duohui Li2, Beini Wang2, Yingfeng Lu1, Peifeng Li1, Fangzheng Yu1, Yonglong Jin1, Xiao Ni1, Yanqing Wu2, Shengnan Yang2, Guanxi Lv2, Xiaokun Li2, Jian Xiao2\* and Jian Wang1\**

*1 Department of Hand Surgery and Peripheral Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 2 Molecular Pharmacology Research Center, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, China, 3 School of Chemistry, Sun Yat-sen University, Guangzhou, China*

### *Edited by:*

*Zhouguang Wang, Albert Einstein College of Medicine, United States*

#### *Reviewed by:*

*Yingnyu Gao, Marshall University, United States Dinghong Zhang, University of California, San Diego,United States*

#### *\*Correspondence:*

*Jian Xiao xfxj2000@126.com Jian Wang jianwang0516@126.com*

*†These authors have contributed equally to this work*

#### *Specialty section:*

*This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology*

*Received: 25 July 2019 Accepted: 23 September 2019 Published: 16 October 2019*

#### *Citation:*

*Dong L, Li R, Li D, Wang B, Lu Y, Li P, Yu F, Jin Y, Ni X, Wu Y, Yang S, Lv G, Li X, Xiao J and Wang J (2019) FGF10 Enhances Peripheral Nerve Regeneration via the Preactivation of the PI3K/Akt Signaling-Mediated Antioxidant Response. Front. Pharmacol. 10:1224. doi: 10.3389/fphar.2019.01224*

The process of axonal regeneration after peripheral nerve injury (PNI) is slow and mostly incomplete. Previous studies have investigated the neuroprotective effects of fibroblast growth factor 10 (FGF10) against spinal cord injury and cerebral ischemia brain injury. However, the role of FGF10 in peripheral nerve regeneration remains unknown. In this study, we aimed to investigate the underlying therapeutic effects of FGF10 on nerve regeneration and functional recovery after PNI and to explore the associated mechanism. Our results showed that FGF10 administration promoted axonal regeneration and functional recovery after nerve damage. Moreover, exogenous FGF10 treatment also prevented SCs from excessive oxidative stress-induced apoptosis, which was probably related to the activation of phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling. The inhibition of the PI3K/Akt pathway by the specific inhibitor LY294002 partially reversed the therapeutic effects of FGF10 both *in vivo* and *in vitro*. Thus, from our perspective, FGF10 may be a promising therapeutic drug for repairing sciatic nerve damage through countering excessive oxidative stress-induced SC apoptosis.

Keywords: fibroblast growth factor 10, axonal regeneration, peripheral nerve injury, oxidative stress, apoptosis

### INTRODUCTION

Unlike the central nervous system (CNS), the peripheral nervous system (PNS) retains a certain regeneration capacity, which enables the regrowth of damaged axons and impaired nerves (Chen et al., 2007). Nevertheless, the regeneration rate of peripheral nerves is slow, and proximal nerve segments rarely regrow over long distances to their target organs. As a result, it affects the quality of life of patients and is a major socioeconomic burden (Sulaiman and Gordon, 2009; Gordon, 2016).

**Abbreviations:** bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; CCK-8, cell counting kit; CNS, central nervous system; DCFH-DA, 2′, 7′‐dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; FGFs, fibroblast growth factors; FGF10, fibroblast growth factor 10; FGF21, fibroblast growth factor 21; HE, hematoxylin and eosin; NGF, nerve growth factor; OCT, optimum cutting temperature; PNI, peripheral nerve injury; PNS, peripheral nervous system; PWT, paw withdrawal threshold; P/S, penicillin/streptomycin; ROS, reactive oxygen species; SCI, spinal cord injury; SCs, Schwann cells; SFI, sciatic function index.

Because nerve regeneration is a complex process that involves interactions among cellular elements, cytokines, extracellular matrix, and growth factors, the complete recovery of injured nerves is difficult (Caillaud et al., 2018).

Schwann cells (SCs), the myelinating cells of the PNS, are essential for nerve regeneration and for the saltatory conduction of action potentials (Scherer and Wrabetz, 2008). Following nerve injury, denervated SCs begin to dedifferentiate, migrate, proliferate, and transform into regeneration-promoting new cells, called repair SCs (Jessen and Mirsky, 2016). This type of SCs is able to generate a favorable microenvironment for axonal growth by clearing degenerated myelin debris and secreting neurotrophic factors (Jessen et al., 2015). Thus, promoting SCs survival and inhibiting apoptosis are vital for maintaining structural and functional integrity following peripheral nerve injury (PNI).

Oxidative stress, characterized by excessive reactive oxygen species (ROS), is a critical initiating factor for PNI (Yang et al., 2016; Ino and Iino, 2017; He et al., 2018). The overproduction of ROS disturbs the oxidation-antioxidant equilibrium and leads to mitochondrial dysfunction, lipid peroxidation, and cell apoptosis. Furthermore, excessive ROS production in SCs causes dysfunction in DNA synthesis, protein expression, and mitochondrial structure (Finkel and Holbrook, 2000; Wang et al., 2013). Therefore, it is essential to inhibit excessive ROS generation to maintain the SC function and the interactions between SCs and other cell types after PNI.

The nuclear factor transcription erythroid-like factor 2 (Nrf2) plays a critical role in regulating redox homoeostasis. The activation of Nrf2 results in the accumulation of some enzymes, such as heme oxygenase‐1 (HO‐1), NAD(P)H:quinone oxidoreductase (NQO1), and superoxide dismutase (SOD2) (Son et al., 2013). Nrf2 is also closely related to cell apoptosis through upregulating the expression of Bcl-2, an antiapoptotic protein. Low levels of Bcl-2 expression and increased expression of proapoptotic proteins, including Bax and caspase-3, are typical markers of cell apoptosis (Niture and Jaiswal, 2011; Niture and Jaiswal, 2012; Niture et al., 2014).

The phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) pathway has been reported to play a major role in the modulation of axonal growth, myelin sheath formation, and SC function in the PNS. The activation of Akt in SCs increases the expression of myelin proteins, including myelin basic protein (MBP) and myelin basic zero (MPZ), which regulates remyelination. In contrast, inhibiting the PI3K/Akt pathway with LY294002 significantly attenuates SCs migration (Lv et al., 2015; Domenech-Estevez et al., 2016). Furthermore, the inhibition of this signaling pathway also obviously decreases the synthesis of proliferating cell nuclear antigen (PCNA; a marker of cell proliferation) in SCs (He et al., 2011). All of these findings show that the PI3K/ Akt pathway modulates the multiple functions of SCs, including migration, proliferation, and remyelination.

FGF10 is a member of the fibroblast growth factors (FGFs) that plays important roles in regulating biological functions such as morphogenesis, proliferation, and the inhibition of apoptosis (Kelleher et al., 2013). FGF10, which was originally identified in rat embryos, mediates biological signaling in a paracrine manner (Itoh, 2016). Furthermore, FGF10 is widely distributed in many organs, such as adipose tissue, the lungs, the limbs, and the prostate, and plays an essential role in regulating cell mitogenesis, proliferation, differentiation, and migration (Min et al., 1998; Thomson and Cunha, 1999; Jimenez and Rampy, 1999; Sakaue et al., 2002). A recent study showed that FGF10 expression in neurons is increased after spinal cord injury (SCI) and that exogenous FGF10 administration induces functional recovery and attenuates the inflammatory response by activating PI3K/Akt signaling in an animal model of SCI (Chen et al., 2017). Another study demonstrated that FGF10 protects neurons and ameliorates cerebral ischemia injury by activating the PI3K/Akt signaling pathway and reducing NF-κB-mediated neuroinflammation (Li et al., 2015b; Li et al., 2016). However, the effects of FGF10 on functional recovery after PNI and the associated molecular mechanism have not been documented to date.

The aim of the present study was to investigate whether FGF10 plays a neuroprotective role in facilitating axonal regeneration and functional recovery after PNI and to explore the related molecular mechanisms. Our results indicate that FGF10 treatment reduces SCs apoptosis, enhances axonal growth and regeneration, and improves functional recovery following PNI. Furthermore, this beneficial effect is most likely regulated by attenuating PI3K/Akt signaling-mediated oxidative stress both *in vivo* and *in vitro*. Collectively, our results suggest that FGF10 performs a certain role and may be a potential agent for the treatment of PNI.

### MATERIALS AND METHODS

### Reagents and Antibodies

FGF10 was obtained from the School of Pharmacy, Wenzhou Medical University (Wenzhou, China). Antibodies against *p*-Akt (#13038), Bax (#14796), Bcl-2 (#2764), and cleaved-caspase-3 (#9664) were obtained from Cell Signaling Technology. Antibodies against MBP (ab40390), NF200 (ab4680), Nrf2 (ab62352), NQO1 (ab34173), MPZ (ab31851), and Akt (ab179463), Histone H3 (ab176842) and the PI3K/Akt inhibitor LY294002 (ab120243), were purchased from Abcam. Antibodies against GAPDH (10494-1-AP), HO-1 (10701-1-AP), SOD2 (24217-1-AP), and PCNA (10205-2-AP) were purchased from Proteintech. An antibody against S100 (sc-53438) was obtained from Santa Cruz Biotechnology.

### Animals

Male SD rats (200~220 g) were purchased from the Laboratory Animal Center of Fujian Medical University (Fujian, China). A temperature of 23 ± 2 °C, a humidity of 35-60%, and a 12:12 h light-dark cycle were applied as standardized laboratory conditions for housing all rats. Meanwhile, they were provided with food and water and were habituated to these conditions for at least 7 days before the experiment. The use of animals in this study was approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University, Wenzhou, China. The living conditions and experimental procedures were conducted in accordance with the National Institutes of Health Guideline concerning the Care and Use of Laboratory Animals.

### PNI Model and Drug Injection

The procedure for generating the animal model was described previously (Li et al., 2017b). In brief, each animal was anaesthetized by an intraperitoneal injection of 10% chloral hydrate (3.5 ml/ kg). An incision was made in the skin to expose the right sciatic nerve. Then, this exposed nerve was crushed with two vascular clips (Oscar, China). The vascular clips clamped the sciatic nerve 7 mm proximal from the sciatic notch at the two ends with 30 g of force for 2 min. Thereafter, the incised skin was sutured with a 4-0 nonabsorbable suture.

Following surgery, all the animals were randomly allocated to four groups (n = 10 for each): the sham group, the PNI group, the FGF10 group, and the FGF10+LY294002 group. The sham group received the same surgical procedure to expose the sciatic nerve but did not undergo compression injury. For the FGF10 group, each rat was injected intramuscularly with 5 μg of FGF10 solution (25 μg/ml) once daily for 28 consecutive days. For the FGF10 + LY294002 group, after the injection of FGF10 solution, each rat was also intravenously injected with LY294002 (a PI3K inhibitor, 0.3 mg/kg/day) (Chen et al., 2017). The rats in the sham and PNI groups were only injected with the same volume of saline solution. After 28 days, all rats were sacrificed to harvest the sciatic nerve for pathology index analysis.

### Von Frey Test and Walking Track Analysis

Rats were habituated to an elevated metal mesh floor for at least 1 h before their responses to mechanical stimulation were tested with von Frey filaments (NC12775; North Coast Medical Inc, CA, USA). von Frey filaments with forces ranging from 2–180 g were applied in ascending order to the third and fourth toes of the plantar surface of the hind paw until they bent. The filaments were repeated five times, and the results of paw withdrawal were recorded.

The gait of the rats along a 100 × 10 × 15 cm corridor, the bottom of which was covered with a 90 × 10 cm piece of white paper, was analyzed. Red ink was painted on the hind paws of the rats. The sciatic functional index (SFI) was calculated based on the colored footprints according to the following formula by Bain et al. (1989): SFI = −38. 3 × (EPL- NPL)/NPL + 109.5 × (ETS -NTS)/ NTS + 13.3 × (EIT - NIT)/NIT-8.8, where E is the right hind limb; N is the left hind limb; PL is the longitudinal distance of the longest footprint; IT is the distance between the second and fourth toes; and TS is the distance between the first and fifth toes. An index of approximately 0 reflects normal function, whereas an index of −100 indicates complete impairment. Two observers who were unaware of the experimental procedures performed the test from day 1 to day 28 following the surgical procedures.

### RSC96 Culture and Treatment

The RSC96 SC line was obtained from ScienCell Research Laboratories. The cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin solution (P/S) in a humidified incubator (37°C, 5% CO2). After two passages, the cells were seeded in 96-well plates (5 × 103 cells/well), and various concentrations of FGF10 (0.043, 0.43, 4.3, 43 nm) were added for 2 h. Then, the medium was supplemented with 100-μm H2O2 for another 2 h. To further evaluate the effect of PI3K/Akt activation on oxidative injury, cells were pretreated with the PI3K inhibitor LY294002 (20 μm) (Wang et al., 2012) for 2 h before the addition of FGF10.

To evaluate cell survival, a cell counting kit (CCK-8, Beyotime Institute of Biotechnology, China) was used. The test was performed according to the manufacturer's instructions. In brief, 10 μl of CCK-8 solution were added to each well, and the cells were incubated for 2 h at 37°C. The optical density was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA). All experiments were repeated at least three times.

### Assays of Intracellular ROS Generation

Intracellular ROS generation was measured by an ROS Assay Kit (DCFH-DA, S0033, Beyotime, China). SCs were plated in 6-well plates at a density of 1×105 cells/ml for 24 h. After treatment with H2O2 with/without FGF10 plus LY294002, 10 μm 2′, 7′‐ dichlorodihydrofluorescein diacetate (DCFH-DA) were added to the culture medium for 20 min. Subsequently, the fluorescence of the cells in five randomly fields for each group was imaged with a Nikon ECLIPSE 80i microscope (Nikon, Japan). ImageJ software was used for quantitative analysis.

To further analyze the change of ROS, the DCF fluorescence was detected by a fluorescence microplate reader (Genios, TECAN) at 485-nm excitation and 53-nm emission.

### Hematoxylin-Eosin (He) Staining

The sciatic nerve tissues were harvested following the established methods and embedded in paraffin (Li et al., 2017b). The nerves were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight and embedded in paraffin the next day. The longitudinal sections were cut into 5-μm thick sections for HE staining; the sections were dyed with hematoxylin for 5 min and with eosin for 10 min. The images were captured using a Nikon ECLIPSE 80i microscope (Nikon, Japan).

### Immunoblotting

Immunoblotting analysis of sciatic nerves and cell extracts were performed as described previously (Zhang et al., 2014; Li et al., 2018b). Briefly, cells and sciatic nerves were lysed using RIPA with protease (Boster, AR0101/AR0103) and phosphatase inhibitors (Applygen, P1260). The lysate was centrifuged at 12 000 g for 20 min. For PCNA detection, the sciatic nerve segments was mechanically homogenized. Then, cytosol and nuclear proteins were extracted using the nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology, Wuhan, China) and centrifuged at 5000 r.p.m. for 10 min at 4°C to extract the nuclear components (Li et al., 2015a). The protein concentration was determined by bicinchoninic acid (BCA) reagents (Thermo Fisher Scientific, Rockford, IL, USA). Sixty micrograms of protein was separated by 8-12% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4°C. The antibodies included *p*-Akt (1:1000), Akt (1:1000), Nrf2 (1:1000), NQO1 (1:1000), HO-1 (1:1000), SOD2 (1:2000), MPZ (1:1000), S100 (1:200), PCNA (1:1000), Bcl-2 (1:1000), Bax (1:1000), Histone H3 (1:1000) and GAPDH (1:10000). The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. The visualization of signals and band intensity were analyzed by chemiluminescence using a gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Three independent samples were analyzed.

### Immunofluorescence Staining

For animal tissues, 5-μm thick longitudinal sections were washed in PBS three times after being embedded in optimum cutting temperature (OCT) compound. For cells, SCs were fixed in 4% paraformaldehyde in PBS for 20 min. Then, the tissue or cell samples were blocked in 5% bovine serum albumin (BSA) containing 0.1% Triton X-100 for 30 min. After incubation with primary antibodies overnight at 4°C, the slides were incubated with a fluorochrome-labeled secondary antibody for 1 h. The primary antibodies included NF200 (1:10000), MBP (1:1000), and Nrf2 (1:1000). The nuclei were stained with DAPI for 7 min. Image acquisition was performed with a Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan).

### Statistical Analysis

All data are expressed as the mean ± SEM. Analysis of statistical significance was performed by one-way ANOVA with Tukey's posttest with GraphPad Prism (GraphPad Software version 6.0, La Jolla, CA, USA). *P*-values less than 0.05 were considered statistically significant.

### RESULTS

### Fgf10 Promotes Functional Recovery

To assess whether the injection of FGF10 increase sensory and motor recovery, walking track analysis and paw withdrawal thresholds were evaluated every week for 4 weeks following surgery. The walking tracks indicated no significant difference among the three groups in the first 2 weeks after surgery. However, the SFI value of the FGF10-treated group began to increase and was significantly greater than that of the PNI group at weeks 3 and 4 (**Figures 1A**, **B**, \*\*\**P* < 0.001). Nevertheless, compared with the group that received FGF10 only, the group that received FGF10 and LY294002 co-administration exhibited motor functional recovery by week 4 (**Figures 1A**, **B**, \*\*\**P* < 0.001).

To evaluate sensory functional recovery to mechanical stimuli, all animals from the four groups were subjected to the von Frey test. As illustrated in **Figure 1C**, the sensory recovery of all groups was rather poor 1 week after crush injury, but recovery in the FGF10 group was better than that in the PNI group (\*\*\**P* < 0.001). The mechanical thresholds gradually decreased in the subsequent weeks. Although the results seemed to show that sensory recovery in the FGF10 group was inferior to that in the PNI group and FGF10 + LY294002 group, the withdrawal threshold in the two latter groups was even lower than that in the control group, suggesting that the PNI rats treated with/without FGF10+LY294002 exhibited hyperalgesia and that FGF10 treatment rescued PNI-induced hyperalgesia at a later stage of nerve recovery. Taken together, these results suggest that FGF10 continuously enhances the recovery of locomotor and sensory function in acute PNI.

### Fgf10 Facilitates Nerve Regeneration

The histological recovery of the injured nerve in each group was evaluated by HE staining and coimmunostaining for neurofilament 200 (NF-200; represents axonal growth) and MBP (a marker of myelination). HE staining revealed that the axonal fibers in the PNI group were scarce and irregular, while the nerve fibers in the FGF10-treated group appeared remarkably regenerated and regular (**Figure 2A**). Double immunofluorescence staining for MBP and NF200 showed that the density of regenerated axons and myelin in the FGF10 group was significantly higher than that in the PNI group and that the degree of regeneration was enough to induce a morphology similar to that of normal nerves (**Figures 2B**–**D**). In contrast, the immunoreactivity of NF200 and MBP was greatly attenuated after the coadministration of LY294002 and FGF10.

\*\*\**P* < 0.001. FGF10 group vs FGF10+LY294002 group: \*\*\**P* < 0.001.

following injury. The data are presented as the mean ± SEM, n = 5. Sham group vs PNI group: \*\*\**P* < 0.001. FGF10 group vs peripheral nerve injury (PNI) group:

### Fgf10 Increases Functional Protein Secretion

S100 is a SC marker that regulates cellular metabolism, motility and proliferation. Myelin protein zero (MPZ; also called P0) is a major extrinsic membrane protein of myelin in the PNS. MPZ function includes forming myelin and maintaining compact myelin morphology. PCNA is a nucleoprotein that is a marker of cell proliferation. The expression of these proteins was quantified using western blotting analysis. As shown in **Figure 3A**, the protein expression of S100, MPZ and PCNA in the FGF10 group was significantly increased compared with that in the PNI group, while this effect was reversed by the injection of LY294002. Quantitative analysis also showed the same trend (**Figures 3B**–**D**). These data reveal that the beneficial effect of FGF10 is able to upregulate the functional expression of these proteins and that this effect further contributes to SC remyelination and axonal regeneration.

### FGF10 Inhibits the Excessive Expression of Oxidative Stress- and Apoptosis-Related Proteins by Activating PI3k/Akt Signaling

To test whether FGF10 treatment inhibits PNI-induced oxidative stress in the sciatic nerve, the expression of oxidative stressrelated proteins, including Nrf2, NQO1, SOD2 and HO-1, was detected by western blotting. The levels of these oxidative stressassociated molecules were slightly increased by PNI. FGF10 treatment further increased the production of these antioxidant proteins to a large degree **(Figures 4A**–**E**).

Previous studies have demonstrated that the recovery of neurological deficits is closely related to the activation of the PI3k/Akt pathway (Wu et al., 2016; Sang et al., 2018). Inspired by this fact, we measured the *p*-Akt and Akt levels using western blotting. Here, we found that the levels of Akt phosphorylation were slightly increased after PNI, and these levels were further

upregulated after PNI rats received FGF10 treatment, which was also confirmed by the *p*-Akt/Akt ratio. However, LY294002 dramatically inhibited this effect on the expression of these proteins (**Figures 5A**, **B**). Nrf2 and HO-1 showed nearly the same trend (**Figures 5A**, **C**, **D**).

We also detected the expression of apoptosis-related proteins (including Bax and Bcl-2) to evaluate whether the anti-oxidative capability of FGF10 helps to decrease cell apoptosis following PNI. Western blotting showed that the level of the pro-apoptotic protein Bax was down-regulated and that the level of the anti-apoptotic protein Bcl-2 was up-regulated in the FGF10 group when compared to the PNI group (**Figures 5E**–**G**). However, the administration of LY294002 partially abolished the effects of FGF10. These results confirm that the antioxidant and anti-apoptotic properties of FGF10 may be involved in the activation of PI3k/Akt signaling.

### Fgf10 Reduces Sc Apoptosis *in Vitro*

To further confirm the protective effect of FGF10, SCs were exposed to 100-μm H2O2 alone or in combination with various concentrations of FGF10. The CCK-8 results showed that cell viability increased as the FGF10 concentration increased and that 4.3-nm FGF10 was the most effective concentration (**Figure 6A**). Therefore, this concentration of FGF10 was chosen for subsequent experiments. Double immunostaining for cleaved-caspase-3 and S100 showed that FGF10 significantly reduced the cleaved-caspase-3 signal intensity, and LY294002 partially reversed this effect (**Figures 6B**, **D**). Consistent with the immunofluorescence, SC apoptosis in all four treatment groups was further confirmed *via* detecting Bax and Bcl-2 expression levels by western blotting (**Figures 6C**, **E**, **F**). These data suggest that FGF10 maintains SC bioactivity under H2O2 exposure.

### Fgf10 Alleviates Oxidative Injury Through Pi3k/Akt Signaling *in Vitro*

To investigate the molecular mechanism by which FGF10 protects SCs against H2O2-induced apoptosis *in vitro*, we first detected the *p*-Akt and Akt levels using western blotting, the change in the *p*-Akt/Akt ratio was slightly increased in the H2O2 group and was further increased in the FGF10-treated

group. This upregulation was reversed by LY294002 treatment. Nrf2 showed nearly the same trend (**Figures 7A**–**C**).

The change in ROS levels in each group since ROS represents the core component of the biology of oxidative stress. Compared with that in the H2O2-treated group, the fluorescence intensity was lower in the FGF10 group but was significantly higher in the FGF10+LY294002 group (**Figures 7D**, **F**). The intracellular ROS level was as similar change as the fluorescence intensity (**Figure 7G**). However, Nrf2 expression was slightly increased in the H2O2 group, which further increased in the FGF10-treated group. This upregulation was markedly reduced after the addition of LY294002 (**Figures 7E**, **H**).These results reveal that FGF10 may have the potential to enhance antioxidant ability *via* activating PI3K/Akt signaling.

### DISCUSSION

Nerve recovery in the PNS is a complex process that triggers a sequence of events, including axonal regeneration, SC proliferation and migration, macrophage infiltration, and angiopoiesis, within the lesion area. During this process, the FGF family is particularly important for directing cellular activity and tissue remodeling. FGFs participate in SC dedifferentiation, proliferation, and remyelination during axonal regrowth (Chen et al., 2007; Jessen et al., 2015). FGF secretion is essential for maintaining neuronal survival after PNI. Furthermore, numerous studies have reported that FGFs, such as basic FGF (bFGF) and nerve growth factor (NGF), are essential for supporting neurite outgrowth and functional recovery during nerve regeneration (Grothe et al., 2006; Sun et al., 2009; Chen et al., 2010; Takagi et al., 2012; Li et al., 2017a). However, there is no knowledge of whether FGF10 promotes nerve regeneration after PNI.

In this article, we reported a new role for FGF10 in improving sensory and motor functional recovery, enhancing axonal regrowth and remyelination, and increasing the expression of functional proteins after traumatic PNI. Moreover, FGF10 induced neuroprotection and neuranagenesis is associated with attenuating the acute activation of oxidative stress and apoptosis in SCs, which is likely regulated by the activation of PI3K/Akt signaling. These findings indicate that FGF10 may be regarded as a potential therapeutic agent for peripheral nerve reconstruction after injury.

FGF10, a typical paracrine growth factor, is also essential for tissue development and regenerative medicine through specifically binding to the epithelial receptor FGFR2b (Zhang et al., 2006). The exogenous supplementation of FGF10 has been shown to prevent the formation and development of numerous diseases, including wound healing deficits, cardiovascular diseases, metabolism syndrome, and acute kidney injury (Konishi et al., 2006; Rochais et al., 2014; Li et al., 2017c; Tan et al., 2018). Previous studies have shown that FGF10 protects neurons against inflammation-induced apoptosis during SCI through the activation of FGFR2/PI3K/Akt signaling (Chen et al., 2017). However, the effect of FGF10 in acute PNI *in vivo* is unknown. In the present study, we reported that FGF10 is sufficient to continuously enhance motor and sensor recovery, improve nerve morphological recovery and reconstruction, and reduce SC apoptosis. These advantages suggest that FGF10 may be a potential therapeutic agent for peripheral nerve repair.

Oxidative stress is a redox-reactive imbalance in which the generation of oxygen-free radicals and ROS is far greater than the formation of antioxidative entities. During PNI, excessive oxidative stress activation is closely linked to SC apoptosis and axonal atrophy, which is unfavorable for nerve repair (Mirzakhani et al., 2018; Ullah et al., 2018; Lu et al., 2019). Numerous lines of evidence have implicated PI3K/Akt signaling in modifying oxidative stress (Pan et al., 2017; Liu et al., 2018; Li et al., 2018a; Gong et al., 2018).

vs H2O2 group: \**P* < 0.05, \*\**P* < 0.01, \*\*\**P* < 0.001. FGF10 group vs H2O2+LY294002 group: \*\*\**P* < 0.001.

Hyperglycemia-induced oxidative stress can be negatively regulated by PI3K/Akt signaling (Pan et al., 2017). Furthermore, previous research has shown that the neuroprotective effect of fibroblast growth factor 21 (FGF21) in promoting neuronal survival and neurofunctional recovery after brain injury is mediated by the

activation of PI3K/Akt signaling (Lu et al., 2019). In addition, the PI3K/Akt signaling pathway also participates in neuronal differentiation, survival and synaptic function (Li et al., 2006; Ketschek and Gallo, 2010; Fu et al., 2014). Given the important role of PI3K/Akt signaling in promoting cell survival and resisting

cellular stress, we hypothesize that PI3K/Akt signaling may act as an important antioxidant mechanism for regulating FGF10-induced neuroprotection and neuranagenesis following PNI.

Here, we found that the levels of stress-related proteins, including Nrf2, NQO1, SOD2, and HO-1, and the ratio of *p*-Akt/ Akt increased markedly; meanwhile, ROS generation and the level of apoptosis were significantly decreased after the administration of FGF10. Suppressing Akt phosphorylation with LY294002 partially reversed these therapeutic effects. This result might be explained that the nerve tissue had the capable of synthetizing and secreting certain amounts of antioxidant enzymes to resist PNI-induced oxidative damage, although this capability was not satisfied the demand of cellular antioxidant defense (Ornitz and Itoh, 2015; Yu et al., 2017). After administration of FGF10 to the PNI rats, the interaction between FGF10-FGFR pairs activated the downstream of signal transduction pathways, such as PI3K/Akt, to dramatically stimulate the expression of antioxidant proteins which further enhanced antioxidative capability and, therefore, promoting SCs proliferation and nerve regeneration. Consequently, FGF10-induced nerve regeneration and functional recovery may occur through PI3K/Akt signalingmediated antioxidant enhancement.

Previous studies reported that oxidative-induced SCs apoptosis is a critical mechanism of neurodegenerative diseases (Purves et al., 2001). In mammalian cells, Bax and Bcl-2 are involved in the regulation of apoptosis. Among them, Bax belongs to proapoptotic gene, whereas Bcl-2 is antiapoptotic gene. Abnormal oxidative stress activity triggers a decreased expression of Bcl-2 and an increased expression of Bax, which severely influences cell survival and induce cell apoptosis (Jezek and Plecitá-Hlavatá, 2009). Moreover, the excessive ROS-induced apoptosis after PNI is closely associated with the ratio of activity of Bax/Bcl-2 (Zhang et al., 2016). In our study, we observed that the downregulation of Bcl-2 and the upregulation of Bax after PNI were reversed after treatment of FGF10, while this effect was partially abolished when combination of FGF10 and LY294002 together. This result indicates that ROS-induced apoptosis after PNI is probably regulated by PI3K/Akt signaling.

In conclusion, our data indicate that the administration of FGF10 can be effective in facilitating SC proliferation, axonal regeneration, and functional recovery after sciatic nerve injury. In addition, the neuroprotective effect of FGF10 treatment is likely associated with suppressing excessive oxidative stress-induced

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cell apoptosis *via* activating PI3K/Akt signaling. Thus, our research may provide an alternative therapeutic strategy for utilizing FGF10 for treating acute traumatic PNI.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the manuscript/supplementary files.

### ETHICS STATEMENT

The animal study was reviewed and approved by Male SD rats (200~220g) were purchased from Laboratory Animal Center of Fujian Medical University (Fujian, China). The temperature between 23 ± 2°C, humidity between 35 and 60%, and a light-dark cycles with the ratio of 12:12h were applied as the standardized laboratory conditions for housing all the rats. Meanwhile, they were provided with food and water, and adjusted to this condition for at least 7 days before experiment. The animals used in this study were approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University, Wenzhou, China. The living condition and experimental procedures were conducted in accordance to the National Institutes of Health Guideline concerning the Care and Use of Laboratory Animals.

### AUTHOR CONTRIBUTIONS

XJ and WJ conceived and designed the research. DL, LD, and WB performed the experiments. DL and LR performed the statistical analysis and wrote the paper. LY, LF, YF, NX, JY, WY, YS, LG, and LX provided assistance with the experiments. All authors discussed the drafting of the manuscript.

### FUNDING

This work was supported by grants from the National Natural Science Foundation of China (81802238,81972150, 81722028), Zhejiang Provincial Natural Science Foundation of China (LR18H50001) and the Project of Wenzhou Science and Technology Bureau(2018Y0498,2015Y0416, 2015Y0235).


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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor is currently organizing a Research Topic with one of the authors, XL, and confirms the absence of any other collaboration.

*Copyright © 2019 Dong, Li, Li, Wang, Lu, Li, Yu, Jin, Ni, Wu, Yang, Lv, Li, Xiao and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Autophagy Activation Is Involved in Acidic Fibroblast Growth Factor Ameliorating Parkinson's Disease via Regulating Tribbles Homologue 3

*Xingfeng Zhong1,2,†, Beini Wang3†, Guanyinsheng Zhang1†, Yuan Yuan3, Xiaoli Hu1, Jun Xiong3, Peipei Zheng3, Yaqian Liu3, Ke Xu4, Jian Xiao3\*, Yanqing Wu4\* and Junming Ye1\**

1 Department of Anesthesia, The First Affiliated Hospital, Gannan Medical University, Ganzhou, China, 2 Department of Anesthesia, Affiliated Hospital of Guizhou Medical University, Guiyang, China, 3 Molecular Pharmacology Research Center, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, China, 4 The Institute of Life Sciences, Wenzhou University, Wenzhou, China

### Edited by:

Zhouguang Wang, Albert Einstein College of Medicine, United States

### Reviewed by:

Yingnyu Gao, Marshall University, United States Dinghong Zhang, University of California, San Diego, United States

#### \*Correspondence:

Jian Xiao xfxj2000@126.com Yanqing Wu yqwu220946@yeah.net Junming Ye yjm7798@sina.com

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 03 September 2019 Accepted: 08 November 2019 Published: 02 December 2019

#### Citation:

Zhong X, Wang B, Zhang G, Yuan Y, Hu X, Xiong J, Zheng P, Liu Y, Xu K, Xiao J, Wu Y and Ye J (2019) Autophagy Activation Is Involved in Acidic Fibroblast Growth Factor Ameliorating Parkinson's Disease via Regulating Tribbles Homologue 3. Front. Pharmacol. 10:1428. doi: 10.3389/fphar.2019.01428

Parkinson's disease (PD) is a degenerative disorder of the central nervous system, resulting in loss of dopamine neurons. Excessive endoplasmic reticulum (ER) stress and autophagy dysfunction play a crucial role on Parkinson's disease (PD) development. It has been showed that acidic fibroblast growth factor (aFGF) alleviates the development of PD by inhibiting ER stress. But the role of autophagy and its relationship with ER stress during aFGF treatment for PD has not been elucidated. We found that both aFGF and rapamycin (Rapa) improved 6-Hydroxy Dopamine (6-OHDA)-induced PD development as shown with histomorphology results in striatum and substantia nigra (SNpc). Additionally, aFGF promoted autophagy with increasing mTOR and decreasing p62 expressions, and then exerts its neuroprotective role in 6-OHDA-treated PC12 cells, which were abolished by chloroquine (CQ) treatment. Moreover, 4-phenylbutyric acid (4-PBA) administration inhibited the expressions of autophagy markers during 6-OHDA-treated PC12 cells, which was similar with aFGF treating PC12 cells under 6-OHDA condition. Furthermore, we had detected the expressions of CHOP and its downstream factor, tribbles homologue 3 (TRB3), a pro-apoptotic protein. We found that TRB3 and CHOP expressions were significantly downregulated after treating with aFGF and 4-PBA in 6-OHDA-treated PC12 cells and PD model. Taken together, this study has demonstrated that aFGF treatment ameliorates 6-OHDA-induced elevated ER stress and subsequently suppression of autophagy via inhibiting TRB3 activation, and consequently ameliorates 6-OHDA-induced neurotoxicity.

Keywords: Parkinson's disease, acidic fibroblast growth factor, autophagy, endoplasmic reticulum stress, tribbles homologue 3

## INTRODUCTION

Parkinson's disease (PD) is a kind of neurodegenerative disease, which is associated with the abnormally accumulated α-synuclein (α-syn) in the substantia nigra pars compacta (SNpc). In the early study of PD development, gene mutation of α-syn on the autosome caused excessive aggregation damage (Fujiwara et al., 2002; Hasegawa et al., 2002; Samuel et al., 2016; Longo et al., 2017). The cellular mechanisms, include autophagy (Magalhaes et al., 2016), endoplasmic reticulum

1 **20** (ER) stress (Kuang et al., 2010; Ning et al., 2019), oxidative stress (Ding et al., 2018; Mamelak, 2018), and others, are involved in the amelioration of misfolded protein. Thus, regulation of cellular stress may be the target for treating PD.

Acidic fibroblast growth factor (aFGF or FGF1) is an important member of fibroblast growth factors, which can be synthesized and released from neuron (Walicke and Baird, 1988). aFGF exerts neuroprotective role in the peripheral and central nervous systems (Tsai et al., 2015; Wu et al., 2017; Aiki et al., 2018; Ko et al., 2018). The previous studies have showed that aFGF can attenuate 6-OHDA-induced dopaminergic neuron toxicity (Wei et al., 2014). In addition, aFGF protects human primary neurons against gp120 toxicity, and overexpression of aFGF alleviates the neurodegeneration of gp120 mice (Everall et al., 2001; Crews et al., 2009). The mechanism underlying aFGF treating for PD is through inhibiting ER stress (Wei et al., 2014). Promotion of autophagy is the mechanism during aFGF treating for spinal cord injury (Li et al., 2018). However, there is no studies to reveal on role of autophagy, and its' relationship with ER stress during aFGF treating for PD.

Autophagy is an adaptive process in body, which degrades and recycles the abnormal cytoplasmic components and organelles to provide nutrients for cells. Hyperactivation of autophagy triggers cell death program. In general, autophagy regulates cell fate depending on cell type, specific environment and predisposing factors (Jin et al., 2012). Accumulating evidences suggest that autophagy is impaired in PD (Lee et al., 2015). Under normal condition, a small amount of aggregated α-syn will be degraded by the giant autophagy pathway (Cao et al., 2017), however, the mutantion of α-syn can inhibit the degradation process of autophagy (Song et al., 2014; Winslow and Rubinsztein, 2014; Lohmann et al., 2019). Previous studies have also found that the enhancement of autophagy can alleviate the toxic effect of α-syn mutation on midbrain dopaminergic neurons (Decressac et al., 2013). These studies indicate that promotion of autophagy is very important for PD treatment.

The endoplasmic reticulum (ER) is responsible for the synthesis and assembly of protein in cell. Once the misfolded protein is accumulated, it triggers an unfolded protein response (UPR), but an excessively sustained response to decompensation leads to the appearance of ER stress, which in turn causes cell death. Recent studies have shown that inhibition of ER stress protects dopaminergic neurons (Shan et al., 2019). Neuronal apoptotic kinase, known as tribbles homologue 3 (TRB3), is a novel protein that induces cell death by ER stress, which is also related to the regulation of autophagy. Normally, the expression of TRB3 in brain is extremely low. Once stress is triggered, TRB3 is sharply increased. Moreover, the expression of TRB3 is significantly increased during development of PD (Aim et al., 2015) and AD (Saleem and Biswas, 2017). However, it is unclear whether aFGF alleviates PD development by suppressing TRB3 expression.

Multiple studies have also shown a crosstalk between ER stress and autophagy (Li et al., 2017; Yin et al., 2017; Zhou et al., 2017). When ER stress occurs, autophagy can be triggered by unfolded protein reactions (UPR) to remove excessive unfolded proteins and damaged organelles, and maintain cell homeostasis.

Moreover, TRB3 plays a key bridge connection between ER stress and autophagy (Salazar et al., 2009; Aim et al., 2015; Tang et al., 2015; Ord and Ord, 2017). Therefore, we have used 6-OHDA to induce PD model or PC12 cells model *in vivo* and *in vitro*, and investigated the role of autophagy regulation during aFGF treating for PD *via* treating with aFGF, rapamycin (Rapa) autophagy inducer and chloroquine (CQ)-autophagy inhibitor. Moreover, 4-phenylbutyrate (4-PBA)-ER stress inhibitor is administrated to investigate the relationship between ER stress and autophagy during these processes.

### MATERIALS AND METHODS

### Animals and Surgical Procedures

One hundred ten adult male SD rats (220–250 g) were purchased from the Animal Center of the Chinese Academy of Science (Shanghai, China). The rats were housed under a 12-h light/dark cycle at 21–23°C and provided access to food and water *ad libitum*. All procedures were approved by Laboratory Animal Ethics Committee of Wenzhou Medical University. After anesthetizing with 10% chloral hydrate, the head of animal was shaved and sterilized. Then, the rat was fixed on the rat brain stereogram (KOPF, Germany), and the 5% compound lidocaine cream was applied to the front of head. After the drug was absorbed, the longitudinal cleavage revealed the fascia, followed by 3% hydrogen peroxide exposing the front point on the skull. The 6-OHDA (10 μl, 2 μg/ul dissolved in 0.2% ascorbic acid, Sigma-Aldrich, St. Louis, MO, USA) was injected into the dental drill hole at the right side of position the striatum (coordinates: A: anterior +0.7 mm, L: 2.8 mm from the midline, H: +5.5 mm) using micro syringe. The injection speed was 10 nl/s, and the needle was left for 10 min after injection. The sham-operation model was performed same surgical procedures and injected an equal volume of 0.2% ascorbic acid.

### Behavioral Test

One week after the right striatum stereotactic injection of 6-OHDA, the animals were tested for rotational behavior. Rats were intraperitoneally injected with apomorphine hydrochloride (0.5 mg/kg), as a dopamine receptor agonist, the dopamine receptor on the injured side is up-regulated, which can cause the rotation of the rats in Parkinson's disease to the healthy side. The device was used to detect the rotational behavior of the rats. The number of rotations of the rats to the opposite side of the injury was recorded within 30 min. The successful PD model rotated to the left side more than 7 turns/min. Then, 72 successful Parkinson's disease rats were randomly divided into six groups (n = 12): PBS, aFGF (80 μg/kg/day, tail vein), CQ (50 mg/kg/ day, intraperitoneally), Rapa (1.5 mg/kg/day, intraperitoneally), aFGF + CQ, and 4-PBA (100 mg/kg/day, intraperitoneally). In addition, the sham-operation group (n = 12) was injected with PBS in the tail vein for 2 weeks. The rotational behavior test of rats was performed at 1, 2 and 3 weeks after surgical procedure and treatment.

## Cell Culture and Treatment

PC12 cells were purchased from the Cell Storage Center of Wuhan University (Wuhan, China). PC12 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin). They were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were seeded in a 96-well plate at density of 1 × 104 , then treated with 6-OHDA at different doses of 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM and 200 μM for 24 h. Then, the cells were incubated with CCK-8 assay (Cell counting kit-8) at 37°C for 2 h, and measured the optical density at 450 nm. The experiments were divided into six groups: PBS, aFGF (20 ng/ ml), CQ (75 μM), aFGF + CQ, Rapa (1 μM), and 4-PBA (1 mM).

### Hematoxylin and Eosin (H&E) Staining and Nissl Staining

After anesthesia, the brain was dissected immediately and placed on the ice, then fixed 4% with paraformaldehyde (PFA) for 24 h in 4 °C. Subsequently, paraffin-embedded brain regions containing substantia nigra and striatum were sliced at 4 um thickness. For Nissl staining, after dewaxed and hydrated, tissue sections were stained with cresol violet and Nissl differentiation solutions according to the instructions (Beyotime) and acquired the image using light microscopy. For H&E staining, the sections were stained with hematoxylin and eosin reagent, and observed under light microscope.

### Immunohistochemical Staining

After anesthesia, the brain was dissected immediately and placed on the ice, then fixed 4% with paraformaldehyde (PFA) for 24 h in 4°C. Subsequently, paraffin-embedded brain regions containing substantia nigra and striatum were sliced at 4 um thickness. After dewaxed and hydrated, the sections were incubated with 3% hydrogen peroxide for 15 min, and antigen retrieval at high temperature and pressure. Then, after blocking with 5% bovine serum albumin (BSA) for 30 min at 37 °C, the sections were incubated overnight at 4 °C with the following primary antibodies: TH (1:500, Abcam), α-syn (1:250, Millipore), CHOP (1:100, Proteintech), TRB3 (1:50, Santa Cruz Biotechnology), mTOR (1:250, Cell Signaling Technology), p62 (1:250, Abcam). Then, the sections were washed three times with PBST and incubated the horseradish peroxidase-conjugated secondary antibody at 37 °C for 2 h. Finally, these sections were reacted with 3,3-diaminobenzidine (DAB, 1:20 dilution) for 5–10 min. Hematoxylin was used to stain the nucleus. The sham operation group was used as a negative control. The signals were analyzed by counting the number of positive cells in region of interest at 400× of microscope of Nikon ECLPSE 80i (Nikon, Tokyo, Japan). The optical density of TH, α-syn, CHOP, TRB3, mTOR and p62 was randomly selected from five samples in each sample.

### Immunofluorescence Staining

The cells were inoculated on coverslips, and then fixed with 4% PFA for 25 min at 4 °C. Subsequently, the cells were washed in PBS three times. The cells were incubated with 5% BSA in PBS containing 0.1% Triton X-100 for 30 min at room temperature. Then, the sections were incubated at 4°C overnight with the following primary antibodies: CHOP (1:50, Proteintech), TRB3 (1:50, Santa Cruz Biotechnology), mTOR (1:250, Cell Signaling Technology), and p62 (1:250, Abcam). After washing with PBS three times for 5 min, the sections were incubated with AlexaFluor-488 or AlexaFluor-647 donkey anti-rabbit/mouse secondary antibodies for 1 h at room temperature. After washed with PBS, the sections were staining the nuclei with DAPI and captured the image on a confocal fluorescence microscope (Nikon, A1 PLUS, Tokyo, Japan).

### Tunel Staining

TUNEL staining was performed using the ApopTag Fluorescein Direct *In Situ* Apoptosis Detection Kit (Roche, Basel, Switzerland). According to the standard protocol, the fixed cell slides were incubated with 20 μg/ml proteinase K solution for 10 min at room temperature. The slides were then rinsed with PBS three times, which was followed by incubation with the TUNEL reaction mixture for 1 h at 37 °C. After rinsing with PBS three times for 5 min, sections were treated with 4 ', 6-diamidino-2 pheny-lindole (DAPI, Beyotime, Shanghai, China) for 5 min at room temperature and mounted with aqueous mounting medium. The results were imaged using a Nikon ECLIPSE 80i microscope (Nikon, Tokyo, Japan). Quantification was performed by counting the TUNEL-positive cells number in five random fields using ImageJ software.

### Western Blot Analysis

The midbrain and PC12 cells were collected and lysed using RIPA buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.5% sodium dexoycholate, 1 mM PMSF and 10 μg/ ml leupeptin). The lysate was centrifuged at 12,000*g* for 20 min at 4°C, and the supernatant was quantified with BCA reagents (Thermo, Rockford, IL, USA). Proteins (30 μg) were separated on a 12% gel and transferred onto a 0.22 μm PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% (w/v) non-fat milk (Bio-Rad) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 0.5 h at room temperature, and then the membranes were incubated overnight at 4°C with the following primary antibodies: TH (1:1000, Abcam), α-syn (1:1,000, Abcam), FGFR1 (1:1,000, CST), GRP78 (1:1,000, Abcam), CHOP (1:500, Proteintech), TRB3 (1:100, Santa Cruz Biotechnology), LC3A/B (1:1,000, CST), mTOR (1:1,000, CST), p-mTOR (1:1,000, CST), and p62 (1:1,000, CST). The membranes were washed with TBST three times and incubated with horseradish peroxidaseconjugated secondary antibodies for 2 h at room temperature. Then, the signals were detected using the Chemi DocXRS + Imaging System (Bio-Rad), and the bands were quantified using densitometric measurement by the Quantity-One software. All experiments were repeated three times.

### Statistical Analysis

Data were presented as mean ± SEM from three individual experiments. All cells culture experiments were conducted in triplicate. Statistical analysis and mapping were performed using GraphPad Prism 5. Except for rat animal behavior using repeated measures analysis of variance, one-way analysis of variance (ANOVA) followed by Tukey's *post hoc* test were used for analyzing significant difference. Multiple comparisons were used to compare between groups. *P <0.05* was used to indicate significant differences in data.

### RESULTS

### aFGF Treatment Ameliorates 6-OHDA-Induced PD Disease via Activating Autophagy

To evaluate the effect of autophagy in aFGF ameliorating 6-OHDA-induced PD, we measured the rotational behavior of APO-induced rat at 7, 14 and 21 days after administration with PBS, aFGF, CQ, aFGF+CQ, and Rapa. The results of rotational behavior showed that from day 0 to 21, the number of turns in 6-OHDA group did not significantly decrease, which is similar with that in CQ group. Compared with that in 6-OHDA group, aFGF treatment significantly decreased the number of turns of 6-OHDA mice, indicating aFGF effectively alleviates the rotational behavior of PD rats. Additionally, the behaviors of PD rats from aFGF + CQ group and Rapa group have significant improved when compared with that in CQ group (**Figure 1A**). These results demonstrated that aFGF may ameliorate PD *via* enhancing autophagy.

We also assessed the striatum morphology and the number of Nissl bodies in nigral to confirm this hypothesis. It was found that the rats in 6-OHDA group and CQ group have showed damaged striatum, irregular hyperplasia and scar, decreased Nissl bodies, which were reversed by aFGF or Rapa treatment. Moreover, compared with CQ group, aFGF + CQ treatment significantly relieved the damage of 6-OHDA on striatum morphology and Nissl bodies (**Figure 1B**), indicating that aFGF treatment can block the inhibition of autophagy and ameliorate the striatum injury and death of SNpc neurons in PD. Furthermore, we had detected the TH and α-syn expressions in the SNpc of rats by immunohistochemical staining (**Figure 1B**). It was observed that the rats from 6-OHDA and CQ group have shown few TH-positive neurons and abundant α-syn deposition in the SNpc, and aFGF and Rapa treatment remarkably block them (**Figures 1C**, **D**). More importantly, aFGF + CQ treatment increased the TH level and reduced α-syn deposition in the substantia nigra region, but its effect was worse than that in aFGF treatment. Taken together, these indicate that aFGF may enhance TH level and decrease α-syn deposition in the substantia nigra neurons of PD rats by activating autophagy, and finally achieve its neuroprotective effect.

### aFGF Treatment Attenuates 6-OHDAinduced PD via Inhibiting ER Stress

We have further confirmed the effect of ER stress on aFGF ameliorating 6-OHDA-induced PD. As shown in **Figure 2A**, aFGF and 4-PBA treatment significantly decreased the number

FIGURE 1 | Effect of autophagy on aFGF attenuating 6-OHDA-induced PD. (A) The number of rotations (turns/min) on the affected side of rats during rotational behavior test, n = 12. \*\*\*\*P <0.0001: aFGF group vs. 6-OHDA group; ####P < 0.0001: aFGF group vs. CQ group; &&&&P < 0.0001: aFGF + CQ group vs. CQ group. (B) H&E staining of striatum region, Nissl staining of nigral region, and immunohistochemistry staining results of TH and α-syn expression in striatum region. Scale bar = 50 μm, n = 6. (C, D) Quantitative analysis of immunohistochemistry staining results of TH and α-syn expression in striatum region, \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001, \*\*\*\*P < 0.0001 vs. the other group, n = 6.

of turns of PD rat, indicating that aFGF may improve the symptoms of PD by inhibiting ER stress. Then, we found that the rats in 6-OHDA group have showed damaged striatum, irregular hyperplasia and scar, decreased Nissl bodies, which are reversed by aFGF or 4-PBA treatment (**Figure 2B**). Moreover, aFGF and 4-PBA treatment blocked 6-OHDA-induced loss of TH-positive neurons and α-syn deposition in the substantia nigra neurons (**Figures 2B–D**). Consistent with the results of immunohistochemical staining, western blot results showed that 4-PBA and aFGF administration increases TH expression and promotes α-syn degradation, and ameliorates 6-OHDA-induced increase FGFR1 expression (**Figures 2E–H**).

### aFGF Administration Activates Autophagy Level by Inhibiting ER Stress During PD Development

Based on our prior study, it was found that ER stress and autophagy are involved in neuroprotective role of aFGF during PD treatment. Next, we have further detected the relationship of ER stress and autophaghy during aFGF treating with PD. Immunohistochemistry results showed that both 4-PBA and aFGF treatement promote autophagic flow by inhibiting mTOR activity and promoting p62 degradation (**Figures 3A–C**). Furthermore, western blot results showed that both 4-PBA and aFGF administration inhibit mTOR activity,

increase LC3B expression and promote p62 degradation (**Figures 3D–G**). All of these studies suggest that aFGF promotes autophagy level and exerts its neuroprotective role during PD *via* suppressing ER stress.

### aFGF Suppresses the Pro-Apoptotic Protein of TRB3 and Subsequently Activates Autophagy via Inhibiting CHOP Expression During PD Treatment

Progressive loss of DAergic is the main pathological manifestation of PD, and previous studies have shown that aFGF can protect neurons by inhibiting ER stress. It is reported that TRB3 is a protein that involved in ER stress-associated apoptosis and autophagic cell death. We speculated that TRB3 is critical factor for ER stress related with autophagy during aFGF treating for PD. Using immunohistochemical staining and western blot assay (**Figures 4A–F**), it was observed that 6-OHDA treatment significantly induces the expression levels of CHOP and TRB3, and both aFGF and 4-PBA treatment reduce the overexpression of these two proteins. Taken together, these studies indicate that aFGF suppresses the pro-apoptotic protein TRB3 and subsequently activates autophagy *via* inhibiting CHOP expression during PD development.

### Effect of Autophagy on aFGF Attenuating 6-OHDA-Induced Apoptosis in PC12 Cells

Here, we had further investigated whether aFGF can attenuate neuronal apoptosis by regulating autophagy *in vitro*. As is shown in **Figures 5A**, **B**, aFGF and Rapa treatment significantly reduced 6-OHDA-triggered apoptotic cells. In addition, TUNEL-positive cells in CQ group was significantly increased compared with that in 6-OHDA group, while the number of apoptotic cells in aFGF + CQ group was significantly lower than that in CQ group (**Figures 5A**, **B**). These data suggest that aFGF alleviates neuronal apoptosis by regulating autophagy under 6-OHDA treatment. Then, we had further detected the expressions of autophagy related protein, and found that 6-OHDA or CQ treatment and significantly decreases the expression of mTOR and increases p62 expression, which are reversed by aFGF and Rapa treatment (**Figures 5C–F**). Moreover, there is significant difference of the expressions of mTOR and p62 between the aFGF + CQ and CQ group (**Figures 5C–F**). All of these studies indicate that aFGF

promotes autophagic circulation and ameliorates 6-OHDAinduced neuronal apoptosis *in vitro*.

### aFGF Administration Activates Autophagy and Protects Neuron by Inhibiting ER Stress-TRB3 Signaling In Vitro

Here, we had further assessed the relationship of ER stress and autophaghy during aFGF treating PC12 cells under 6-OHDA condition. Consistent with the results *in vivo*, 6-OHDA treatment significantly decreased the expressions of mTOR and LCB, and increased p62 expression in PC12 cells, and both aFGF and 4-PBA treatment remarkably blocked them (**Figures 6A–H**), indicating that aFGF activated autophagy by inhibiting ER stress in PC12 cells

Then, we had investigated whether inhibition of ER stress-TRB3 signaling is involved in aFGF ameliorating 6-OHDAinduced neuronal apoptosis. TUNEL staining results showed that the numbers of TUNEL-positive PC12 cells in aFGF and 4-PBA treated-group are significantly reduced when compared with that in 6-OHDA group (**Figures 7A** and **B**), indicating that aFGF exerts its neuroprotective role by inhibiting ER stress. Then, using immunofluorescence staining and western blot, we detected the expressions of GRP78, CHOP and TRB3 in PC12 cells, and found that both aFGF and 4-PBA treatment decrease 6-OHDA-induced increases of GRP78, CHOP and TRB3 expressions (**Figures 7C–J**). These studies confirm that aFGF administration activates autophagy and exerts its neuroprotective role by inhibiting ER stress-TRB3 signaling *in vitro*.

### DISCUSSION

With the rapid growth of the elderly population, the incidence of PD is also increasing. However, the effective treatments for PD are still limited. At present, there are many studies focused on investigating the neuroprotective drugs, and neurotrophic factors have attracted much attention in recent years. aFGF, a important

Scale bar = 50 μm. \*\*P < 0.01, \*\*\*P < 0.001, \*\*\*\*P < 0.0001 vs. the other group, n = 5.

member of FGFs, has been reported to have neuroprotective effect (Tsai et al., 2015; Wu et al., 2017). Increased expression of aFGF was observed during central neuronal injury (Walicke and Baird, 1988), which indirectly indicates that aFGF is important for neuronal survival. Previous studies have shown that aFGF has a protective effect on 6-OHDA-induced DAergic injury (Wei et al., 2014). As a disease associated with excessive accumulation of misfolded proteins, α-syn accumulation promotes PD progression. ER and autophagy are essential for the normal protein synthesis and translocation in body. Our current study has shown that aFGF ameliorates the development of PD by regulating autophagy and ER stress. The specific mechanism may be related to aFGF down-regulating TRB3 *via* inhibiting ER stress, and then activating autophagy to protect DAergic from 6-OHDA.

6-OHDA is a neurotoxin that selectively damages DAergic in SNpc. A single injection of 6-OHDA into striatum can be used to simulate the chronic progression of classical PD model. Autophagy is generally an adaptive mechanism that allows for the orderly degradation and recycling of cellular components. It has shown that the autophagy level is significantly decreased in serious of degenerative diseases (Pickford et al., 2008; Spencer et al., 2009; Menzies and Rubinsztein, 2010), indicating that promotion of autophagy may protect DAergic during PD development (Darabi et al., 2018). In our current study, a rat model of PD was established using 6-OHDA induction and behavioral evaluation indicators. According to behavioral indicators, it was found that on the 7th and 14th days after administration, the behavior of PD rat was significantly improved after treating with aFGF and Rapa, while CQ treatment not only did have no remission, but

also promoted the neuronal death of PD rats, which is due to the inhibition of autophagy level in PD (Michiorri et al., 2010). In addition, morphological staining results are consistent with that in behavioral test. However, the CQ group behavior was significantly improved after CQ with drawal for 1 week, which may be related to drug half-life. Tyrosine hydroxylase (TH) is a maker protein in DAergic. Immunohistochemical staining results have shown that both aFGF and autophagy activatorcould alleviate the loss of TH, while autophagy inhibitor aggravated TH loss and α-syn accumulation. aFGF and autophagy agonist alleviated the abnormal expression of protein. These results

demonstrated that aFGF can alleviate PD development by regulating autophagy.

In current study, using autophagy inhibitors and agonists, it was found that aFGF can alleviate PD development by regulating autophagy. DAergic death contributes to the development of PD. *In vitro*, TUNEL staining result have further demonstrated that aFGF attenuates apoptosis of PC12 cells, which is consistent with the results that in autophagy agonist group. It has been reported that aFGF promotes autophagy level and consequently contributes to the recovery of spinal cord injury. Conversely, our and others' studies have shown that autophagy level is enhanced

during PD, and some LC3B positive cells colocalize with TUNEL positive signals (Niu et al., 2018), which may suggest that regulatory mechanisms are different in different type of nerve injuries.

6-OHDA-induced PD rats model are considered to be a standard model, and studies have confirmed that 6-OHDA can act as an ER stress inducer (Yamamuro et al., 2006). Therefore, ER stress activators were not added in this study. Previous studies have shown that aFGF protects DAergic by inhibiting ER stress (Wei et al., 2014). Both ER stress and autophagy are involved in the pathological process of PD. But the relationship between them is still unclear in PD. Previous studies have shown ER stress can regulate autophagy (Hetz et al., 2009; Senft and Ronai, 2015). In this study, the ER stress inhibitor was added to investigate the regulation of autophagy by ER stress. The studies *in vivo* and *in vitro* have showed that inhibition of ER stress promoted autophagy. It is indirectly shown that aFGF can protect neurons by inhibiting ER stress to promote autophagy.

Since the neuroprotective effect of aFGF is mainly through binding with FGFR1 (Li, 2019). Specifically, FGFR1 is expressed on TH-positive cells of the SNpc and terminals in striatal (Boshoff et al., 2018). Our study also showed that FGFR1 level was increased after PD (**Figure 2**), while the expression of FGFR1 was similar in the sham group and aFGF treatment group, which may suggest that FGFR1 will transfer into the nucleus due to injury. After administration, it promotes the transfer of FGFR1 to the membrane, thereby promotes the repair of neuronal damage. In contrast, studies have shown that inhibition of the FGFR1 signaling pathway in the pathogenesis of temporomandibular joint osteoarthritis activates autophagy level, thereby ameliorates disease (Wang et al., 2018), probably due to FGFR1 having the different effects on different tissue.

The PD is characterized by degenerative DAergic degeneration, which induces excessive neuronal apoptosis. CHOP is a critical protein that involved in ER stress-induced apoptosis. During prolong ER stress, CHOP expression rapidly increases and leads to apoptosis. TRB3 is an inducing factor associated with neuronal death, and it is associated with ER stress-induced apoptosis (Ohoka et al., 2005). Misfolded protein Aβ induces increases of TRB3 in neurons during PD development (Saleem and Biswas, 2017). It is shown that elevated expression of TRB3 is associated with DAergic death (Aim et al., 2015). Recently, it is unclear the relationship of ER stress and autophagy during aFGF protecting DAergic. Therefore, this study explored the role of TRB3 on this process. The results showed that the 6-OHDA treatement induces TRB3 expression and decreases the expressions of the autophagy-related indicators, mTOR and LC3B/A, which was reversed by aFGF treatment. Previous studies have shown that TRB3 blocks autophagic flow (Saleem and Biswas, 2017), and this study also confirmed that elevated TRB3 also causes increased p62 (an indicator of autophagosome clearance), indicating that autophagic flow is impaired in PD, and TRB3 binding to p62 affects the clearance of autophagosomes (Huang et al., 2015).

Our current study had showed that aFGF and autophagy inducer treatment increased the survival rate of PC12 cells. Moreover, TRB3 was significantly increased in 6-OHDAinduced PD animals and cell models, and aFGF reduced TRB3 expression. Conversely, studies have shown that TRB3 enhances autophagy (Tsai et al., 2018), leading to autophagic death of cells, which may cause excessive expression of TRB3 with varying degrees of stress, thereby aggravating cell death. Moreover, the FGF signaling pathway is involved in the activation of blastocysts by activating autophagy (Shin et al., 2017). These results indicate that aFGF ameliorates 6-OHDAinduced neuronal death *via* down-regulating the pro-apoptotic protein of TRB3 *in vivo* and *in vitro*.

## CONCLUSION

This study has demonstrated that aFGF administration ameliorates 6-OHDA-induced PD development. Mechanistic study reveals that aFGF activates autophagy level by inhibiting ER stress-induced TRB3 overexpression during PD development, and subsequently ameliorates 6-OHDA-induced neuronal apoptosis. Our current study firstly elucidates the role of autophagy and its relationship with ER stress during aFGF treatment for PD, which may provide a new potential target for clinical treatment of PD.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ supplementary material.

## ETHICS STATEMENT

The animal study was reviewed and approved by Laboratory Animal Ethics Committee of Wenzhou Medical University.

## AUTHOR CONTRIBUTIONS

JY and YW conceived and designed the experiments. XZ, BW, and GZ performed the experiments. XZ, YW, and JiX performed statistical analysis and wrote the paper. YY, XH, JuX, PZ, YL, and KX provided assistance with experiments. All authors discussed the results and approved the final manuscript.

### ACKNOWLEDGMENTS

This work was supported by research grants from the National Natural Science Foundation of China (81560375, 81760230, 81801233 and 81802251), Zhejiang Provincial Natural Science Foundation (LQ18H090011 and LQ18H150003).

### REFERENCES


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overexpression: a process modulated by the natural autophagy inducer corynoxine B. *Autophagy* 10, 144–154. doi: 10.4161/auto.26751


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Zhong, Wang, Zhang, Yuan, Hu, Xiong, Zheng, Liu, Xu, Xiao, Wu and Ye. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Paracrine Fibroblast Growth Factor 1 Functions as Potent Therapeutic Agent for Intrahepatic Cholestasis by Downregulating Synthesis of Bile Acid

#### Edited by:

Huan Lin1,2†

, Chuanren Zhou1†

Zhifeng Huang1\* and Jianlou Niu1\*

Zhouguang Wang, Albert Einstein College of Medicine, United States

#### Reviewed by:

Chen Li, Charité Medical University of Berlin, Germany Yi Kou, University of Southern California, United States

#### \*Correspondence:

Zhifeng Huang hzf@wmu.edu.com Jianlou Niu niujianlou@126.com

† These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 22 October 2019 Accepted: 22 November 2019 Published: 20 December 2019

#### Citation:

Lin H, Zhou C, Hou Y, Li Q, Qiao G, Wang Y, Huang Z and Niu J (2019) Paracrine Fibroblast Growth Factor 1 Functions as Potent Therapeutic Agent for Intrahepatic Cholestasis by Downregulating Synthesis of Bile Acid. Front. Pharmacol. 10:1515. doi: 10.3389/fphar.2019.01515 <sup>1</sup> School of Pharmacy, Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Engineering Laboratory of Zhejiang Province for Pharmaceutical Development of Growth Factors, Biomedical Collaborative Innovation Center of Wenzhou, Wenzhou, China, <sup>3</sup> School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China

, Qi Li <sup>1</sup>

, Guanting Qiao<sup>1</sup>

, Yang Wang<sup>3</sup>

,

, Yushu Hou1†

Endocrine fibroblast growth factor (FGF) 19 has been shown to be capable of maintaining bile acid (BA) homeostasis and thus hold promise to be a potential therapeutic agent for cholestasis liver disease. However, whether paracrine FGFs possess this BA regulatory activity remains to be determined. In our study, we identified that paracrine fibroblast growth factor 1 (FGF1) was selectively downregulated in the liver of alpha naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis mice, suggesting a pathological relevance of this paracrine FGF with abnormal BA metabolism. Therefore, we evaluated the effects of engineered FGF1 mutant - FGF1DHBS on the metabolism of hepatic BA and found that this protein showed a more potent inhibitory effect of BA biosynthesis than FGF19 without any hepatic mitogenic activity. Moreover, the chronic administration of FGF1DHBS protected liver against ANIT-induced injury by reducing hepatic BA accumulation. Taken together, these data suggest that FGF1DHBS may function as a potent therapeutic agent for intrahepatic cholestasis liver disease.

Keywords: fibroblast growth factor 1, bile acid, cholestatic liver disease, alpha naphthylisothiocyanate, negative regulator, intrahepatic cholestasis

### INTRODUCTION

As synthesized from cholesterol in the liver, bile acids (BAs) are normally stored in the gallbladder. After meal, it is released into the small intestine to facilitate the digestion and absorption of dietary lipids (de Aguiar Vallim et al., 2013). However, the disturbance of this physiological homeostasis will threaten human health, due to the inherent detergent properties of BAs that are potentially cytotoxic to tissues (Kaplan and Gershwin, 2005).

Cholestasis is one of the most life-threatening liver diseases that is characterized with impaired bile flow and intrahepatic retention of toxic BAs. The occurrence and development of this disease frequently leads to liver fibrosis, cirrhosis and even liver failure (Luo et al., 2014). To date, the main prescribed drugs to treat cholestasis are ursodeoxycholic acid (UDCA) and obelicholic acid (OCA). However, up to 40% of patients' response poorly to UDCA therapy (Lindor, 2007; Hirschfield et al., 2018). For patients with late-stage cholestasis, the only available option is liver transplantation (Lindor, 2007). Thus, there is still an unmet need for more effective therapeutic agents to relief cholestatic liver disease.

There are accumulating evidences demonstrating that endocrine fibroblast growth factor 15 (FGF15)/FGF19 signals from the intestine to the liver to suppress the expression of cholesterol 7a-hydroxylase (Cyp7A1) (the first rate-limiting enzyme in the classical BA synthetic pathway) as a negative feedback loop on BA synthesis (Inagaki et al., 2005; Goetz et al., 2007; Degirolamo et al., 2016). The discovery of these gut-liver hormonal axes raises the prospect that modulating BA metabolism by FGF1 (Nicholes et al., 2002) may be a novel approach for treating cholestasis. However, the development of a FGF19-based therapy is challenged with regard to its unwanted mitogenic activity and tumorgenicity (Nicholes et al., 2002; Wu et al., 2010). It has been attempted by several groups to uncouple the BA regulatory capacity and tumorigenic activity of FGF19 by engineering a M70 mutant (a FGF19 variant) that retains BA regulatory activity without causing hepatocellular carcinoma (HCC) (Zhou et al., 2014). This nontumorigenic FGF19 variant is shown to reduce hepatic BA accumulation and protect mice from liver injury caused by either intrahepatic or extrahepatic cholestasis, which suggests that it is an applicable approach for the treatment of cholestatic liver disease (Luo et al., 2014; Zhou et al., 2016).

More recently, serum level of FGF21 (another member of FGF endocrine subfamily besides FGF19) is found to be highly upregulated in patients with biliary atresia, suggesting a compensatory response may be initiated (Li et al., 2016). However, the therapeutic effect of this endocrine hormone on cholestasis is still controversial; both its positive and negative regulations on BA synthesis have been reported (Zhang et al., 2017; Chen et al., 2018).

The mammalian FGFs are grouped into five paracrine subfamilies and one endocrine subfamily (Beenken and Mohammadi, 2009; Degirolamo et al., 2016). These FGFs trigger a few common sets of downstream signaling pathways, including PLCg/PKC, FRS2a/RAS-MAPK, Gab1/PI3 kinase/ Akt, and CrkL/Cdc42-Rac (Kouhara et al., 1997; Larsson et al., 1999; Dailey et al., 2005; Eswarakumar et al., 2005; Seo et al., 2009; Chau et al., 2010), via activations of FGF receptors (FGFRs) in either an HS-dependent (in the case of paracrine FGFs) or a Klotho coreceptor dependent (in the case of endocrine FGFs) manner (Yayon et al., 1991; Kurosu et al., 2007; Goetz and Mohammadi, 2013). Due to the fact that both endocrine FGF19 and FGF21 are capable of controlling BA metabolism (Inagaki et al., 2005; Zhang et al., 2017; Chen et al., 2018), it is logical to postulate that paracrine FGFs may also possess this ability. Thus, we systematically screened expressions of all paracrine FGFs in alpha naphthylisothiocyanate (ANIT)-induced cholestasis mice and found that only FGF1 was downregulated compared to others, indicating that this paracrine FGF may be associated with the pathogenesis of BA disorders. We further confirmed that FGF1 could suppress BA synthesis both in vitro and in vivo. Moreover, the chronic administration of FGF1DHBS protected liver against ANIT-induced injury by reducing hepatic BA accumulation. Taken together, all the data suggest that FGF1-based therapy may be an effective way to treat cholestatic liver disease.

### MATERIALS AND METHODS

### Materials

Full-length wide type recombinant human FGF19 (FGF19WT) and the FGF1 variant (FGF1<sup>D</sup>HBS) carrying triple mutations (Lys127Asp, Lys128Gln, and Lys133Val) were expressed in E.coli (BL21) and purified as described in the previous studies (Goetz et al., 2007; Huang et al., 2017). Mouse Total BA Assay Kit was purchased from Crystal Chem INC. Alphanaphthylisothiocyanate (ANIT) was purchased from Sigma Aldrich (St. Louis, MO, USA). Alanine transaminase (ALT) and aspartate transaminase (AST) Assay Kits were purchased from Sigma Aldrich (St. Louis, MO, USA) and performed in accordance with the protocols provided with manufacturers. All other reagents were of analytical grade.

### Identification of Differential Paracrine FGFs Between ANIT-Induced Cholestasis Model and Vehicle Group

Male C57BL/6J mice (20–25 g) were acquired from the Chinese Academy of Science-Shanghai Laboratory Animal Center. All animals were maintained on a 12-h light/dark cycle and a temperature of 25°C ± 2°C. Animal care and all animal experiments conformed to the Guide for the Care and Use of Laboratory Animals provided by U.S. National Institutes of Health and were approved by the Animal Care and Use Committee of Wenzhou Medical University, China.

ANIT-induced intrahepatic cholestasis was established according to previous reports (Dai et al., 2017). Briefly, mice were randomized into two groups based on body weight. One group was orally administered with a single dose of ANIT (75 mg/kg, dissolved in olive oil), termed as ANIT-induced cholestasis model group; another group was treated with the same volume of olive oil, serving as the vehicle group. Mice were euthanized 48 h after administration. Blood of mice was collected to determine serum levels of liver enzymes (ALT and AST). Liver tissues of both groups were collected and total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. The differential mRNA levels of all paracrine FGFs between ANIT-induced cholestasis model and the vehicle group were determined by RT-PCR and relative mRNA levels were calculated by comparative threshold cycle method using b-actin as the internal standard. The primers of all paracrine FGFs were listed in the (Table S1).

### Primary Hepatocyte Experiments

Primary mouse hepatocytes were isolated and cultured according to the methods described previously (Zhou et al., 2017). Primary mouse hepatocytes were plated on rat tail collagen-coated dishes at a density of 1.5×10<sup>5</sup> cells/cm<sup>2</sup> and were allowed to attach for 24 h in a 37°C incubator with 5% CO2. Culture medium was consisted of 2% fetal bovine serum Williams' E medium supplemented with 10 nM dexamethasone, 1% penicillin/ streptomycin, L-glutamine. Twenty-four hours after isolation, cells were treated with the indicated concentration of FGF1DHBS or FGF19WT (0.2, 2.0, or 20 nM) for 6 h. Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. The relative mRNA levels of Cyp7A1, Cyp8B1, Cyp27A1, and Cyp7B1 were determined by real-time polymerase chain reaction (RT-PCR).

For knockdown of FGF receptor 4 (FGFR4) expression by siRNA, cells were seeded in 6-well plates for 24 h. Transient transfections were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA) according to manufacturer's protocol. After transfection with either siRNA (control or FGFR4 siRNA) (all from Santa Cruz Biotechnology, Dallas, Texas, USA) for 24 h, cells were treated with FGF1DHBS (20 nM) for 6 h. Then, cells were extracted and analyzed the Cyp7A1 mRNA levels by RT-PCR.

### Acute Effects of FGF1DHBS on the Genes That Involved in the BA Metabolism

To directly and comprehensively evaluate the effects of FGF1DHBS on BA metabolism, 6–8 weeks' C57BL/6J mice were administered with a single intraperitoneal injection of PBS, FGF1<sup>D</sup>HBS, or FGF19WT at increasing doses (0.01, 0.1, and 1.0 mg/kg) respectively. After 4 h of administration, liver tissues were collected and snap-frozen in liquid nitrogen for analysis of the key genes involved in BA metabolism including BA biosynthesis and transport.

### Effects of Subchronic Administration of FGF1DHBS on the BA Homeostasis

A 6-day subchronic study was performed in 6–8-week-old C57BL/6J mice. Male mice were administered with vehicle, FGF1DHBS (0.1 and 1.0 mg/kg, once daily) or FGF19WT (0.1 and 1.0 mg/kg, twice daily BID), PBS and FGF19WT-treated mice served as negative and positive control respectively. On the sixth day, the mice received the last dose and were placed in a new cage without food. Tissue samples including liver, gallbladder, and small intestine were collected 4 h after the last administration for genes and BA content analysis. Collect the small intestine and retain the complete contents.

Total BAs of liver, gallbladder, and small intestine were determined using Mouse Total BA Assay Kit (Crystal Chem INC). The extraction method from tissues was according to methods described previously (Chen et al., 2018). Briefly, the froze liver, gallbladder, and small intestine from subchronic treated mice were individually homogenized with 75% ethanol and total BAs were extracted by incubating the tissue homogenate in a 50°C shaker for 2 h. Supernatants from the extraction were collected after centrifugation and diluted in PBS for analysis. The dilution factors for each tissue extract were estimated to ensure that BA measurements fell in the linear range of the standard curve using mouse BA kit. The BA pool size was determined as sum of the amount of total BAs in the liver, gallbladder, and small intestine and its contents.

### Real-Time Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from treated primary hepatocytes or frozen tissues, such as the liver, intestine, and gallbladder using TransZol Up Kit (TransGen Biotech). The total RNA was reverse-transcribed into complementary DNA with One-Step gDNA Removal Kit (TransGen Biotech). Quantitative real-time polymerase chain reaction (RT-PCR) was done using ChamQ Universal SYBR qPCR Master Mix (Vazyme) with specific primers (listed in the Table S1) on a Step One Plus Real-Time PCR system (Applied Biosystems® Quant Studio® 3). b-actin was used as an endogenous control to normalize for differences in the amount of total RNA added to each reaction. For mRNAs levels of paracrine FGFs with the cycle threshold (CT) value equal to or greater than 35 are considered undetectable.

### Western Blot Analysis of Protein Expression

Liver tissues were then homogenized in protein extraction buffer (Sigma-Aldrich, St. Louis, MO) with protease and phosphatase inhibitors, and collected by centrifuging. The protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Waltham, MA), and equal amounts of samples were mixed with loading buffer, subjected to SDS-polyacrylamide gels at 110 V for 100 min, and then the protein was transferred onto polyvinylidine fluoride membranes (PVDF) (Millipore-Billerica, MA, USA). After blocking with 5% nonfat milk for 2 h at room temperature, the PVDF membranes were washed three times with Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBST) and then incubated at 4°C overnight with primary antibody to mouse FGF1 (1:1000) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000), all the antibodies were obtained from Abcam (Cambridge, MA). The membranes were washed and later incubated with HRP-conjugated secondary antibody. Immuno-reactive protein bands were visualized using the ChemiDocTM XRS with Imaging LabTM Software (Bio-Rad, Hercules, CA) and quantified by optical densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA).

### Therapeutic Effects of FGF1DHBS on the ANIT-Induced Interhepatic Cholestasis

Male C57BL/6J mice (6–8 weeks old; n = 10 mice per group) were injected intraperitoneally with PBS, FGF1DHBS (0.1 mg/kg) or FGF1DHBS (1.0 mg/kg) once daily for 6 days. On the fourth day, a single dose of ANIT (75 mg/kg, dissolved in olive oil) was administered via oral gavage. Mice were euthanized on the sixth day, 4 h after the final dosage. Serum, liver, gallbladder, and intestine samples were collected for analysis. Perform the analysis of serum liver function index (ALT and AST). Detect the BA content in serum, liver, gallbladder, and small intestine.

### Histological Analysis

Hematoxylin and Eosin (H&E) staining was performed to detect the protection of FGF1DHBS for liver injury in the ANIT-induced intrahepatic cholestasis. Briefly, liver tissues of each group were collected and kept in 4% paraformaldehyde for a post-fix overnight, and the paraffin-embedded liver tissues were then sectioned into 5 mm thickness sections by LEICA RM2235 (LEICA, Germany). The sections were dried at 40°C, deparaffinized, and then rehydrated using a series of ethanol solutions. The rehydrated sections were subjected to H&E solution based on the manufacture's protocol. After the slides were gently washed with tap water and subsequently rinsed in distilled water, the slides were mounted with mounting medium. Finally, the sections were observed and imaged using a Nikon ECLIPSE NI fluorescence microscope (Nikon, Japan).

### Statistical Analysis

All results are expressed as means ± SEM. One-way ANOVA followed by Dunnett's posttest was used to compare data from multiple groups (GraphPad Prism). When indicated, Student's t test or Mann-Whitney test was used to compare two treatment groups. A P-value of 0.05 or less was considered statistically significant.

### RESULTS

### FGF1 Is Selectively Downregulated in the Liver of ANIT-Induced Intrahepatic Cholestasis Mice

To explore the pathological relevance of paracrine FGFs with cholestatic liver disease, C57BL/6J mice were orally challenged with a single dose of anaphthylisothiocyanate (ANIT - a xenobiotic compound) (75 mg/kg of body weight) for 48 h to induce intrahepatic cholestasis with olive oil as (Yu et al., 2017). We found that serum levels of ALT, AST, and total BA were markedly upregulated with concomitant increases of hepatic BAs in these mice (Figures 1A, D). To our surprise, we found that only mRNA level of FGF1 in the liver was selectively downregulated (51% reduction relative to vehicle group) compared to other paracrine FGFs (Figure 1E; Table S2). This was further confirmed by the fact that the protein expression of hepatic FGF1 was also significantly abrogated (Figure 1F). Taken together, these results suggest that FGF1 may be involved in the control of BA metabolism.

### FGF1DHBS Suppresses Biosynthesis Pathways of BA in Primary Mouse Hepatocyte

Considering the potential safety of wild type FGF1 (FGF1WT), we used an FGF1 variant (FGF1<sup>D</sup>HBS) in the following study. As reported in our previous study, this mutant exhibits a markedly reduced proliferative potential while preserving the full metabolic activity of FGF1WT (Huang et al., 2017).

The synthesis of BAs in the liver are tightly controlled by two pathways to match the physiological needs, which are comprised of one classic pathway catalyzed by Cyp7A1 and an alternative pathway regulated by two key enzymes (Cyp27A1 and Cyp7B1) (Russell, 2009).

To assess the capability of FGF1DHBS to regulate the biosynthesis of BA, primary mouse hepatocytes were stimulated with increasing concentrations of FGF1DHBS with vehicle and FGF19WT as controls. Consistent with previous reports (Holt et al., 2003; Luo et al., 2014), FGF19 selectively inhibited the mRNA levels of Cyp7A1 (52% reduction verse vehicle group) in the primary mouse hepatocytes without affecting those of Cyp8B1, Cyp27A1, and Cyp7B1 (Figures 2A–D). Notably, FGF1DHBS could inhibited the mRNA levels of Cyp7A1, Cyp8B1, Cyp27A1, and Cyp7B1, showing a greater potency on the inhibition of Cyp7A1 mRNA levels compared to that FGF19WT (Figures 2E–H) and these inhibitory effects were compromised by the knockdown of FGF receptor 4 (FGFR4) expression by siRNA transfection (Figures 2I–L). Taken together, these data indicate that FGF1DHBS directly downregulates BA biosynthesis by FGFR4-mediated inhibiting of the classical and alternative pathways.

### FGF1DHBS Acutely Inhibits BA Synthesis Pathways In Vivo

To evaluate the inhibitory effect of FGF1DHBS on hepatic BA synthesis in vivo, C57BL/6J mice were received with a single intraperitoneal (i.p.) injection of FGF1DHBS at the dose of 0.01, 0.1 or 1.0 mg/kg of body weight. As previous reported (Luo et al., 2014), FGF19WT remarkedly suppressed the expression of Cyp7A1 mRNA levels 4 h after injection (1.0 mg/kg, 39% reduction relative to vehicle group) (Figure 3A). In the case of FGF1<sup>D</sup>HBS, its acute administration dose-dependently inhibited hepatic mRNA expressions of Cyp7A1, Cyp8B1, Cyp27A1, and Cyp7B1 (Figures 3B–E), suggesting that FGF1DHBS might reduce BA biosynthesis through both the classic and alternative pathways, which was consistent with the in vitro data. Notably, FGF1DHBS more potently inhibited the mRNA level of Cyp7A1 than FGF19WT (99% reduction relative to vehicle group at 1.0 mg/kg dose), probably due to that FGFR4-binding affinity of FGF1DHBS is much higher than that of FGF19WT (Figure 4B).

In addition to BA synthesis, the maintenance of BA homeostasis relies on various hepatic membrane transporters (Zollner et al., 2006; Halilbasic et al., 2013). In the liver, the uptake of BA from hepatic portal vein is mainly mediated by basolateral uptake transporters (Ntcp, Oatp1, and Oatp2), while its excretion through microbilliary ducts is regulated by BA canalicular efflux transporters (canalicular bile salt export pump Bsep, Mrp2, and Mdr2) (Zollner et al., 2006; Halilbasic et al., 2013). To comprehensively investigate the effects of FGF1DHBS on BA intracellular and extracellular transports, mRNA levels of both basolateral uptake transporters and BA canalicular efflux transporters in the liver were determined. As

FIGURE 1 | The mRNA and protein levels of FGF1 in liver were downregulated in the ANIT-induced intrahepatic cholestasis mouse model. Eight-week-old C57BL/ 6J mice were orally administrated with olive oil (control group) or 75 mg/kg ANIT (cholestasis mouse model) for once and then liver tissues were collected and analyzed after 48 h. (A, B) Evaluation of liver function as determined by serum levels of ALT and AST. (C, D) Serum BA concentrations (C) and hepatic BA pool (D) of each group were measured. (E) The levels of hepatic FGF1 gene expression of each group were examined by real-time polymerase chain reaction (RT-PCR); (F) Representative western blot analysis (up panel) and densitometric quantification (down panel) of FGF1 protein expression in liver tissues from ANIT-induce intrahepatic cholestasis mouse model and control group; data are normalized to GAPDH. Data are presented as mean ± SEM; \*\*\*\*p < 0.0001 versus control group; n = 8-12. ANIT-model, ANIT-induced intrahepatic cholestasis mouse model; Ctrl, control group; FGF1, fibroblast growth factor 1; GAPDH, glyceraldehyde-3 phosphate dehydrogenase.

shown in Figures 3F–H, expressions of Bsep and Mrp2 were only modestly suppressed by FGF1DHBS treatment without affecting that of Mdr2. For basolateral uptake transporters, FGF1DHBS significantly reduced mRNA levels of Ntcp, Oatp1, Oacp2, and especially Ntcp (89% reduction verse vehicle group), indicating that FGF1DHBS may prevent the uptake of BA from hepatic portal vein (Figures 3I–K). Moreover, the measurement of hepatic BA pool showed that there was no remarkable difference between FGF1<sup>D</sup>HBS-treated mice with control group (Figure 3L). Taken together, these data suggest that FGF1DHBS is a more potent inhibitor on BA biosynthesis than FGF19 in vivo and prevent the uptake of BA from hepatic portal vein.

### Chronic Administration of FGF1DHBS Inhibits Biosynthesis of BA Without Inducing Hepatic Proliferation

A 6-day study was conducted to evaluate the long-term effect of FGF1DHBS on BA biosynthesis. The dosage and frequency used

control group. \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001.

was according to previous report (Chen et al., 2018), mice were i.p. administered with FGF19WT (twice daily) or FGF1DHBS (once daily) at 0.1 or 1.0 mg/kg of body weight (Figure 4A). Consistent with its acute effects, FGF1DHBS at 1.0 mg/kg significantly inhibited hepatic expressions of Cyp7A1, Cyp8B1, Cyp27A1, and Cyp7B1 (Figures 4B–E) with a remarkable reduction of BA content in the liver and gallbladder (Figures 4F–H). Besides, total BA pool was also largely reduced (Figure 4I). These data further demonstrate that FGF1DHBS can inhibit BA biosynthesis and reduce BA pool by downregulating BA synthetic pathways. Thus, we speculate that FGF1DHBS may be a potential therapeutic agent for treating cholestasis.

In addition, we evaluated the safety of chronic FGF1DHBS treatment. Serum levels of ALT and AST were not altered by FGF1<sup>D</sup>HBS, suggesting that liver was not injured (Figures 5A, B). We further assessed the mitogenic activity of FGF1DHBS and FGF19WT in the 6-day study by determining the hepatic mRNA levels of proliferation markers including Ki67, alpha fetaprotein (AFP), and proliferating cell nuclear antigen (PCNA). Consistent with previous studies (Wu et al., 2010), FGF19WT significantly upregulates these proliferating signals in the liver (Figures 5C– E). However, mice treated with FGF1DHBS did not exhibit any hepatic proliferation activity (Figures 5C–E). Moreover, signs of hyperplasia by immune-histochemical staining using PCNA showed that FGF19WT caused a clear increase in the number of hyperproliferating cells in the liver of mice. As expected, there was no increase in the number of hyperproliferating cells in the livers of FGF1<sup>D</sup>HBS-treated mice over that of the PBS-treated control (Figure 5F). This consistence with our previous study further confirms the safety of chronic FGF1DHBS treatment.

### FGF1DHBS Ameliorates ANIT-Induced Intrahepatic Cholestasis

We further assessed the potential therapeutic efficacy of FGF1DHBS on ANIT-induced intrahepatic cholestasis. Consistent with previous studies, increased intrahepatic BA content and reduced the intestine BA pool were observed in the mice treated by ANIT, compared with that of the vehicle group, confirming the establishment of intrahepatic cholestasis

intraperitoneally with FGF1DHBS or FGF19WT at dose of 0.01, 0.1 and 1.0 mg/kg, FGF19WT-treated mice served as a positive control. Liver tissues were collected 4 h after dosing and the hepatic genes involved in BA biosynthesis and transporter were evaluated by real-time polymerase chain reaction (RT-PCR). (A) Effect of FGF19WT on Cyp7A1 mRNA; (B–E) Hepatic mRNA levels of Cyp7A1 (B), Cyp8B1 (C), Cyp27A1 (D), and Cyp7B1 (E) were determined by RT-PCR. (F–H) RT-PCR analysis of canalicular efflux transporters (Bsep, Mrp2 and Mdr2). (I–K) RT-PCR analysis of basolateral uptake transporters (Ntcp, Oatp1, and Oatp2). (L) Hepatic BA pool after acute administration of FGF1<sup>D</sup>HBS. Data was normalized to actin mRNA levels and expressed as fold change relative to control group (PBS-treated mice). Data are presented as mean ± SEM; \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\* p < 0.0001 versus control group; n = 8.

model (Figures 6A–D). Notably, we observed that FGF1DHBS (1.0 mg/kg of body weight) treatment significantly reduced BA contents in the liver, gallbladder, and small intestine and thus lowered the total BA pool (Figures 6A–D).

The elevated hepatic BA accumulation in ANIT-induced intrahepatic cholestasis mice induced a remarkable liver damage as revealed by increasing serum levels of ALT and AST (Figures 6E–G). We found that the chronic treatment of FGF1DHBS (1.0 mg/kg of body weight) significantly lowered serum levels of total BA, ALT, and AST, indicating a protective effect of FGF1DHBS on ANIT-induced liver damage. The histological analysis suggested that FGF1DHBS treatment reduced cholestasis associated necrotic lesions in the liver, compared to the vehicle treatment (Figure 6H). Taken together, all the data demonstrate that FGF1DHBS protects against liver damage in ANIT-induced intrahepatic cholestasis by reducing hepatic BA accumulation through the inhibition of both the classic and alternative pathways.

### DISCUSSION

The homeostasis of BA metabolism is tightly regulated by numerous enzymes, transporters, and nuclear receptors (Halilbasic et al., 2013). Previous studies have shown that both FGF19 and FGF21 are capable of controlling its metabolism (Schaap et al., 2009; Luo et al., 2014; Wunsch et al., 2015; Zhang et al., 2017; Chen et al., 2018). Here, we firstly identified that paracrine FGF1 was selectively downregulated in the liver of ANIT-induced intrahepatic cholestasis mice (Figures 1E–G). Then, we confirmed that the engineered FGF1DHBS could suppress BA biosynthesis by downregulating both of the classical and alternative pathway in vitro and in vivo (Figures 2 and 3). We further demonstrate that exogenous administration of FGF1 could protect ANIT-induced cholestasis liver injury by inhibiting biosynthesis of BA (Figures 2–4).

FGF1 is a prototype of FGF family that has been implicated in various physiological processes including wound healing, neuroprotection and adipogenesis (Beenken and Mohammadi, 2009). Recently, Johan W. Jonker et al. identified that PPARg-

The effect of chronic FGF1DHBS and FGF19WT treatment on the mRNA levels of Cyp7A1 (B), Cyp8B1 (C), Cyp27A1 (D), and Cyp7B1 (E) in the liver of C57BL/6J mice. (F-I) BA pool of liver (F), small intestine (G), and gallbladder (H). Total BA pool size (I) were calculated. Data are presented as mean ± SEM; \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*p < 0.0001 versus Control group; n = 8.

FGF1 axis is critical for maintaining metabolic homeostasis and insulin sensitization (Jonker et al., 2012). Although known as a mitogenic factor, exogenous administration of FGF1 results in potent and insulin-dependent glucose lowering effect in insulin resistance mice. This discovery uncovers an unexpected, neomorphic insulin-sensitizing effect of FGF1 to treat type 2 diabetes (Suh et al., 2014) and expands the functions of this classically known mitogen. In our study, we firstly show another novel function of FGF1 to protect cholestasis liver disease by regulating BA biosynthesis.

The signaling and functions of FGFs are tightly regulated by spatial and temporal expressions of FGFs, FGFRs, and coreceptors (heparin sulfate and klotho) and most importantly by binding specificity of FGF-FGFRs (Beenken and Mohammadi, 2009). As a unique member of FGFs, FGF1 is termed as "the universal FGFR ligand" because it overrides the barrier of FGF-FGFRs binding specificity (Beenken et al., 2012), which makes it binding with liver-enriched FGFR4 and forming the structural basis for BA regulatory activity (Figure 2). Notably, the engineered FGF1 partial agonist (FGF1<sup>D</sup>HBS) carrying triple mutations located on the heparin sulfate binding regions, which diminishes the ability of FGF1DHBS to induce heparin sulfate-assisted FGFR dimerization (Huang et al., 2017) but did not affect its binding specificity with FGFRs. This may be the reason that FGF1DHBS still exhibits BA regulatory activity (Figures 2 and 3).

One plausible explanation for FGF1DHBS not inducing hepatic proliferation may rely on the "threshold model" published in our previous study; as the dimerization strength of FGFR decreases, the mitogenic potential of FGF1 is dissipated much earlier before the decrease of its glucose-regulatory activity. Thus, the mitogenic and glucose-lowering activity of FGF1 could be uncoupled by dampening FGF-FGFR dimerization through triple mutations that diminish FGF1-heparin binding affinity

including Ki-67, AFP, and PCNA in C57BL/6J mice after chronic treatment, data was normalized to actin mRNA levels and expressed as fold change relative to control group (PBS-treated mice) (F) immune-histochemical staining using PCNA. Data are presented as mean ± SEM; \*P < 0.05; \*\*\*P < 0.001; versus control group; n = 8.

(Huang et al., 2017). In the study, we found that FGF1DHBS could regulate BA metabolism without exhibiting any hepatic proliferation activity, suggesting that the BA regulatory activity and mitogenic activity of FGF1 could be also separated in this condition.

Interestingly, acute administration of FGF1DHBS not only reduced the BA biosynthesis by downregulating Cyp7A1 expression, but also suppressed the uptake of BA from hepatic portal vein by reducing the expression of basolateral uptake transporters (Ntcp, Oatp1, and Oatp2) (Figure 3). These dual effects can limit the accumulation of BA in the liver and facilitate the protective effects of FGF1 on ANIT-induced cholestasis. To further determine whether the changes of these genes are due to the direct effect of drug stimulation or the negative feedback upon changes in BA content, we measured the content of total BA in mouse liver after single injection. The results showed that synthesis of BA genes was inhibited whereas the hepatic BA content did not change, suggesting that changed expressions of these enzyme and transporters may be directly associated with drug stimulation but not negative feedback.

Although the results presented in the study demonstrate that FGF1DHBS suppresses hepatic BA accumulation and protect liver against ANIT-induced injury, several limitations need to be addressed. Firstly, the promiscuity of FGF1 with all FGFRs and its mitogenic activity representing safety concerns hurdles the development of FGF1-based therapy. Although no significant proliferation activity and tumorgenicity in the liver were observed after chronic treatment of FGF1<sup>D</sup>HBS, further investigations are needed to comprehensively assess of safety of long-term administration of FGF1DHBS or engineer FGF1 variants specifically binding with FGFR4. Secondly, the ANITinduced intrahepatic cholestaisis mouse model used in the present study only depicts certain aspects of cholestatic liver disease, extra experimental models are also required to evaluate the potentially therapeutic value of FGF1 mutants.

### CONCLUSION

0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001.

In conclusion, our study uncovers the association of paracrine FGF1 with BA metabolism and demonstrated that FGF1 functions as a negative regulator of BA synthesis through FGFR4-mediated inhibition of the classical and alternative pathway. Thus, FGF1 can function as a protector against ANIT-induced liver injury under cholestasis. Moreover, the study from engineered FGF1DHBS holds a promise of FGF1 to be a therapeutic candidate for cholestatic liver disease or other BA metabolism syndromes.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ Supplementary Material.

### ETHICS STATEMENT

The animal study was reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University.

### AUTHOR CONTRIBUTIONS

HL, CZ, YH, QL, GQ, YW, ZH, and JN researched the data. ZH and JN contributed to the initial discussion and design of the project. HL and JN wrote the manuscript. JN is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

### FUNDING

This work was supported by grants from the Natural Science Foundation of China (81803415, to JN) and the Natural Science

### REFERENCES


Foundation of Zhejiang (LY18H070002 and LQ19H300002, to YW and JW), and the Science and Technology Project of Wenzhou (Y20180176, to JW).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2019.01515/full#supplementary-material


Zollner, G., Marschall, H. U., Wagner, M., and Trauner, M. (2006). Role of nuclear receptors in the adaptive response to bile acids and cholestasis: pathogenetic and therapeutic considerations. Mol. Pharm. 3 (3), 231–251. doi: 10.1021/ mp060010s

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lin, Zhou, Hou, Li, Qiao, Wang, Huang and Niu. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Using Recombinant Human Collagen With Basic Fibroblast Growth Factor to Provide a Simulated Extracellular Matrix Microenvironment for the Revascularization and Attachment of Islets to the Transplantation Region

#### Edited by: Zhouguang Wang, Albert Einstein College of Medicine, United States

### Reviewed by:

Yang Mei, Northwestern University, United States Yu Kang, University of Pennsylvania, United States

#### \*Correspondence:

Lei Peng 15607662705@163.com Hongxing Fu hx\_fu79@163.com Yingzheng Zhao pharmtds@163.com

† These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 09 October 2019 Accepted: 27 November 2019 Published: 10 January 2020

#### Citation:

Zhu Q, Lu C, Jiang X, Yao Q, Jiang X, Huang Z, Jiang Y, Peng L, Fu H and Zhao Y (2020) Using Recombinant Human Collagen With Basic Fibroblast Growth Factor to Provide a Simulated Extracellular Matrix Microenvironment for the Revascularization and Attachment of Islets to the Transplantation Region. Front. Pharmacol. 10:1536. doi: 10.3389/fphar.2019.01536 Qunyan Zhu1,2,3† , Cuitao Lu1† , Xuan Jiang<sup>1</sup> , Qing Yao<sup>1</sup> , Xue Jiang<sup>1</sup> , Zhiwei Huang<sup>1</sup> , Yina Jiang<sup>1</sup> , Lei Peng1\*, Hongxing Fu1\* and Yingzheng Zhao1,2,3\*

<sup>1</sup> College of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Engineering Laboratory of Zhejiang Province for Pharmaceutical Development of Growth Factors, Biomedical Collaborative Innovation Center of Wenzhou, Wenzhou, China, <sup>3</sup> Trauma Center, The First Affiliated Hospital of Hainan Medical College, Haikou, China

Islet transplantation is considered a potential therapeutic option to reverse diabetes. The pancreatic basement membrane contains a variety of extracellular matrix (ECM) proteins. The abundant ECM is essential for the survival of transplanted islets. However, the ECM proteins necessary for maintaining islet vascularization and innervation are impaired by enzymatic digestion in the isolation process before islet transplantation, leading to destruction of islet microvessels. These are the primary concern and major barrier for long-term islet survival and function. Thus, it is crucial to create an appropriate microenvironment for improving revascularization and islet function to achieve better transplantation outcome. Given the importance of the presence of ECM proteins for islets, we introduce recombinant human collagen (RHC) to construct a simulated ECM microenvironment. To accelerate revascularization and reduce islet injury, we add basic fibroblast growth factor (bFGF) to RHC, a growth factor that has been shown to promote angiogenesis. In order to verify the outcome, islets were treated with RHC combination containing bFGF and then implanted into kidney capsule in type 1 diabetic mouse models. After transplantation, 30-day-long monitoring displayed that 16 mg–60 ng RHC-bFGF group could serve as superior transplantation outcome. It reversed the hyperglycemia condition in host rapidly, and the OGTT (oral glucosetolerance test) showed a similar pattern withthe control group. Histological assessment showed that 16 mg–60 ng RHC-bFGF group attenuated apoptosis, promoted cellular proliferation, triggered vascularization, and inhibited inflammation reaction. In summary, this work demonstrates that application of 16 mg–60 ng RHC-bFGF and islets composite enhance the islet survival, function, and long-term transplantation efficiency.

Keywords: diabetes, recombinant human collagen (RHC), basic fibroblast growth factor (bFGF), islet–matrix attachments, islet transplantation

## INTRODUCTION

Diabetes is one of the most common serious chronic diseases and is associated with several serious complications, including cardiovascular, kidney, eye, nerve, cerebrovascular, and peripheral vascular diseases (Nathan, 1993; Bader et al., 2016; Wang et al., 2017). Type 1 diabetes (T1D) is an autoimmunemediated metabolic disease that is characterized by permanent destruction of insulin-producing beta cells, which results in absolute insulin deficiency (Davis et al., 2012). The main therapies for controlling glycemic levels are exogenous insulin administration and oral hypoglycemic agents. Unfortunately, most of the time, these treatments fail to control the blood glucose level, which results in severe hypoglycemia and complications (Bogdani et al., 2014). Islet transplantation offers therapeutic potential for patients with T1D to normalize glucose metabolism and prevent the complications of the disease (Davis et al., 2012). Despite the rapid progress that has been made in islet transplantation after the disclosure of the "Edmonton protocol," multiple donors are needed to achieve long-term insulin independence, which limits the large-scale application of this protocol. The disruption of islet–matrix attachments or the extracellular matrix (ECM) components between endocrine and exocrine cells during isolation decreases islet function and viability before and after transplantation, which severely hinders the efficiency of transplantation (Barrett-Connor and Orchard, 1985). Furthermore, numerous studies demonstrated that 20~40% of islets undergo apoptosis during the culture. However, pre-conditioning in culture prior to islet transplantation, such as the supplementation of angiogenic agents (Uzunalli et al., 2015; Qiu et al., 2017), antioxidant agents, or oxygen carriers in the culture medium, has enabled islets to obtain longer (Emamaullee et al., 2008; Maillard et al., 2011; Brandhorst et al., 2013; Schaschkow et al., 2015), better graft function and greater efficiency (Kin et al., 2008; Schaschkow et al., 2015).

ECMs, such as collagen, have been developed to address apoptosis during islet culture, to preserve islet morphology, to promote cell survival, and to provide essential physical scaffolding for cellular constituents, which can enhance islet insulin secretion and reinforce islet structure to resist mechanical stress during transplantation (Salvay et al., 2008; Schaschkow et al., 2015). Collagen type III also has been demonstrated to enhance microvascular strength and elasticity, provide adequate nutrients in cells, and directly bind to hemangioblasts, thereby promoting the formation of new blood vessels (Sailakshmi et al., 2011). In this study, recombinant human collagen (RHC), collagen type III, was obtained from a high-density fermented supernatant of GS115/ Ppic9KG6 fermentation, which solved the problems of conventional extraction methods, such as poor hydrophilicity and immune rejection. Growth factors, such as basic fibroblast growth factor (bFGF), are natural substances that are capable of stimulating cellular growth, proliferation, healing, and cellular differentiation (Barrientos et al., 2008; Xiang et al., 2011). Our previous study demonstrated that bFGF had potent angiogenetic capabilities in fibroblasts and epithelial cells, which thus promote angiogenesis and are beneficial to islet revascularization (Zhao et al., 2014).

In this study, we investigated a co-culture system of combined bFGF and RHC to provide a simulated ECM microenvironment for islets. We hypothesize that such a microenvironment can rebuild the islet–matrix attachments and, thus, promote the revascularization of islets, which will benefit islet culture both in vitro and in vivo (as illustrated in Figure 1).

### MATERIALS AND METHODS

### Animals and Reagents

BALB/C male mice that were 8–10 weeks old were obtained from Shanghai, China. All the mice were housed with a 12 h light/dark cycle at 24 ± 1°C and provided with food ad libitum for a week before initiation of the study. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Collagenase type V, streptozotocin (STZ), fluorescein diacetate (FDA), and propidium iodide (PI) were purchased from Sigma Inc., St. Louis, MO, USA. bFGF was ordered from Gelusite Biology Technology Company, Zhejiang, China. RHC was kindly provided by Nanjing University of Science and Technology, Jiangsu JL, and Biotech Co., Ltd. Ficoll-1077, Ficoll-1119, Hanks' solution, and CMRL-1066 were purchased from Wenzhou Icelltrans Technologies Co., Ltd., Wenzhou, China.

### Islet Isolation

Islets were isolated from the pancreas of mice according to methods that were modified from other studies (Zmuda et al., 2011). Briefly, the mice were anesthetized by inhaling isoflurane and were placed in the supine position on the dissecting table. The abdomen was sprayed with 75% ethanol, followed by opening it as a U-incision; the entrance of the common bile duct into the duodenum was located and ligated; and 2.5 ml collagenase type V (1 mg/ml, dissolved in cold Hank's solution) was injected into the common bile duct by retrograde intubation. Once the pancreas was perfused, it was removed from the mouse by pulling it free from the points of contact with the intestines, stomach, and spleen. The pancreas was transferred in a 50 ml centrifuge tube. Then, the appropriate amount of collagenase type V was added and subjected to digestion in a 37 ± 0.5°C water bath for 3–5 min with gentle shaking. The digested tissue was washed three times with cold Hank's solution. Subsequently, the islets were purified with a discontinuous density gradient (Ficoll-1119, Ficoll-1077, and Hanks' solution) by centrifuging at 2,000 rpm for 5 min with the brake off. The supernatant containing purified islets was washed three times with cold Hank's solution. If necessary, the islets were further purified by handpicking.

### Islet Counting and Equivalent Calculation

The cultured islets were transferred into a culture dish with 0.5 mm lattices for their quantification under the microscope. The number of islets were counted and calculated to the islet

equivalents (IEQs; diameter standardizing to 150 mm) (Lembert et al., 2003).

### Islet Culture

The purified islets were divided into six groups according to different culture conditions: 1) FREE; 2) 60 ng bFGF; 3) 1 mg RHC; 4) 16 mg RHC; 5) 1 mg–60 ng RHC-bFGF; and 6) 16 mg– 60 ng RHC-bFGF. Each group contained approximately 200–300 purified islets. In the "FREE" group, the islets were cultured in CMRL-1066 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen, Inc., USA) and 1% antibiotics (100 U/ ml penicillin, 100 ng/ml streptomycin). In the "60 ng bFGF" group, the islets were cultured in CMRL-1066 medium containing bFGF (60 ng/ml). In the "1 mg RHC" group, the islets were cultured in CMRL-1066 medium containing RHC (1 mg/ml). In the "16 mg RHC" group, the islets were cultured in CMRL-1066 medium containing RHC (16 mg/ml). In the "1 mg–60 ng RHC-bFGF" group, the islets were cultured in CMRL-1066 medium containing RHC (1 mg/ml) and bFGF (60 ng/ml). In the "16 mg–60 ng RHC-bFGF" group, the islets were cultured in CMRL-1066 medium containing RHC (16 mg/ml) and bFGF (60 ng/ml). The concentration of bFGF 60 ng/ml was chosen according to our previous study (Qiu et al., 2017).

### Islet Viability and Reactive Oxygen Species In Vitro

After being cultured with the different concentration of RHC (0, 1, 16, and 25 mg/ml) and bFGF (60 ng/ml) in six groups for 24 h at 37°C in a humidified 5% CO2, the viability of the islets was analyzed by FDA/PI staining. The ratio of green to green plus red cells provided the percentage of viability. Images were examined and recorded with an inverted microscope (Nikon ECLIPSE Ti-S; Ruikezhongyi, Beijing, China).

To visualize the reactive oxygen species, cultured islets were harvested and washed, followed by incubating with dihydroethidium (DHE, Sigma) for 30 min at 37°C, washing with phosphate-buffered saline (PBS, Gibco), and monitoring by an inverted fluorescence microscope.

### Transmission Electron Microscopy Examination

To determine whether the endocrine granules in the beta cells were preserved or released, the ultrastructure of islets was observed by transmission electron microscopy (TEM) (Daoud et al., 2010). The islets in different groups were collected after culturing for 24 h and were fixed immediately in 2.5% (w/v) glutaraldehyde at 4°C overnight. After dehydration in a series of acetone–water solutions, the islets were embedded in Epon. Semi-thin section and toluidine blue staining were performed to observe the islets locations. Finally, ultra-thin sections of at least six blocks per sample were cut and examined with the electron microscope (H-7500, Hitachi, Ibaraki, Japan).

### The Stability of bFGF in the Culture System

To investigate the stability of bFGF in RHC, the degradation of bFGF in vitro was conducted. Three bFGF formulations (60 ng/ ml bFGF alone, 60 ng/ml bFGF with 1 mg/ml RHC, and 60 ng/ ml bFGF with 16 mg/ml RHC) were prepared and incubated at 37°C. At 0, 12, 24, 48, 72, 120, and 168 h, 100 ml of aliquot was extracted and stored at −80°C before detection. The concentration of bFGF was detected by an ELISA kit.

### Induction of Diabetic Mellitus

Irreversible diabetes was chemically induced in mice by single intraperitoneal injections (IP) of 150 mg/kg STZ. Non-fasting blood glucose levels were measured by using a glucometer (Pro doctor®, Tai Doc Technology Corp, Beijing, China) from whole blood samples that were obtained by tail snipping. Mice with two consecutive non-fasting blood glucose levels higher than 22.2 mmol/L were used for transplantation. The diabetic mice were randomly divided into six groups of six. The blood glucose levels of donor mice were also confirmed before islet isolation to verify that they were metabolically normal.

### Islet Transplantation

After 6 h culture, the islets in the six groups were collected and prepared for transplantation. The number of transplanted islets was 200 IEQ for each group. Transplantation was performed as reported (Zmuda et al., 2011; Fu et al., 2016). Mice were anesthetized with isoflurane, and the right back was shaved and sterilized with 75% ethyl alcohol. A small incision was made, and the kidney was exposed. The islets in the syringe were injected slowly and scattered under the kidney capsule. Then, the skin was sutured carefully. It is good to ensure that the kidney is kept moist with saline during the entire process. After transplantation, antibiotics were administered every day for a week to reduce the inflammatory response.

## Function Assessment In Vivo

After transplantation, non-fasting blood glucose levels and the weights of animals were measured regularly until the end of the study. The graft was considered to be functional only if the nonfasting blood glucose levels were stably maintained at less than 11.1 mmol/L, with no reoccurrence of hyperglycinemia. An oral glucose tolerance test (OGTT) was conducted on day 30 post– islet transplantation to assess metabolic capacity. Mice were placed in fresh cages with access to water but no food for 12 h. Then, 2 mg/ml glucose in 200 mg/ml solution was administered to the mice orally. Blood glucose was evaluated at time 0 (prior to glucose administration) and then at 20, 40, 60, 90, and 120 min after glucose administration. The area under the curve (AUC) of blood glucose was also calculated.

### Histologic and Immunohistochemical Analyses

At 30 days after transplantation, mice were anesthetized and were transcranially perfused with saline. Kidney with islets were resected and fixed in 4% paraformaldehyde overnight, followed by embedding in paraffin and sectioning at 5 mm thickness. The tissue was dewaxed and rehydrated in xylene and gradient alcohol for hematoxylin and eosin (H&E) staining for the evaluation of general morphology and Masson's trichrome staining for the evaluation of fibrosis. To identify proteins in grafts, immunohistochemistry staining was performed, primary anti-insulin (15848-1-AP, 1:400, Proteintech), anti-HIF-1a (ab1, 1:200, Abcam), anti-Caspase-3 (ab13847, 1:200, Abcam), anti-Ki67 (ab15580, 1:400, Abcam), anti-CD31 (ab182981, 1:200, Abcam), and anti-IL-6 (DF6087, 1:300, Affinity) staining. Digital images were acquired by using a light microscope or confocal microscope.

### Statistics Analysis

All analyses were performed using Graph Pad Prism 7 software (Graph Pad Software, Inc., La Jolla, CA, USA). All results are expressed as the mean ± SD for each group. The one-way ANOVA followed by Tukey's test was for comparing the multi-group data. P-values less than 0.05 were considered statistically significant.

## RESULTS

### Islet Viability In Vitro

To calculate the viability of islets under the different conditions, FDA/PI staining was performed at 0, 7, and 24 h after culturing. The results are shown in Figure 2.

From the results (Figure 2), it can be seen that after 7 h coculture, the viability of the 1 mg RHC group, the 1 mg–60 ng RHC-bFGF group, and the 16 mg–60 ng RHC-bFGF group increased, while the 16 mg RHC group had the opposite tendency. After culturing for 24 h, the viability of both the 1 mg–60 ng RHC-bFGF group and the 16 mg–60 ng RHC-bFGF group were over 90%, which was better than the viability of the bFGF-alone or RHC-alone groups. We suspected that simultaneous application bFGF and RHC can improve the viability of islets. However, we also found that the viability of the "25 mg RHC" and "25 mg to 60 ng RHC-bFGF" groups was inferior, at either 7 h or 24 h (Figure 1S).

The ultrastructure of the islets was observed by TEM (Figure 2E). It can be seen that the endocrine granules in the beta cells were preserved, which means that the islets did not degranulate in different culture groups. The mitochondria also did not swell, and the cristae were clear. There was no evidence of membrane disruption in the islets after cultivation for 24 h.

### Islet Hypoxia In Vitro

After co-culturing the islets in each group for 24 h, the fluorescence staining of DHE of the 1 mg RHC group, the 1 mg–60 ng RHC-bFGF group, and the 16 mg–60 ng RHC-bFGF group significantly decreased compared to that of the FREE group, 60 ng group, and 16 mg RHC group (Figure 3A), which suggested that the ROS levels significantly decreased when the islets were co-cultured with RHC and/or bFGF, compared with those of the bFGF-alone or FREE islets groups.

HIF-1a was a crucial marker for cellular proliferation in a hypoxic condition (Tsuchiya et al., 2015). After islet transplantation for 30 days, the grafts were removed, and the HIF-1a immunohistochemical staining results are shown in Figure 3B. As shown in Figure 3B, there was almost no HIF-

The three columns of each group represent 0, 7, and 24 h from left to right (\*\*\*\*P < 0.0001, \*\*\*P < 0.001, 0 vs 24 h; ####P < 0.0001, 0 vs 7 h). (E) TEM of each group after 24 h culturing.a and A: FREE group; b and B: 60 ng bFGF group; c and C: 1 mg RHC group; d and D: 16 mg RHC group; e and E: 1 mg–60 ng RHCbFGF group; f and F: 16 mg–60 ng RHC-bFGF group; N: nucleus; M: mitochondria; \*: secretory granules.

1a expression in the islets in FREE, and HIF-1a was subtly expressed in the 60 ng bFGF and 16 mg RHC groups. For the 1 mg–60 ng RHC-bFGF group and the 16 mg–60 ng RHC-bFGF group, the expression of HIF-1a had strikingly increased. The previous reports indicated that HIF-1a expression was strongly expressed when the islets were cultured with ECM in a hypoxic condition (Maillard et al., 2011). Based on the results, RHC combined with bFGF would promote cellular proliferation and survival, whereas RHC or bFGF alone would not.

## The Degradation Curve of bFGF

The impact of RHC on bFGF stability in vitro was assessed. The results (Figure 4) showed that free bFGF degraded the fastest and decreased to 40% within 12 h. In contrast, the bFGF with the 16 mg RHC group was maintained at approximately 60% bFGF within 12 h. After 72 h, the residual bFGF in the free bFGF group was lower than 5%, while the bFGF concentrations in the 1 mg RHC and 16 mg RHC groups were 10% and 25%, respectively. Even at 168 h, there was still residual bFGF in the 16 mg/ml RHC

FIGURE 3 | Hypoxia. (A) Dihydroethidium (DHE) staining was performed on the islets after 24 h cultivation for all groups. (B) Representatives of HIF-1a immunohistochemical staining of the islet graft in each group. a: FREE group; b: 60 ng bFGF group; c: 1 mg RHC group; d: 16 mg RHC group; e: 1 mg–60 ng RHC-bFGF group; f: 16 mg–60 ng RHC-bFGF group.

group. Above all, RHC could increase the stability of bFGF and prolong the action time of bFGF in the islets.

### bFGF With RHC Improved Islet Function In Vivo

As shown in Figure 5A, the mice transplanted with 200 IEQ 16 mg–60 ng RHC-bFGF islets achieved euglycemia the fastest. Meanwhile, the body weight of the group increased (Figure 5B). The body weight of the other two groups (transplanted with 200 IEQ 1 mg RHC islets or 200 IEQ 1 mg–60 ng RHC-bFGF islets) also achieved euglycemia by the end of the experiment (Figures 5A, B). However, the other three groups (transplanted with 200 IEQ islets, 200 IEQ 60 ng bFGF islets, or 200 IEQ 16 mg RHC islets) maintained the hyperglycemic condition and exhibited weight loss. Above all, 200 IEQ islets transplanted after culture with 16 mg–60 ng RHC-bFGF achieved the best efficiency, while a previous report demonstrated that 400 IEQ were normally required to achieve the same outcome (Qiu et al., 2017).

To further investigate islet function in vivo, an OGTT were performed at 30 days after islet transplantation. As shown in Figure 5C, the 16 mg–60 ng RHC-bFGF group shared a similar blood glucose level pattern with that of the normal CON group (Figure 5C). Glucose levels in the 1 mg RHC group were significantly higher than those of the 1 mg–60 ng RHC-bFGF group and did not return to the baseline at 120 min. However, the blood glucose levels of the 60 ng bFGF group and the DM group remained at high levels at 120 min after glucose administration. The AUCs of the 1 mg RHC and FREE groups were similar to that of the DM group, while that of the 16 mg–60 ng RHC-bFGF group was similar to that of the CON group (Figure 5D). These results suggest that RHC containing bFGF contributed to better islet function.

### bFGF With RHC Maintained Islet Morphology, Enhanced the Reestablishment of the Islet–ECM and Attenuated Apoptosis

The islets had a tendency to form clusters, which can lead to hypoxia and necrosis of the islets and affect the function of the islets (Uzunalli et al., 2015). To monitor the morphology of transplanted islets 30 days after transplantation, (H&E staining (Figure 6A), insulin-immunofluorescence staining (Figure 6B), and Masson's trichrome staining (Figure 6C) were performed on the paraffin section. H&E and positive insulinimmunofluorescence staining showed that the islets were alive, which indicated that the islets remained functional on day 30 post-transplantation. However, as shown in Figure 6B, the islets of the FREE and 60 ng bFGF groups gathered and fused into a large cell cluster. There was also a slight aggregation of the islets in group 16 mg RHC, and the islets boundary was blurred. While in the 1 mg RHC, 1 mg–60 ng RHC-bFGF, and 16 mg–60 ng RHCbFGF groups, the islets had a clear boundary and were single and not clustered, and in the 16 mg–60 ng RHC-bFGF group, the islets were the most intact.

Moreover, collagen is a major component of the ECM, which indicated early attenuation of apoptosis of implanted islets in preliminary studies (Salvay et al., 2008; Davis et al., 2012). Masson's trichrome staining showed collagen existed in the region of transplanted islets of all the groups and collagen was most abundant in the 16 mg–60 ng RHC-bFGF group (Figure 6C). Thus, it was speculated that the successful reestablishment of the islet–ECM was crucial for minimizing islet apoptosis and preserving islet survival and function, and in turn, accounted for the better transplantation outcome (Wang and Rosenberg, 1999; Kim et al., 2012; Mao et al., 2017). Hence, it was speculated that bFGF-RHC was crucial in facilitating transplantation efficiency.

In addition, Figure 6D shows the caspase-3 immunohistochemistry staining of the graft on day 30 posttransplantation. The FREE group had an obvious positive staining compared with that of the other groups, especially in the central region of the islet clusters, and apoptosis was

FIGURE 7 | Improved engraftment of transplanted islets by promoting proliferation and angiogenesis. Representative immunohistochemical staining of (A) Ki67 and (B) CD31 expression. (C) IL-6 immunohistochemistry staining was performed to monitor the inflammatory response. a: FREE group; b: 60 ng bFGF group; c: 1 mg RHC group; d: 16 mg RHC group; e: 1 mg–60 ng RHC-bFGF group; f: 16 mg–60 ng RHC-bFGF group.

obvious, which was related to the tendency of the islets to form clusters causing the central region to be susceptible to hypoxia and necrosis (Figure 6D). The lack of islet–matrix attachments led to rapid cell death, suggesting that the new contact attenuated apoptosis and the islets mass (Schaschkow et al., 2015). In this work, we utilized RHC and bFGF to achieve this purpose. It was proven that bFGF can promote angiogenesis, which was beneficial to the engraftment of islets (Zhao et al., 2014; Qiu et al., 2017), and ECM was shown to be capable of creating contacts.

### RHC Supplemented With bFGF Facilitated Graft Function by Minimizing the Post-Transplantation Inflammatory Reaction, Enhancing the Vascular Density and Increasing the Proliferation

The ECM proteins could promote cell survival and proliferation, which resulted in the functionality of b-cells (Lai et al., 2005). In addition, Ki67 is a biomarker for evaluating cell proliferation around the graft. Figure 7A shows that a low level of Ki67 was found in the FREE group and the 60 ng bFGF group, while significantly higher expression was found in other groups. Moreover, the level of Ki67-positive cells was significantly higher in the 1 mg–60 ng RHC-bFGF group compared with the 1 mg RHC group. These data suggest that RHC alone or RHC supplemented with bFGF facilitated islet proliferation.

The engraftment efficiency was greatly hindered by the destruction of the intra-islet blood vessel network and poor diffusion of nutrients. Therefore, it is critical to address the revascularization issue, which will improve the islet survival and functionality, and ultimately the transplantation outcome (Mao et al., 2017). To investigate the effect of pre-treated islets on islet revascularization, immunohistochemistry staining of CD31 was performed (Figure 7B). Based on the results, the density of the CD31 hot point was significantly higher in the high-RHC group than in the low-RHC group in the RHC-alone groups. By adding bFGF, the density of the CD31 hot point of the high concentration group was still higher than that of the low concentration group, and the expression was higher than that of the RHC-alone group, and there was a remarkable difference compared with the levels observed in the bFGF group alone. These data indicate that RHC supplemented with bFGF specifically increased the revascularization after transplantation.

We examined the inflammatory reaction to the implanted islets. IL-6 immunohistochemistry staining revealed that bFGF could inhibit the inflammatory reaction surrounding the implanted islets compared with the staining observed in the FREE group (Figure 7C). The RHC-alone group exhibited subtle expression of IL-6. However, IL-6 was almost not observed when RHC was combined with bFGF compared with the expression in the RHC-alone and FREE groups. As previously reported, the 60% of islets that underwent apoptosis during early transplantation was due to the inflammatory reaction (Barshes et al., 2005). In this work, RHC combined with bFGF could minimize the inflammatory reaction to improve islet survival and viability in early transplantation. Eventually, this treatment enhanced the islet transplantation outcome.

## CONCLUSION

Islet transplantation is the most promising potential therapeutic option for reversing diabetes. However, despite the remarkable progress that has been made to improve the post-transplantation outcome, due to the destruction of islet–matrix attachments and the intra-islet vessel network during the isolation, the reestablishment of islet–matrix attachments and revascularization must be solved to achieve long-term graft function. Herein, we introduce RHC and bFGF into study. RHC is one of the components of the ECM. A previous study showed that the ECM that surrounded the transplanted islet in vitro and in vivo is a key factor in maintaining vascularization, cell interaction, and the innervations of islets, and further enhances islet survival and function (Salvay et al., 2008; Pepper et al., 2015; Mao et al., 2017). Growth factors are naturally occurring substances that can stimulate cellular growth, proliferation, healing, and cellular differentiation. Our previous study demonstrated that bFGF had potent angiogenetic capabilities in fibroblasts and epithelial cells, thus promoting angiogenesis, which was beneficial in strengthening the graft revascularization (Barrientos et al., 2008; Xiang et al., 2011).

Our study (Figure 8) showed that RHC surrounding the islets reconstitutes islet–matrix attachments, promotes islet proliferation, and slows the degradation of bFGF. Meanwhile, bFGF eliminates inflammatory cells around the islet transplanted site. Together, these factors provide a simulated ECM microenvironment for islet survival and act on intra-islet, thereby reconstructing the damaged microvessel. Through a combination of RHC and bFGF, the supply of oxygen and nutrients inside the islets was restored, and apoptosis of the intra-islet was inhibited. The dual effects of RHC and bFGF on the inter-islet and external microenvironment of islets can decrease the quantity of islets needed to reverse diabetes compared with that indicated by the previous study, which, to a certain extent, alleviates the problem of insufficient donors.

Superior transplantation efficiency is evident from the 16 mg– 60 ng RHC-bFGF group, which proved to have remarkably better blood glucose responsiveness and blood glucose control. These results suggest that the RHC-bFGF combination enables a simulated ECM microenvironment that facilitates better transplantation efficiency.

### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

### REFERENCES


### ETHICS STATEMENT

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University.

### AUTHOR CONTRIBUTIONS

QZ, CL, YZ, and HF participated in research design. QZ, XuaJ, XueJ, ZH, and YJ were responsible for performing the experiments. QZ, QY, YZ, LP, and HF contributed to the writing and editing of the manuscript. All authors have read and approved this article.

### FUNDING

This research was supported by the National Natural Science Foundation of China (grant no. 81772316, 81603036, and 81903551), Key Research and Development Program of Zhejiang Province (grant no. 2018C03013), Zhejiang Provincial Natural Science Foundation (grant no. LY19H180001, LQ19H300001, LY17H180008), Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents (Y-ZZ), 151 Talent Project of Zhejiang Province, Zhejiang Provincial Foundation for Health Department (grant no. 2019322086), Wenzhou Municipal Science and Technology Bureau (grant no. Y20190177), and School Talent Start Fund of Wenzhou Medical University (grant no. QTJ15020).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2019. 01536/full#supplementary-material

FIGURE 1S | Islet viability in vitro. Islets were cultured for 7 (A, B) and 24 h (C, D).


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Zhu, Lu, Jiang, Yao, Jiang, Huang, Jiang, Peng, Fu and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Assessment of the Preventive Effect Against Diabetic Cardiomyopathy of FGF1-Loaded Nanoliposomes Combined With Microbubble Cavitation by Ultrasound

Lei Zheng1,2, Chuan-Li Shen<sup>2</sup> , Jian-Min Li <sup>3</sup> , Yu-Lei Ma<sup>1</sup> , Ning Yan<sup>1</sup> , Xin-Qiao Tian1\* and Ying-Zheng Zhao4,5\*

#### Edited by:

Zhouguang Wang, Albert Einstein College of Medicine, United States

#### Reviewed by:

Cheng-Hai Zhang, Harvard Medical School, United States Shuhui Liu, Icahn School of Medicine at Mount Sinai, United States

> \*Correspondence: Xin-Qiao Tian tianxq2005@163.com Ying-Zheng Zhao pharmtds@163.com

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 09 October 2019 Accepted: 27 November 2019 Published: 10 January 2020

#### Citation:

Zheng L, Shen C-L, Li J-M, Ma Y-L, Yan N, Tian X-Q and Zhao Y-Z (2020) Assessment of the Preventive Effect Against Diabetic Cardiomyopathy of FGF1-Loaded Nanoliposomes Combined With Microbubble Cavitation by Ultrasound. Front. Pharmacol. 10:1535. doi: 10.3389/fphar.2019.01535 <sup>1</sup> Department of Ultrasonography, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Department of Ultrasonography of Central China Fuwai Hospital, Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, China, <sup>2</sup> Department of Ultrasonography, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, <sup>3</sup> Department of Pathology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, <sup>4</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>5</sup> Engineering Laboratory of Zhejiang Province for Pharmaceutical Development of 6 Growth Factors, Biomedical Collaborative Innovation Center of Wenzhou, Wenzhou, China

Acidic fibroblast growth factor (FGF1) has great potential in preventing diabetic cardiomyopathy. This study aimed to evaluate the preventive effect of FGF1-loaded nanoliposomes (FGF1-nlip) combined with ultrasound-targeted microbubble destruction (UTMD) on diabetic cardiomyopathy (DCM) using ultrasound examination. Nanoliposomes encapsulating FGF1 were prepared by reverse phase evaporation. DM model rats were established by intraperitoneal injection of streptozotocin (STZ), and different forms of FGF1 (FGF1 solution, FGF1-nlip, and FGF1-nlip+UTMD) were used for a 12-week intervention. According to the transthoracic echocardiography and velocity vector imaging (VVI) indexes, the LVEF, LVFS, and VVI indexes (Vs, Sr, SRr) in the FGF1-nlip+UTMD group were significantly higher than those in the DM model group and other FGF1 intervention groups. From the realtime myocardial contrast echocardiography (RT-MCE) indexes, the FGF1-nlip+UTMD group A and A×b showed significant differences from the DM model group and other FGF1 intervention groups. Cardiac catheter hemodynamic testing, CD31 immunohistochemical staining, and electron microscopy also confirmed the same conclusion. These results confirmed that the abnormalities, including myocardial dysfunction and perfusion impairment, could be suppressed to different extents by the twice weekly FGF1 treatments for 12 consecutive weeks (free FGF1, FGF1-nlip, and FGF1-nlip+UTMD), with the strongest improvements observed in the FGF1-nlip+UTMD group. In conclusion, the VVI and RT-MCE techniques can detect left ventricular systolic function and perfusion changes in DM rats, providing a more effective experimental basis for the early detection and treatment evaluation of DCM, which is of great significance for the prevention of DCM.

Keywords: diabetic cardiomyopathy, acidic fibroblast growth factor, ultrasound-targeted microbubble destruction, liposomes, preventive effect

### INTRODUCTION

With the development of the economy and society, as well as changes in people's dietary habits and lifestyles, the incidence of diabetes mellitus (DM) is increasing annually, and the age of onset has become younger. According to statistics, there are approximately 425 million DM patients worldwide in 2017, and the number is expected to reach 700 million by 2045 (Cho et al., 2018). The acute and chronic complications caused by DM not only endanger the physical and mental health of patients but also bring a huge economic burden to patients' families and the health industry. Diabetes cardiomyopathy (DCM) is a series of changes in the myocardial structure and function caused by DM (Borghetti et al., 2018; Parim et al., 2019) that are not related to coronary atherosclerosis, hypertension, and valvular heart disease (Rubler et al., 1972). At present, DCM has been identified as the main cause of heart failure and death in patients with DM (Evangelista et al., 2019; Gulsin et al., 2019; Zamora and Villena, 2019). Therefore, early detection and timely intervention therapy are of great significance for the prognosis of DM patients.

Fibroblast growth factors (FGFs) have at least 23 members and are widely distributed in the body. Each member has a certain structural similarity (Beenken and Mohammadi, 2009; Itoh and Ornitz, 2011; Zhang et al., 2013; Li, 2019). Among them, acidic fibroblast growth factor (FGF1) is one of the earlier founded members of the fibroblast growth factor family. Studies have shown that FGF1 has a wide range of physiological functions, such as antioxidant damage (Pena et al., 2017), induction of endothelial and smooth muscle cell proliferation and angiogenesis (Zhao et al., 2016), promotion of tissue wound repair (Xu et al., 2018), neurotrophic regeneration (Tsai et al., 2015), etc. Therefore, FGF1 has a great potential in the prevention and treatment of DCM. However, the lack of an efficient and safe delivery system limits FGF1 application in vivo.

Nanoliposomes are double-layer vesicle structures composed of hydrated lipids (Abu and Ishida, 2017; Kapoor et al., 2017). The particle sizes range from 10 to 1000 nm. Nanoliposomes have the characteristics of high drug loading, sustained-release drugs, high stability in vivo, non-toxicity, non-immunogenicity, and biodegradability (Chen and Stephen, 2019). Therefore, using nanoliposomes as drug carriers can not only effectively protect the drug from the influence of environmental conditionsin vivo but also can penetrate the endothelial gap and complete capillaries to reach the target tissue to be absorbed by most cells, thus playing a corresponding biological effect. However, it is difficult for nanoliposomes to locally aggregate at high concentrations in vivo to achieve highly effective targeted therapy.

Ultrasound targeted microbubble destruction (UTMD) provides a new method for myocardial targeted delivery of FGF1. Under the energy of diagnostic or therapeutic ultrasound, ultrasound microbubbles can explode in the region of interest or target tissues. Cavitation and mechanical effects of blasting can increase the permeability of local vascular walls or cell membranes, thereby increasing the dose of drugs/genes in target organs or target tissues and their corresponding biological effects (Frenkel, 2008; Chen et al., 2018; Liang et al., 2018; Lin et al., 2018;Yang et al., 2019).

Echocardiography, as a practical tool for the non-invasive evaluation of cardiac function, has been widely applied in clinical and animal experiments. Velocity vector imaging (VVI) is based on two-dimensional gray-scale ultrasound images with a high frame rate. It uses spatial coherence, speckle and boundary tracking techniques of ultrasound pixels to automatically track and recognize the motion of echo spots in the region of interest in each frame image and quantitatively analyzes the structural mechanics of myocardial tissue motion to obtain a reflection of the myocardium. Compared with traditional techniques, VVI has no angle dependence, and its strain and strain rate measurements are relatively unaffected by respiration (Azam et al., 2012; Li et al., 2012; Zhou et al., 2015). Therefore, it is superior to conventional ultrasound and tissue imaging and its derivative technology in cardiac function abnormalities and heart disease treatment effect evaluation. Real-time myocardial contrast echocardiography (RT-MCE) technology is used to inject ultrasound contrast agents containing microbubbles into the body through peripheral veins. Because the size of microbubbles is the same as that of red blood cells, the hemodynamics is similar to that of red blood cells. Microbubbles can freely distribute in myocardial tissue through cardiac capillaries. Microbubbles produce a large number of liquidgas interfaces in the blood, thus reflecting a large number of ultrasound signals and increasing the video density of myocardial microcirculation. By observing the contrast enhancement of the myocardium with echocardiography, the tissue perfusion information can be evaluated at the microvascular level. The whole and local perfusion of myocardium can be observed and analyzed non-invasively. The volume, velocity, and flow of myocardium can be measured quantitatively (Wei et al., 2012; Jiang et al., 2017; Danijela et al., 2018; Geng et al., 2018). Although the application of VVI and RT-MCE technology is increasing, few studies have evaluated the efficacy of DCM in prevention and treatment.

Therefore, FGF1 loaded nanoliposomes (FGF1-nlip) combined with UTMD technology were used in this study to intervene in early DM rats. The effects of this method on left ventricular function and blood flow perfusion in DM rats were evaluated by conventional echocardiography, velocity vector imaging (VVI), real-time myocardial contrast echocardiography (RT-MCE), and histomorphology.

### MATERIALS AND METHODS

### Preparation and Properties of FGF1-nlip

FGF1-nlip were prepared by reverse phase evaporation. The specific preparation process was as follows: FGF1 lyophilized powder (Guangzhou Jinan University Medical Biotechnology Research and Development Center) was dissolved in physiological saline to form FGF1 (1 mg/ml) solution. Next, 90 mg of natural phospholipids (Shanghai AVT) and 10 mg of cholesterol (Shanghai AVT) were weighed and dissolved in 4 ml of dichloromethane (Guangdong Guanghua Technology Co., Ltd.) for use. The FGF1 solution was slowly added dropwise to the phospholipid-dissolved methylene chloride and rapidly sonicated (100 W, 15 s) to form a stable w/o emulsion. The methylene chloride was removed by rotary evaporation under reduced pressure. After the dichloromethane was completely removed, the mixture was hydrated with an appropriate amount of physiological saline and ultrasonically dispersed (100 W, 10 min) in an ice bath to give a final FGF1 concentration of 10 mg/ml. Blank nanoliposomes were prepared by replacing the FGF1 solution with an equal volume of distilled water. The characterization of FGF1-nlip mainly includes morphological characteristics, zeta potential, particle size, and the encapsulation efficiency of the particles. In this study, the particle size and morphological characteristics of FGF1-nlip were determined by transmission electron microscopy. The hydration particle size and zeta potential of FGF1-nlip were determined by dynamic light scattering. The determination method of the encapsulation efficiency of FGF1-nlip was as follows: 1.0 ml of liposome suspension containing FGF1 was accurately measured in an ultrafiltration tube, centrifuged at 10,000 r/min for 30 min, the filtrate was removed and diluted properly, and the FGF1 ELISA reagent was used. The cassette was assayed for FGF1 protein concentration. Encapsulation ratio (%) = (total protein amount − amount of protein in the filtrate)/total protein amount × 100%.

### Experimental Animals

Seventy male Sprague-Dawley (SD) rats, aged 40 to 50 days, with body weight 180–220 g, were provided by the Experimental Animal Center of Wenzhou Medical University (Experimental Animal License No.: SYXK (Zhejiang] 2015-0009). All animal experiments were performed with the permission of the Animal Ethics Committee of Wenzhou Medical University (approval number: wydw2016-0025).

### Experimental Animal Groups

After 7 days of adaptive feeding, all rats were fasted for 12 hours overnight. On the second day, 56 rats were randomly selected, and each rat was intraperitoneally injected with a 1% solution of streptozotocin 70 mg/kg. The blood glucose concentration was measured by tail vein sampling on the 1st, 3rd, and 7th days after injection. The rats with blood glucose levels exceeding 16.7 mmol/L and with higher food intake, polydipsia, and polyuria were selected as DM rats (52 rats were modeled, 4 were excluded without being modeled). Subsequently, DM rats were randomized into 4 groups with 13 rats in each group: DM model group, FGF1 solution group, FGF1-nlip group, and FGF1-nlip+UTMD group. The remaining 14 were used as the normal control group, and the same amount of citrate buffer was intraperitoneally injected and they were fed under the same conditions.

All experimental rats were housed in the SPF animal room (the average indoor temperature was approximately 20–24°C, the average humidity was approximately 50–60%), and the on-time changes were simulated by turning on and off the lights regularly. All rats were freely supplied with sterile water during the feeding process.

## Experimental Animal Intervention

After intraperitoneal anesthesia with 10% chloral hydrate solution, rats in the DM model group were injected with saline via the caudal vein; rats in the FGF1 solution group were injected with FGF1 solution via the caudal vein; and rats in the FGF1-nlip group were injected with FGF1-nlip solution via the caudal vein. After anesthesia, rats in the FGF1-nlip+UTMD group were shaved in the anterior cardiac region and fixed in the left lateral decubitus position. FGF1-nlip solution was well mixed with SonoVue microbubbles (Bracco, Italy) before administration. The Acuson Sequoia 512C system (Siemens, Germany) was used, and the RT-MCE imagingmodewas set up. The probefrequencywas set to 12 to 14 MHz (15L8-w linear array probe). The probe was placed in the precordial area of the rats, and the probe and skin were filled with coupling agents. The ultrasound instrument was converted into contrast mode. Left ventricular short axis view at the papillary muscle level was taken, and the focusing depth was 3.5–4.0 cm. The mixture of FGF1-nlip and SonoVue microbubbles was slowly injected into rats. When the image showed that a large number of contrast agents were filling the myocardium, MBD function (mechanical index MI = 1.9) was used to blast the microbubbles repeatedly until the microbubbles completely disappeared. The dose of fibroblast growth factor 1 in each intervention group was 15 µg/kg (Zhao et al., 2016). Drug intervention lasted 12 weeks, twice a week, for a total of 24 times.

### Ultrasound Image Acquisition

Before and 12 weeks after the intervention (De Blasio et al., 2015; Atta et al., 2018), rats in each group were anesthetized with 10% chloral hydrate intraperitoneally and shaved in the anterior cardiac region. The probe was placed in the anterior cardiac region of rats, and the coupling agent was filled between the probe and the skin. The left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were measured by conventional M-mode echocardiography in all rats.

VVI imaging mode was started, and the RES button on the machine was pressed to enlarge the image while reducing the spatial resolution appropriately to obtain the highest frame rate possible. The frame rate of this study was adjusted to between 60 and 90 Hz. Acoustic acquisition of two-dimensional gray-scale dynamic images of the papillary muscle horizontal left ventricular short axis view for three or more cardiac cycles was used. All dynamic images were stored on MO discs and imported into Siemens Sygno US Workplace 3.01 analysis software for offline analysis.

The cadence key was pressed to enter the contrast mode. The depth of the image was 3–4 cm. The RES key was pressed to enlarge the region of interest locally. The mechanical index was 0.35. The gain was adjusted so that there was no obvious acoustic signal in the myocardial tissue and remained constant throughout the process. Contrast medium was infused slowly through the caudal vein. Left ventricular short axis myocardiography at the papillary muscle level was performed at rest to observe the filling of contrast media in the cardiac cavity and myocardium. After the filling of contrast media in the myocardium was sufficient, the contrast media in the myocardium were destroyed by high energy pulse, and then the instrument was automatically converted to low energy imaging to obtain real-time dynamic imaging. All real-time dynamic images were stored on MO discs and then imported into Siemens Sygno US Workplace 3.01 analysis software for offline auto-tracing contrast quantitative technique (ACQ) analysis.

### VVI Analysis

In the VVI analysis software, a frame image of the clearest endocardium was selected, and the boundary of the left ventricular endocardium and epicardiumwas drawn clockwise.According to the standard 16-segment method developed by the American Academy of Echocardiography, the left ventricular wall was automatically divided into six segments: anterior wall (AW), lateral wall (LW), posterior wall (PW), inferior wall (IW), posterior septum (PS), and anterior septum (AS). The position of each segment was monitored, and the myocardial velocity, radial strain, and strain rate curves of 6 segments were obtained automatically by software. Themean systolic peak velocity (Vs), radial peak strain (Sr), and radial peak strain rate (SRr) were measured in 6 segments of the left ventricle.

### MCE Analysis

In the analysis software, the left ventricular short-axis view of the anterior wall myocardial tissue was selected to outline the region of interest (in the process of delineation, the interference of factors such as endometrial, epicardial, and papillary muscles was avoided). The software automatically tracks the region of interest in each frame of the image and allows the operator to adjust the location of the region of interest at any frame so that it is always within the corresponding myocardial tissue. The analysis software measures the average acoustic intensity of the region of interest of each frame of image and fits it to the acoustic intensitytime curve y = A × (1−e -bt ),where A is the peak acoustic intensity of the region of interest, reflecting the blood volume of the local myocardium, b is the contrast agent perfusion rate, reflecting the blood flow velocity of the myocardium, the product of the two (A×b) represents the regional myocardial blood flow, and each rat is analyzed 2~3 times and the average taken to reduce the error. All of the analysis curves with a goodness offit (GOF) < 0.9 were excluded.

## Hemodynamic Evaluation

All the experimental rats were anesthetized with 10% chloral hydrate after intraperitoneal injection, and then they were routinely disinfected. The skin of the anterior cervical region was cut, the trachea was separated, the animal ventilator was opened, the thoracic cavity was opened, and the cardiac catheter was penetrated into the left ventricle of the rat with heparin saline after filling the heart catheter with heparin saline. After the biological signal was transformed, Chart5 for Windows analysis software was synchronized. Left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and the maximum rise and fall rate of left ventricular internal pressure (LV ± dp/dtmax) were recorded.

### CD31 Immunohistochemical Staining and Cardiac Capillary Density Measurement

The rats were sacrificed, the heart was removed, and the left ventricular myocardium was taken from the papillary muscles, fixed with 10% formaldehyde, and embedded in paraffin. The paraffin sections of myocardial tissue were stained with CD31 (Abcam, ab28364, USA) for immunohistochemistry and observed under an optical microscope. Eight slices were taken from each group, and 10 high-power fields (hpf) (400×) were randomly selected from each slice. The number of microvascular sections was calculated and averaged as the microvascular density (MVD) in the myocardial tissue.

### Transmission Electron Microscopy

A portion of the left ventricle was cut into 1-mm tissue blocks and fixed in 2.5% glutaraldehyde for more than 4 h for electron microscopy. Myocardial tissue was then immobilized with 1% osmium acid for 1 h and then rinsed and stained with 1% uranium acetate for 1 h. After dehydration with acetone and embedding with epoxy resin, myocardial tissue was cut into 1 µm sections and stained with toluidine blue. The ultrathin sections were cut from the block and studied under a JEM-1230 transmission electron microscope (JEOL of Japan).

### Statistical Analysis

Data analysis was performed using IBM SPSS Statistics 25.0 statistical software. The normal distribution of the measurement indicators was expressed as the mean ± SD. The multivariate sample means were compared for variance homogeneity test. The mean comparison between groups was analyzed by one-way ANOVA. The LSD-t test was used to compare the two groups. Dunnett's T3 test was performed on the variance, and the difference was statistically significant at P < 0.05.

## RESULT

## Characterization of FGF1-nlip

Observation of blank nanoliposomes and FGF1-nlip by transmission electron microscopy showed that the particle size distribution of the two was uniform, some adhesions existed, and the lipid bilayers were visible in the spherical shape (Figure 1). The average particle size of liposomes and FGF1-loaded liposomes was determined by the dynamic light scattering method to be 68.24 ± 1.61 nm and 96.23 ± 6.56 nm, respectively. The polydispersity index of the particle size was 0.092 ± 0.04 and 0.143 ± 0.02, respectively. Zeta potential measurements showed that the zeta potentials of the blank and drug-loaded liposomes were −1.885 ± 0.075 and −3.06 ± 0.01 mV, respectively. Further, the encapsulation efficiency measurement showed that the encapsulation efficiency of the FGF1 liposome was 73.52 ± 3.25%.

### General Condition of Experimental Rats

After 12 weeks of intervention, 4 rats in the DM model group and in the FGF1-nlip group died, 3 rats in the FGF1 solution group and the FGF1-nlip+UTMD group died, and no rats in the normal control group died. The number of rats in each group was 9 in the DM model group, 10 in the FGF1 solution group, 9 in the FGF1-nlip group, 10 in the FGF1-nlip+UTMD group, and 14 in the normal control group.

### Echocardiography Evaluation

Before intervention, there was no significant difference in LVEF andLVFS between the ratsineachgroup (P >0.05).After12weeks of intervention, the LVEF and LVFS in the DM model group were significantlylower than thosein the normal control group (P < 0.05). Compared with the DM model group, the LVEF and LVFS of the FGF1 intervention group were significantly increased; compared

with the other intervention groups, the rats in the FGF1-nlip +UTMD group were significantly increased (P < 0.05) (Table 1).

### VVI Evaluation

As shown in Figure 2, the myocardial velocity, radial strain, and strain rate curves of 6 segments were obtained automatically by software. Before the intervention, there was no significant difference in Vs, Sr, and SRr among the groups (P > 0.05). After 12 weeks of intervention, Vs, Sr, and SRr in the DM model group were significantly lower than those in the normal control group (P < 0.05); Vs, Sr, and Sr in the FGF1 solution group, FGF1-nlip group, and FGF1-nlip+UTMD group were significantly higher than those in the model group (P < 0.05); those in the FGF1-nlip+UTMD group were higher than those in the other intervention groups (P < 0.05) (Table 2).

### MCE Evaluation

Figure 3 shows the average acoustic intensity of the region of interest of each image frame. Before the experimental intervention, there was no significant difference in A, b, and A × b between the groups (P > 0.05). After 12 weeks of intervention, A, b, and A × b in the DM model group were significantly lower than those in the normal control group (P < 0.05). Compared with the DM model group, the FGF1 solution group and the FGF1-nlip group A andA × b were significantly increased (P < 0.05). Although there was an increasing trend of b, the difference was not statistically significant (P > 0.05), while the FGF1-nlip+UTMD group A, b, and A×b were significantly increased (P < 0.05); compared with the FGF1 solution group and FGF1-nlip group, the FGF1-nlip+UTMD group A and A × b were significantly increased (P < 0.05), and b increased slightly (P > 0.05) (Table 3).

### Hemodynamic Analysis

After 12 weeks of intervention, the LVESP, +dp/dtmax, and −dp/ dtmaxin the DMmodel groupwere significantlylower than thosein the normal control group (P < 0.05), and LVEDP was significantly higher than thatin the normal controlgroup (P<0.05).LVESP,+dp/ dtmax, and −dp/dtmax in the FGF1 intervention groups were significantly higher than those in the DM model group (P < 0.05), and the levelswere significantly lower than those in themodel group (P<0.05).TheLVESP,+dp/dtmax,and−dp/dtmaxin theFGF1-nlip +UTMD group were significantly higher than those in other FGF1 intervention groups (P < 0.05), and the LVEDP in the FGF1-nlip

#### TABLE 1 | Results of LVEF and LVFS (mean ± SD).


LVEF, left ventricular ejection fraction; LVFS, left ventricular fraction shortening. Data are Mean ± SD.

a P < 0.05 vs the normal control group.

b P < 0.05 vs DM model group.

c P < 0.05 vs FGF1-nlip+UTMD group.

TABLE 2 | Results of peak velocity, radical strain, and radical strain rate in the control and study groups (mean ± SD).


Vs = systolic peak velocity; Sr = radial peak strain; SRr = radial peak strain rate.

a P < 0.05 vs the normal control group.

b P < 0.05 vs DM model group.

c P < 0.05 vs FGF1-nlip+UTMD group.

+UTMD group was significantly lower than that in the other FGF1 intervention groups (P < 0.05) (Table 4).

### Myocardial Capillary Density

CD31 immunohistochemical staining showed that the vascular endothelial cells were brown. The MVD of myocardial tissue in the DM model group was significantly lower than that in the normal control group (P < 0.05). The MVD in each FGF1 intervention group was significantly higher than that in the DM model group (P < 0.05). The MVD in the FGF1-nlip +UTMD group was significantly higher than that in the other FGF1 intervention groups (P < 0.05) (Figure 4).

### Electron Microscopic Findings

As shown in Figure 5, compared with the normal group, in the DM model group after 12 weeks, the Z line of cardiac muscle and myocutaneous fibers was arranged in a disordered manner, partly ruptured, with swollen aggregated mitochondria, and some vacuoles appeared. However, after 12 weeks of various forms of FGF1 prevention, the ultrastructure of the heart improved, especially in the fibrous filamentts and muscle fibers of the heart of rats in the FGF1-nlip+UTMD group, which were arranged neatly, and most of the mitochondria were normal. The results showed that themyocardial ultrastructure of DM rats was improved after different forms of FGF1 prevention, but compared with other forms of prevention, FGF1-nlip combined with UTMD technology had the most obvious preventive effect.

## DISCUSSION

In this study, the ratmodel of DMwas established byintraperitoneal injection of STZ. Routine echocardiography, the VVI technique, and RT-MCE were performed before drug intervention. No significant difference was found in cardiac function and the blood perfusion index among each group. After 12 weeks offeeding, DM rats underwent routine echocardiography, VVI, and cardiac catheterization techniques. The results showed that the LVEF, LVFS, and VVI indexes (Vs, Sr, and SRr), LVESP, and ± dp/


A = peak acoustic intensity; b = contrast agent perfusion rate; A×b = myocardial blood flow

a P < 0.05 vs the normal control group.

b P < 0.05 vs DM model group.

c P < 0.05 vs FGF1-nlip+UTMD group.

#### TABLE 4 | The hemodynamic data in the in vivo experiment (mean ± SD).


Note : LVESP = left ventricular systolic pressure; LVEDP = left ventricular end diastolic pressure; ± dp/dt max = maximum rate of the rise and fall of left ventricular pressure. a P< 0.05 vs the normal control group.

b P< 0.05 vs DM model group.

c P< 0.05 vs FGF1-nlip+UTMD group.

P < 0.05 vs the DM model group, <sup>c</sup>

group, <sup>b</sup>

group; (B) DM model group; (C) FGF1 solution group; (D) FGF1-nlip group; (E) FGF1-nlip+UTMD group; (F) Statistical histogram. <sup>a</sup>

P < 0.05 vs the FGF1-nlip+UTMD group).

P < 0.05 vs the normal control

FIGURE 5 | Representative pictures of electron micrographs (magnification 10000×) of left ventricular heart muscle sections from the rats of each group (A) normal control group; (B) DM model group; (C) FGF1 solution; (D) FGF1-nlip group; (E) FGF1-nlip+UTMD group)].

dtmax in the DM model group were significantly lower than those in the normal control group (P < 0.05), while LVESP in the DM model group was higher than those in the normal control group (P < 0.05). RT-MCE showed that A, b, and A × b in the DM model group were significantly lower than those in the control group. This indicated that obvious abnormalities in left ventricular systolic function and blood flow perfusion appeared in DM rats at the end of 12 weeks. Histological observation also confirmed that the MVD value of myocardial tissue in DM rats was significantly lower than that in the control group, and there were pathological changes such as disordered filament arrangement, mitochondrial swelling and degeneration. Therefore, it is very important to prevent the occurrence of DCM by intervening before the obvious pathological changes in myocardium in DM rats.

FGF1 is a type of cell growth factor with many biological activities. It has a high affinity for heparin. FGF1 can promote the proliferation of vascular endothelial cells and smooth muscle cells, thereby promoting angiogenesis, alleviating myocardial ischemia, and improving cardiac function (Cochain et al., 2013; Zhang et al., 2016). In previous studies, in various animal models of myocardial ischemia or infarction, FGF1 increased the regional myocardial blood flow and the density of capillaries and arterioles and improved ventricular function (Zhao et al., 2012). Other studies have shown that non-mitotic FGF1 can prevent DCM (Zhang et al., 2013) by inhibiting oxidative stress and injury. Therefore, FGF1 is a potentially valuable therapeutic agent for the prevention and treatment of DCM.

In order to increase the stability of the drug, the reverse phase evaporation method (Yue et al., 2014; Mehrabi et al., 2016) was used to prepare nanoliposomes encapsulated with FGF1. Through the analysis of its characteristics, it was confirmed that the drug-loaded lipids had a round appearance, small particle size (<100 nm), good dispersion (polydispersityindex <0.15),and a certainZeta potential, which could maintain good stability (Figure 1). Compared with traditionalliposome preparation technology, themethodadoptedin this study has a shorter preparation time, and the average temperature of the whole preparation process is controlled below 25°C,which canmaintain the biological activity of protein drugs to a great extent. This shows that the preparation method can efficiently achieve the inclusion of FGF1 and is suitable forin vivo application. Inorder toincrease thelocalconcentrationofFGF1in theheart,exert the therapeutic effects toagreater extent, and reduce theinfluence on the whole body (e.g., liver, spleen, and kidney), this study injected FGF1-nlip mixed with SonoVue microbubbles into DM rats via the caudal vein. After myocardial development, UTMD was used to explodemicrobubbles toincrease thedoseofFGF1-nlip releasedinto the myocardium and exert its corresponding biological effects.

After 12 weeks of intervention, routine echocardiography and VVI (Tables 1–2) showed that LVEF, LVFS, and VVI (Vs, Sr, and SRr) in the FGF1 intervention group were significantly higher than those in the DM model group (P < 0.05), while LVEF, LVFS, and VVI (Vs, Sr, and SRr) in the FGF1-nlip +UTMD group were significantly higher than those in the other FGF1 intervention groups (P < 0.05), which was confirmed by hemodynamic studies (Table 4). The results showed that the FGF1 nlip+UTMD group had the least damage to left ventricular systolic function in DM rats. In addition, the ultrastructural changes of the FGF1-nlip+UTMD group were the least. Therefore, this method can be used as an effective strategy to prevent the deterioration of cardiac function in DM rats.

The results of RT-MCE in this study (Table 3) showed that after 12 weeks of intervention, the levels of A and A × b in the FGF1 solution group and FGF1-nlip group were significantly higher than those in the DM model group (P < 0.05), while the levels of A, b, and A × b in the FGF1-nlip+UTMD group were significantly higher than those in the DM model group (P < 0.05), and A and A × b were significantly higher than those in the other FGF1 intervention groups. To improve myocardial microcirculation, the application of FGF1-nlip combined with UTMD has the best preventive effect. This was confirmed by CD31 immunohistochemical assay (Figure 4).

In addition, there was no significant difference in left ventricular function and blood perfusion between the FGF1 solution group and the FGF1-nlip group. The reason is that the drug encapsulated by liposomes alone lacks targeting ability and is easily captured by the reticuloendothelial system in vivo (Ishihara et al., 2012; Hsu et al., 2014). It is difficult to maximize the efficacy of the drug in cardiac aggregation. Combined with UTMD, the cavitation effect and mechanical action produced by the instantaneous blasting of ultrasound microbubbles can cause temporary open pores (Chen et al., 2013; Qian et al., 2018; Zhao et al., 2018) on the endothelial cell membrane and capillaries of the myocardium. The target delivery of FGF1-nlip to the myocardium can realize the rapid release of the target and rapid penetration, thus realizing the myocardial transmission of FGF1 and exerting its biological function.

In the present study, although each intervention group had a significant improvement in cardiac function, the survival rate was lower than that in the normal control group. The cause of death in rats is related to the deterioration of the physical condition of rats caused by diabetes injury, and may be related to the influence of anesthetic drugs. Because the animals could not cooperate effectively, the rats in the intervention group had higher requirements for anesthesia depth to ensure the stability and effectiveness of the intervention. The normal control group did not undergo relevant intervention, so there was no death.

In conclusion, the application of FGF1-nlip combined with UTMD technology can efficiently deliver FGF1 to the heart of DM rats and prevent abnormal left ventricular systolicfunction and myocardial perfusion in DM rats. The changes in left ventricular systolic function and blood perfusion in DM rats can be detected by VVI and RT-MCE technology, which provides a more effective basis for the evaluation of DCM therapeutic effects.

The limitations of this study are as follows: First, UTMD technology is used to promote the entry of FGF1-nlip into cells, thus causing a certain degree of damage to cells. It is necessary to further optimize the parameters of ultrasound irradiation for local release of the highest drug concentration in the case of minimal tissue damage. Second, because the pathogenesis of DCM is complex and the effect of FGF1 alone is limited, whether it is necessary to combine other drugs to improve the efficacy needs further study.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ supplementary material.

### ETHICS STATEMENT

The animal study was reviewed and approved by the Animal Ethics Committee of Wenzhou Medical University.

## AUTHOR CONTRIBUTIONS

LZ: Overall experimental design, data analysis, paper writing. C-LS: Acquisition and analysis of ultrasound Image of animals. J-ML: Histopathological detection and analysis, wzyxyljmin@ 163.com. Y-LM: Feeding of experimental animals, imayulei@ 163.com. NY: Pharmaceutical intervention in laboratory animals. X-QT: Research overall guidance, paper revision, funding support. Y-ZZ: Preparation of nanoliposomes, revision of papers, financial support.

## FUNDING

This research was supported by the Chinese National Natural Science Funds (Grant No. 81571696, U1704175), Medicine Grant from Wenzhou Bureau of Science and Technology (Y20170049), Key Research and Development Program of Zhejiang province (Grant No. 2018C03013).

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Zhou, J., Pu, D. R., Tian, L. Q., Tong, H., Liu, H. Y., Tan, Y., et al. (2015). Noninvasive assessment of myocardial mechanics of the left ventricle in rabbits using velocity vector imaging. Med. Sci. Monit. Basic Res. 21, 109–115. doi: 10.12659/MSMBR.894053

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Zheng, Shen, Li, Ma, Yan, Tian and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Compound 15c, a Novel Dual Inhibitor of EGFRL858R/T790M and FGFR1, Efficiently Overcomes Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance of Non-Small-Cell Lung Cancers

#### Gaozhi Chen1,2† , Yuyan Bao3† , Qiaoyou Weng<sup>4</sup> , Yingxin Zhao<sup>1</sup> , Xiaoyao Lu<sup>1</sup> , Lili Fu<sup>1</sup> , Lingfeng Chen<sup>1</sup> , Zhiguo Liu<sup>1</sup> , Xiaomin Zhang<sup>3</sup> and Guang Liang1\*

<sup>1</sup> Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Engineering Laboratory of Zhejiang Province for Pharmaceutical Development of Growth Factors, Biomedical Collaborative Innovation Center of Wenzhou, Wenzhou, China, <sup>3</sup> Department of Pharmacy, Sanmen People's Hospital of Zhejiang, Sanmen, China, <sup>4</sup> Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention, The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui, China

In the past decades, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved as an effective treatment strategy for the patients with EGFRmutated non-small-cell lung cancer (NSCLC). However, the tolerance for the EGFR-TKI always occurred after continuous administration for a period of time and limiting the application of these drugs. Activation of FGFR1 signaling pathway was one of the important escape mechanisms for EGFR-TKI resistant in NSCLC. Here, a novel dual inhibitor of EGFRL858R/T790M and FGFR1, compound15c, was found and can efficiently overcame the EGFR-TKI resistance via its simultaneous inhibition of their kinase activities. Comparison with EGFRL858R/T790M and FGFR1 inhibitor treatment alone or combined revealed that the inhibition of EGFRL858R/T790M and FGFR1 activity by 15c was responsible for surmounting the intrinsic EGFR-TKI resistance in EGFRL858R/T790M-mutated H1975 cells and the acquired resistance in Afatinib-tolerant PC9 cells (AFA-PC9). Flow Cytometry and Caspase3 activity analysis assay showed that 15c induced significant the early apoptosis of H1975 cells. Xenograft tumor formation in BALB/c mice induced by a H1975 cells was suppressed by 15c treatment, with no changes in animal body weight. Generally, 15c may act as a new-generation EGFR-TKI for the therapy of NSCLC patients suffering a resistance to current TKI.

Keywords: NSCLC, EGFR, FGFR1, drug resistance, kinase inhibitor

### Edited by:

Zhouguang Wang, Albert Einstein College of Medicine, United States

### Reviewed by:

Aiping Jiang, Harvard University, United States Shuai Wu, Wistar Institute, United States

#### \*Correspondence:

Guang Liang wzmcliangguang@163.com

These authors have contributed equally to this work

†

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 22 October 2019 Accepted: 27 November 2019 Published: 10 January 2020

#### Citation:

Chen G, Bao Y, Weng Q, Zhao Y, Lu X, Fu L, Chen L, Liu Z, Zhang X and Liang G (2020) Compound 15c, a Novel Dual Inhibitor of EGFRL858R/T790M and FGFR1, Efficiently Overcomes Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance of Non-Small-Cell Lung Cancers. Front. Pharmacol. 10:1533. doi: 10.3389/fphar.2019.01533

### INTRODUCTION

In the past few decades, to improve therapeutic activity and selectivity while developing antitumor drugs is always a great challenge. By targeting the genetic differences of cancer cells, several molecule-targeting drugs, such as Afatinib and Gefitinib, had been approved by U.S. FDA and shown their promising therapeutic activity against various cancers (Cataldo et al., 2011; Hirsch and Bunn, 2012). However, the therapeutic strategies of "one drug-hits-one target-treats-one disease" still face significant challenges as the drug resistance (To et al., 2019; Wojtaszek et al., 2019). Recent studies suggested that targeting the multiple targets might be a practicable approach to ameliorate the therapeutic activity and selectivity, meanwhile preventing the drug resistance (To et al., 2019).

Among cancer-related deaths, lung cancer is the leading cause that contributed to 27% of cancer-related mortality (Quintanal-Villalonga et al., 2019) and almost 85% of lung cancers were identified as non-small cell lung cancer (NSCLC) (Chang, 2011). The high dynamic of epidermal growth factor receptor (EGFR), one of the classic receptor tyrosine kinase (RTK), in NSCLC (Hirsch et al., 2003) recommended it as an attractive target for the development of small-molecule tyrosine kinase inhibitors (TKIs) to treat with NSCLC. At the beginning, several first-generation EGFR-targeted TKIs, such as Erlotinib and Gefitinib, had been approved by U.S. FDA for the treatment of NSCLC patients that harbor activating mutations in the EGFR (L858R or delE746-A750) (Cohen et al., 2003; Cohen et al., 2005; Cataldo et al., 2011). However, resistance to those inhibitors can be acquired due to secondary mutations (Bean et al., 2007; Kuang et al., 2009). The most common secondary mutations in the EGFR is T790M at exon 20, which is named as "gatekeeper" mutation (Zhou et al., 2009). Subsequently, a series of second-generation and third-generation EGFR-TKIs had being developed to overcome the resistance, especially T790M-associated resistance (Riely, 2008; Niederst et al., 2015; Chen et al., 2017b). Unfortunately, tolerance still occurred as a second mechanism that activation of alternative pathways, such as cMet, IGF-1R, and FGFR (Kono et al., 2009). Unlike the widely reported cMet and IGF-1R, the FGFR-dependent signaling act as an escape mechanism for EGFR-TKI resistance still have long way to go (Kono et al., 2009).

Fibroblast growth factor receptors (FGFRs) are a family of RTKs with four different members (FGFR1–4), and each of the FGFR contain an extracellular immunoglobulin domains, a single transmembrane domain and an intercellular kinase domain (Li, 2019). Moreover, genetic abnormalities or aberrant activation of the FGFR signal transduction have been implicated with the pathogenesis of the various disease and disorders including cancer, Alzheimer's disease, diabetes and its complications (Ferrer and Marti, 1998; Gozgit et al., 2012; Harrison, 2012; Gallagher and LeRoith, 2013; Touat et al., 2015; Li, 2019). Among these receptors, FGFR1 amplification is identified in about 20% of NSCLC. In addition, Azuma et al., reported that the enhanced expression of FGFR1 conducted as an escape mechanism for cell survival of Afatinib-resistant cancer cells and may compensate the loss of EGFR-driven signaling pathway (Azuma et al., 2014). Further, Quintanal-Villalonga and his colleagues found that highlyexpressed FGFR1 may result in a higher resistance to EGFR-TKI in the patients with EGFR-mutated lung cancer and those patients may benefit from combined EGFR/FGFR inhibition (Quintanal-Villalonga et al., 2019). Raoof and her colleagues performed wholegenome CRISPR screening and identified FGFR3 as the top target facilitating the survival of mesenchymal EGFR mutant cancers (Raoof et al., 2019). Therefore, dual EGFR-FGFR1 blockade may be a promising clinical strategy to overcome EGFRmutated NSCLC.

Nowadays, drugs with polypharmacological activities are shown to be advantageous over combination therapy as their lower incidences of side effects and more resilient therapies (Anighoro et al., 2014). Thus, in the current study, we find that compound 15c, an EGFRL858R/T790M selective inhibitor in our previous study (Chen et al., 2017a), exhibited a dual inhibitory activity against EGFRL858R/T790M and FGFR1 and efficiently overcame the EGFR-TKI tolerance in EGFR-mutated NSCLC cells via concurrently inhibiting these two kinases activity. We suggest that 15c may act as a new-generation EGFR-TKI for the therapy of NSCLC patients suffering a resistance to current TKI.

### MATERIALS AND METHODS

### Cell Culture and Reagents

WZ4002 (#S1173) and AZD4547 (#S2801) were purchased from Selleck Chemicals. Human bronchial epithelial cell line BEAS-2B, human lung squamous cancer cell line H520, and human NSCLC cell lines H1975 and PC9 were procured from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences and tested for mycoplasma contamination by DAPI staining before experiment. All the cells were maintained in RPMI-1640 medium (#C11875500BT, Gibco) with 10% FBS (#10270-106, Gibco), 100 mg/ml streptomycin, and 100 U/ml penicillin (#15140122, Gibco) and placed in a humidified cell incubator (5% CO2, 37°C). Antibodies including anti-p-EGFR (#3777S), anti-p-FGFR1 (#2544S), anti-EGFR (#2646S), anti-FGFR (#9740S), anti-GAPDH (#5174S), and HRP-linked antirabbit IgG (#7074S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

### Kinase Inhibition Assay

The kinase inhibitory activities of candidate and positive inhibitors were tested via a Caliper Mobility Shift Assay. The difference between substrate and its phosphorylated product was detected to characterize the activity. Shortly, the reaction solution containing compounds, substrates, ATP, and enzymes was mixed well and transferred to a 384-well plate for the experiment. EDTA was introduced to terminate the process after incubate for 1h at room temperature. The data was collected on an EZ Reader II (Caliper Life Sciences, MA). The inhibitory rates of tested compounds were calculated depending on the negative control wells (without ATP) and positive control wells (without compounds). The recombinant kinases, including EGFRWT (#08-115), EGFRL858R/T790M (#08-510), and FGFR1WT (#08-133) were acquired from Carna Biosciences (Kobe, Japan). All the independent experiments were executed in duplicate and three times at six concentrations (0.001, 0.01, 0.1, 1, 10, and 100 mM) and IC50 value was calculated.

### Anti-Proliferation Assay (MTS Assay)

All kinds of cells (4×10<sup>3</sup> ) were planted in 96-well plate and cultured overnight before examination. The protocol was formulated according to CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) Technical Bulletin. Briefly, after treated the cells with different compounds for 72 h, 20 ml CellTiter 96® AQueous One Solution Reagent (MTS, #G3580, Promega, San Luis Obispo, CA) was added and the system was incubated for another 4 h at 37°C. The absorbance at 490 nm was recorded by using a microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA). The results of three independent assays were exhibited as IC50 value (mean ± SEM).

### Western Blot Analysis

After treated with compounds, cells or tumor tissues were harvested and lysed in protein lysate buffer followed by centrifugation (12,000 rpm, 10 min, 4°C), supernatants were collected. The protein concentrations were measured using the Quick Start™ Bradford Protein Assay Kit (#5000201, Bio-Rad, Hercules, CA). Equivalent amount of protein samples were separated by 12% SDS-polyacrylamide gel (SDS-PAGE) and then transferred to PVDF membrane. The blotting was blocked with 5% nonfat milk at room temperature for 2 h and then incubated with primary antibody at 4°C for overnight. At last, anti-rabbit HRP-conjugated secondary antibody was added and incubated with membrane for 1 h. Between every two steps, the membrane will be washed with TBST for three times. The immune-reactive bands were detected via Clarity Max Western ECL Substrate Kit (#1705062, Bio-Rad, Hercules, CA). The density of the immune-reactive bands was analyzed by Image J (National Institute of Health, MD).

### Cell Apoptosis Analysis

H1975 cells were seeded on 60-mm dishes for 12 h, and then treated with DMSO (vehicle), 15c (0.5, 2.5, 5, or 10 mM), WZ4002 (10mM), AZD4547 (10mM), or WZ4002 (10 mM) combined with AZD4547 (10 mM) for 24 h. After harvested and washed with PBS, cells were double stained with FITC Annexin V Apoptosis Detection Kit I (#556547, BD Biosciences, CA). The apoptosis were evaluated by using a FACSCalibur flow cytometer (BD Biosciences, CA).

### Analysis of Caspase-3 Activity

The activity of Caspase-3 in H1975 cell lysates was detected by using a Caspase-3 Activity Kit (C1115, Beyotime Institute of Biotechnology, Nantong, China). The caspase-3 activity was normalized and calculated as percentage of control group.

### Establishment of Afatinib-Resistant PC9 Cells

PC9 cells cultured in RPMI 1640 with 10% FBS were continuously exposed to Afatinib at the stepwise increased concentrations up to 1 µM over the following 3 months. After that, cells remained were cultured in Afatinib-free growth medium until the stable growth was restored. The isolated Afatinib-resistant cell line was named as AFA-PC9.

### In Vivo Antitumor Study

Protocols for animal studies were approved by the Wenzhou Medical University Animal Policy and Welfare Committee. Male BALB/c nu/nu mice (6 weeks old, 18–22 g) were acquired from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were housed at a constant room temperature with a 12 h: 12 h light/dark cycle and fed with a standard rodent diet and water. H1975 cells were injected subcutaneously into the right flank (1×10<sup>7</sup> cells in 200 ml of PBS). 40 mice were divided into five groups (n = 8/group): control (vehicle), 15c (10 mg/kg/ day), WZ4002 (10 mg/kg/day), AZD4547 (10 mg/kg/day), and WZ4002 (10 mg/kg/day) combined with AZD4547 (10 mg/kg/ day). The compounds were given by intraperitoneal (i.p.) injection. The tumor volumes were determined at the indicated time points (V = 0.5 × L × W<sup>2</sup> ; L: length, W: width, V: volume). After sacrificed, the tumors were weighed and collected for studies on the protein expression.

### Statistical Analysis

At least three independent experiments were performed for each assay (n≥3). Data were shown as mean ± SEM. GraphPad Prism 5.0 (GraphPad, San Diego, CA) was used for the statistical analyses. Student's t-test and two-way ANOVA were employed to analyze the differences between different sets of data. P < 0.05 was considered statistically significant and exhibited by \* or # characters.

### RESULTS

### Compound 15c Acted as an EGFRL858R/ T790M/FGFR1 Dual Inhibitor and Selectively Kills Human Lung Cancer Cells

In our previous work (Chen et al., 2017a), we designed and optimized a series analogue of WZ4002 and evaluated their inhibition activity against EGFRL858R/T790M, and found that several derivatives exhibited a high potency and selectivity between EGFRL858R/T790M and EGFRWT. In order to explore whether those compounds can inhibit the FGFR1 kinase activity, all the compounds were measured the kinase inhibitory effect against EGFRWT, EGFRL858R/T790M, and FGFR1WT in a cell-free system called Caliper Mobility Shift Assay. As shown in Figures 1A, B, 15c exhibited the high inhibition towards EGFRL858R/T790M and FGFR1WT, and relatively lower inhibition against EGFRWT. Thus, 15c can act as an EGFRL858R/T790M and FGFR1WT dual inhibitor.

Kinases such as EGFR and FGFR play a critical role in the proliferation of cancer cells. Thus, we next detected the antiproliferation effects of 15c on cultured human lung cancer cells and normal cells. Four cell lines, lung cancer PC9 cells (EGFRL858R+ and FGFR1<sup>−</sup> ), lung cancer H520 cells (EGFR<sup>−</sup>

FIGURE 1 | Compound 15c acted as an EGFRL858R/T790M/FGFR1 dual inhibitor and selectively kills human lung cancer cells. (A) The chemical structure of WZ4002 and 15c. (B) The kinase inhibitory activity against EGFRWT, EGFRL858R/T790M, and FGFR1WT of 15c. (C) Immunoblots of the expression levels of EGFR and FGFR1 protein in H1975, PC9, H520, and Beas-2B cell lines. (D) Anti-proliferation effect of 15c against H1975, PC9, H520, and Beas-2B cell lines. The IC50 values were reported as mM values (n = 3 independent experiments).

and FGFR1+ ), lung cancer H1975 cells (EGFRL858R/T790M+ and FGFR1<sup>+</sup> ), and normal lung epithelial cells Beas-2B cells (EGFR<sup>+</sup> and FGFR1+ ), were chosen for the cytotoxic assay (Figure 1C). 15c exhibited high anti-proliferation activity against lung cancer cells rather than normal Beas-2B cells (highest concentration at <sup>30</sup> <sup>m</sup>M) (Figure 1D). Those findings indicated that 15c acted as an EGFRL858R/T790M/FGFR1 dual inhibitor in cell-free system and selectively inhibited the proliferation of human lung cancer cells.

### Compound 15c Inhibited the Phosphorylation of EGFR and FGFR1 in the Cell System

In order to further confirm whether compound 15c can impede the phosphorylation of EGFR and FGFR1 in cells, the kinase inhibitory activity of 15c was detected in various ligand stimulated cancer cells. As mentioned before, PC9 is a human lung adenocarcinoma cell line with EGFRL858R mutation without FGFR1 expression, while H520 is a human lung squamous cell carcinoma cell line with the high expression of FGFR1 but without EGFR expression. Thus, the p-EGFR levels in EGFinduced PC9 cells and the p-FGFR1 levels in FGF-stimulated H520 cells were determined, respectively. The third-generation EGFR-TKI WZ4002 and the FGFR1-TKI AZD4547 were selected for comparison. The results were summarized in Figures 2A, B. Compound 15c, similar to the positive control, can inhibit the phosphorylation of FGFR1 and EGFR in a dosedependent manner in different cell lines.

In addition, H1975 is a human adenocarcinoma cell line with EGFRL858R/T790M mutation and FGFR1 expression, which can be used as an EGFR-TKI acquired resistant cell line. In this part, we treated the H1975 cells with 1 mM 15c, WZ4002, AZD4547, or WZ4002 and AZD4547 combination followed by bFGF or EGF stimulated. The p-FGFR1 and p-EGFR levels were determined by Western blot. In the EGF-stimulated H1975 cells (Figure 2C), 15c and WZ4002 exhibited the similar inhibitory activity against the phosphorylation of EGFR and AZD4547, a FGFR1 inhibitor, showed no effect on the phosphorylation of EGFR. The inhibitory effect of WZ4002 and AZD4547 combination was slightly better than 15c or WZ4002 alone, but no significant difference was noted. Similarly, in the bFGF-induced H1975 cells (Figure 2D), 15c and AZD4547 exhibited the comparable inhibitory activity against the phosphorylation of FGFR1 and WZ4002 showed no effect. The inhibitory impact of WZ4002 and AZD4547 combination was almost the same to 15c or WZ4002 alone. Those results revealed that 15c was a potential EGFRL858R/T790M/FGFR1 dual inhibitor in the cell system.

### Compound 15c Induced Caspase-Dependent Apoptosis in H1975 Cells

To explore whether 15c can accelerate H1975 cells apoptosis, a series apoptosis related experiments were introduced. H1975 cells that starved for 12 h were treated with variable level of test compounds, positive inhibitors, or vehicle for 24 h. After harvested and washed with PBS, cells were double stained with FITC Annexin V Apoptosis Detection Kit I. The apoptotic cells were detected by flow cytometry analysis and the results were also illustrated in the column chart (Figures 3A, B). Significant apoptosis was observed in 15c treated H1975 cells in a dosedependent manner compared with the vehicle group. Moreover, the apoptosis rates of 15c at 5mM and 10mMweremuch higher than that WZ4002 or AZD4547 treated alone or combined at 10 mM. In addition, caspase 3 activity assay were further applied to verify the effect of 15c on inducing H1975 apoptosis (Figure 3C). The results

level in EGF-stimulated H1975 cells. (D) Analysis of the p-FGFR1 level in bFGF-stimulated H1975 cells. The statistical results were shown as means ± SD (n = 3) on the right of each panels. Significant difference was indicated as \* or # character (\*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001 versus vehicle group, # p < 0.05, ##p < 0.01, and ###p < 0.001 versus control group).

suggested that 15c could induced the apoptosis of H1975 cells via activating caspase-dependent apoptosis.

### Compound 15c Overcame Afatinib-Tolerant in Established Acquired Resistant PC9 Cell Line

The effect of 15c on reversing drug resistant in lung cancer cells was further studied by domesticated a cell line resistant to Afatinib. The PC9 cells were continuously exposed to Afatinib at the stepwise increased concentrations from 0.025 to 1 µM over the following 3 months to construct the Afatinib-resistant cell lines AFA-PC9. Western blot analyses of PC9 and AFA-PC9 were shown in Figure 4A, and the FGFR1 expression was significantly amplified in AFA-PC9 and the EGFR expression was not affected. The morphology change of the AFA-PC9 was demonstrated in Figure 4B. Compared to PC9, AFA-PC9 cells started to grow in clusters, which was completely consistent with the conclusions in previous studies. MTS analysis was also carried out on the drug-resistant AFA-PC9 (Figure 4C). Compared with the PC9 cells, the sensitivity of AFA-PC9 cells to Afatinib was reduced by more than 1000 times. Then, we compared the anti-proliferative effects of compound 15c and WZ4002 on AFA-PC9 and PC9 cells. It turned out that the sensitivity of AFA-PC9 to WZ4002 decreased more than 100

times (IC50 was changed from 0.025 to 3.2 mM) while only decreased two times against 15c (IC50 was changed from 0.32 to 0.66 mM), indicating that 15c can overcame the acquired resistant to Afatinib in PC9 cell line.

### Compound 15c Attenuated H1975 Xenograft Tumor Growth In Vivo by Suppressing p-EGFR and p-FGFR1 Levels

Subcutaneous xenograft model of H1975 cells in immunodeficient mice was applied to evaluate the in vivo antitumor activity of 15c. Male BALB/c nu/nu mice (6 weeks old, 18– 22 g) were subcutaneously injected with H1975 cells (1×10<sup>7</sup> cells in 200 ml of PBS). When the tumor volume was around 80–100 mm3 , the mice were randomly divided into five groups (eight in each) including negative control group (Vehicle group), 15c group (10 mg/kg/day, ip), WZ4002 group (10 mg/kg/day, ip), AZD4547 group (10 mg/kg/day, ip), and WZ4002 combined with AZD4547 group (10 mg/kg/day, ip). After intraperitoneal administration of compound for 20 days, the tumor volume and weight of each group were measured and shown in Figures 5A–C. 15c significantly reduced H1975 tumor volume and weight versus vehicle control. Even compared with the positive inhibitor alone, 15c still exhibited a better tumor-inhibition effect. Only WZ4002 and AZD4547 combination administration can restrain the H1975 tumor to the same size of the tumor treated by 15c. Importantly, these is no significant weight loss of mice even treated 15c for 20 days (Figure 5D). Mechanistically, western blot analysis of tumor tissues revealed that 15c attenuated H1975 xenograft tumor growth in vivo by suppressing the phosphorylation of FGFR1 and EGFR (Figures 5E, F).

### DISCUSSION

Lung cancer is the leading cause of cancer-related death worldwide with NSCLC accounting for 85% of cases. Therefore, it is of great theoretical and practical significance to study the pathogenesis of lung cancer and develop the corresponding therapeutic drugs. In the past few decades, it has been determined that the activation of EGFR is closely

Afatinib at stepwise increased concentrations up to 1 µM over the following 3 months to establish an Afatinib-resistant PC9 cell line named ADA-PC9. (A) Western Blot analysis of the EGFR and FGFR1 expression in PC9 cells and AFA-PC9 cells. (B) Morphological changes of AFA-PC9 cells compared to PC9 cells; (C) Antiproliferation effect of 15c, WZ4002, and Afatinib against PC9 and AFA-PC9 cell lines. The IC50 values were reported as mM values.

related to the genesis and progress of lung cancer (Lynch et al., 2004; Paez et al., 2004; Pao et al., 2004; Sharma et al., 2007). Thus, EGFR has attracted great attentions as it is an ideal target for the cancer therapy. There are a series of small molecular inhibitors that can inhibit the kinase activity of EGFR had been found and named as EGFR-TKIs (Sequist et al., 2007; Pao and Chmielecki, 2010). Gefitinib and Erotinib are the firstgeneration EGFR-TKIs that have been studied clinically with a great advance. However, patients generally develop secondary mutations, such as T790M gatekeeper, and lead to drug resistance after EGFR-TKIs treated for 12 months (Chen et al., 2017b). As a result, second and third-generation EGFR-TKIs, e.g. Afatinib, WZ4002, and Osimertinib, are developed to overcome the T790M mutation-associated resistance (Zhou et al., 2009; Mok et al., 2017). The poor therapeutic window of Afatinib limited its clinical application (Chen et al., 2017b). Osimertinib exhibits an excellent treatment effect for NSCLC patients with EGFR T790M-mutated, but acquired resistance is still inevitable (Oxnard et al., 2018). Thus, there is still an urgent demand to establish an effective antitumor strategy for those NSCLC patients with acquired resistance to second or third-generation EGFR-TKIs.

Müller-Tidow and his colleagues identified the expression of 56 RTKs in primary NSCLC tumors and found that 33 RTKs are expressed at the mRNA level in 25% samples (Müller-Tidow et al., 2005), indicating that those RTKs may function as an alternatives to EGFR signal pathway in NSCLC, such as cMet, Axl, IGF-1R, and PDGFR. The amplification of cMet is a major factor of acquired resistance in lung cancer and the combination of Gefitinib and cMet antibody can significantly enhance the growth inhibition in cMet over-expressing cell lines (Bean et al., 2007; Zucali et al., 2008). Similarly, the IGF-1R survival pathway contribute to Gefitinib resistance and synergistically inhibition of IGF-1R could strengthen the antitumor effect of Gefinitib (Desbois-Mouthon et al., 2006). In addition, Axl lead to EGFR-TKIs resistant by increasing expression and forming a hetero-dimerization with EGFR and inhibition of Axl could prevent or overcome acquired resistance to EGFR-TKIs in EGFR-mutated lung cancer (Zhang et al., 2012; Vouri et al., 2016). Recently, several researches mentioned that the activation of the autocrine ring of FGF/FGFR, one of the typical RTKs, could act as a compensatory mechanism to promote the survival and growth of EGFR-TKIs resistantlung cancer cells. These findings declared that simultaneous inhibition of EGFR and FGFR1 activity may be a novel and effective strategy to overcome FGFR1 related EGFR-TKIs resistant in NSCLC.

Nowadays, combination therapy of various drugs with different targets has become an important strategy for cancer treatment (Bell, 2013). Combined drugs can improve the curative effect as the synergism of different activity. However, increased off-target effect and metabolic pressure restricted the application of combined administration. Moreover, drugs with polypharmacological activities are shown to be advantageous over combination therapy as their lower incidences of side effects and more resilient therapies. Thus, developing dual-inhibitors that target FGFR1 and EGFR will be an efficient way to reverse FGFR1-related EGFR-TKIs resistant in NSCLC.

Here, we presented an EGFRL858R/T790M/FGFR1 dualinhibitor 15c, which can simultaneous inhibited the kinase activity of EGFRL858R/T790M and FGFR1 both in kinase assay and cell study, for the treatment of FGFR1-amplified EGFR-TKIs resistant lung cancers. The anti-proliferation effects of 15c on NSCLC cell lines and normal BEAS-2B cell line showed that 15c could effectively hold back the proliferation of NSCLC cells rather

15c group. ip, intraperitoneally injected.

than normal cells. Particularly, 15c exhibited a better inhibitory effect to H1975 cells and AFA-PC9 cells, which contained EGFR mutation and FGFR1 overexpression, than WZ4002 and AZD4547. Further flow cytometry and Caspase 3 activity analysis demonstrated that 15c could induce the apoptosis in a dose-dependent manner and the effect was better than WZ4002 combined AZD4547 treatment. Finally, the in vivo study as well revealed that the anti-tumor ability of 15c was comparable to the combination administration of WZ4002 and AZD4547 but better than the separate administration.

In summary, acquired resistant is the major reason for EGFR-TKI tolerant NSCLCs and FGFR1 amplification has been proved that contribute to the EGFR-TKI resistant. Thus, EGFRL858R/ T790M/FGFR1 dual inhibitor 15c can be developed as an ideal candidate drug for FGFR1-amplified EGFR-TKIs resistant lung cancers.

### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

### ETHICS STATEMENT

The animal study was reviewed and approved by the Wenzhou Medical University Animal Policy and Welfare Committee.

### REFERENCES


### AUTHOR CONTRIBUTIONS

Participated in research design: GC, QW, GL. Cell and animal study: GC, YB, QW, YZ, and XL. Kinase study: LF. Performed chemical synthesis of 15c: LC and ZL. Contributed to the writing of the manuscript: GC, XZ, GL.

### FUNDING

This work was supported by the National Key R&D Program of China (2017YFA0506000), the National Natural Science Foundation of China (Grants No. 81703352 and 81773579), the Zhejiang Province Natural Science Foundation (LY17B020008), the Zhejiang Medical Health Technology Platform Project (2018KY183) and the Zhejiang Natural Science Foundation Pharmaceutical Society Joint Fund Project (LYY19H310004).


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Chen, Bao, Weng, Zhao, Lu, Fu, Chen, Liu, Zhang and Liang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Fibroblast Growth Factor 10 Attenuates Renal Damage by Regulating Endoplasmic Reticulum Stress After Ischemia–Reperfusion Injury

### Edited by:

Zhouguang Wang, Albert Einstein College of Medicine, United States

#### Reviewed by:

Yan Cui, UCLA Brain Research Institute, United States Yue Zhang, University of Southern California, United States

\*Correspondence:

Jian Xiao xfxj2000@126.com Jin-San Zhang Zhang\_JinSan@wmu.edu.cn

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 17 October 2019 Accepted: 14 January 2020 Published: 07 February 2020

#### Citation:

Tan X, Yu L, Yang R, Tao Q, Xiang L, Xiao J and Zhang J-S (2020) Fibroblast Growth Factor 10 Attenuates Renal Damage by Regulating Endoplasmic Reticulum Stress After Ischemia–Reperfusion Injury. Front. Pharmacol. 11:39. doi: 10.3389/fphar.2020.00039 Xiaohua Tan1,2, Lixia Yu3 , Ruo Yang<sup>2</sup> , Qianyu Tao<sup>2</sup> , Lijun Xiang<sup>2</sup> , Jian Xiao2\* and Jin-San Zhang2,4\*

<sup>1</sup> Department of Pathology, School of Basic Medicine, Qingdao University, Qingdao, China, <sup>2</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>3</sup> Department of Pharmacy, Xixi Hospital of Hangzhou, Hangzhou, China, <sup>4</sup> Institute of Life Sciences, Wenzhou University, Wenzhou, China

Renal ischemia–reperfusion (I/R) injury is a predominant cause of acute kidney injury (AKI), the pathologic mechanism of which is highly complex involving reactive oxygen species (ROS) accumulation, inflammatory response, autophagy, apoptosis as well as endoplasmic reticulum (ER) stress. Fibroblast growth factor 10 (FGF10), as a multifunctional growth factor, plays crucial roles in embryonic development, adult homeostasis, and regenerative medicine. Herein, we investigated the molecular pathways underlying the protective effect of FGF10 on renal I/R injury using Sprague– Dawley rats. Results showed that administration of FGF10 not only effectively inhibited I/Rinduced activation of Caspase-3 and expression of Bax, but also alleviated I/R evoked expression of ER stress-related proteins in the kidney including CHOP, GRP78, XBP-1, and ATF-4 and ATF-6. The protective effect of FGF10 against apoptosis and ER stress was recapitulated by in vitro experiments using oxidative damaged NRK-52E cells induced by tert-Butyl hydroperoxide (TBHP). Significantly, U0126, a selective noncompetitive inhibitor of MAP kinase kinases (MKK), largely abolished the protective role of FGF10. Taken together, both in vivo and in vitro experiments indicated that FGF10 attenuates I/R-induced renal epithelial apoptosis by suppressing excessive ER stress, which is, at least partially, mediated by the activation of the MEK–ERK1/2 signaling pathway. Therefore, our present study revealed the therapeutic potential of FGF10 on renal I/R injury.

Keywords: fibroblast growth factor 10, acute kidney injury, endoplasmic reticulum stress, ischemia–reperfusion, ERK1/2

### INTRODUCTION

Acute kidney injury (AKI), characterized by rapidly declining glomerular filtration rate (GFR), is a clinical lethal symptom mainly caused by renal ischemia–reperfusion (I/R) injury, sepsis, and nephrotoxic drugs (Mehta et al., 2007; Bonventre and Yang, 2011). AKI is considered as a nosocomial disease with an incidence of 2–7% in hospitalized patients. Despite advances of therapeutic strategies in the past decades, the morbidity and mortality of AKI remain very high (Chertow et al., 2005; Uchino et al., 2005; Bonventre and Yang, 2011; Basile et al., 2012). Renal I/R injury, commonly caused by shock, surgery interventions, kidney transplantation, and toxic insults, accounts for the majority of AKI (Chertow et al., 2005; Ishani et al., 2009). Many previous studies have shown that the pathological mechanism of renal I/R injury is often associated with excessive reactive oxygen species (ROS), oxidative stress, autophagy, inflammation, apoptosis as well as ER stress (Paller et al., 1984; Thadhani et al., 1996; Inagi, 2009; Hotamisligil, 2010; Tan et al., 2017). Although several drugs and therapeutic treatments that could ameliorate renal ischemia injury in animal models have been reported, few of them have been successfully utilized in clinical therapies (Tsuda et al., 2012; Tan et al., 2013). Rapid restoration of renal blood flow after ischemia remains the quickest way to lessen renal tissue damage and functional deterioration caused by ischemia (Paller et al., 1984). However, reperfusion itself also has the potential to elicit additional damage, mainly caused by over-production of ROS, mitochondrial dysfunction, and inflammatory response, which further leads to apoptosis or necrosis (Tsuda et al., 2012; Tan et al., 2013; Inoue et al., 2019). Therefore, effective treatment for AKI is desperately needed.

Endoplasmic reticulum (ER) is a specialized organelle for the synthesis, folding, and trafficking of proteins (Cao and Kaufman, 2012). Many studies have shown that ER is highly sensitive to the changes of the intracellular microenvironment (Cao and Kaufman, 2014; Walter and Ron, 2011). Hypoxia and oxidative stress intrinsic to I/R injury could impair the protein folding of ER. Overaccumulation of unfolded and misfolded proteins triggers the Unfolded Protein Response (UPR) to resolve the excessive ER stress. It has been demonstrated that UPR could expand the ER membrane and thus improve the efficiency of ER for protein folding. UPR could also decrease mRNA translation and reduce protein expression (Schuck et al., 2009). It has been reported that ER stress plays an important role in cell growth, differentiation, and apoptosis. However, excessive activation of ER stress and UPR could activate apoptotic signaling pathways (Hetz et al,. 2012; Tabas and Ron, 2011). Studies have revealed that C/EBP homologous proteins (CHOP), also known as DNA damage inducible transcript 3 (DDIT3), is a master regulator of maladaptive ER stress-induced apoptosis (Rutkowski et al., 2006). Therefore, a strategy focusing on the inhibition of maladaptive ER stress may facilitate the treatment of renal I/ R injury.

Fibroblast growth factor 10 (FGF10) is an important member of the FGF family, which mediates mesenchymal to epithelial signaling in a paracrine manner. FGF10 plays a crucial role in embryonic development, wound healing, and tissue regeneration with binding and activating FGF receptor (FGFR) on the cell surface (Itoh and Ohta, 2014). FGF10 highly specifically binds to FGFR2b and initiates the activation of intracellular signaling cascades, including the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway (Zhang et al., 2006; Cho et al., 2009; Wang et al., 2009; Itoh, 2015). Many experimental studies using Fgf10 gene knockout mice have confirmed the crucial role of FGF10 in the development and homeostasis of multiple organs such as the kidney, lung, limb, and pancreatic gland (De Moerlooze et al., 2000; Ohuchi et al., 2000; Beenken and Mohammadi, 2009; Michos et al., 2010; El Agha et al., 2012; Itoh, 2015). It has been reported that FGF10 could accelerate the regeneration of myocardium after myocardial I/R injury (Rochais et al., 2014). Recombinant FGF10 has also been utilized for the treatment of ulcerative colitis and mucositis (Sandborn et al., 2003; Freytes et al., 2004). However, the protective mechanism of FGF10 on renal I/R injury has not yet been fully confirmed. In the present study, we hypothesized that FGF10 could attenuate renal I/R injury by suppressing excessive ER stress and inhibiting renal tubular epithelial cell apoptosis. The protective effect of FGF10 on AKI may be related to the activation of MEK–ERK1/2 signaling pathway. We verified our hypothesis with Sprague–Dawley (SD) rats subjected to renal I/R injury. Rat renal tubular epithelial cell line NRK-52E was also utilized to clarify the protective mechanism of FGF10 in the present study. Results demonstrated that the protective effect of FGF10 on AKI is intimately connected to ER stress which is, at least partially, mediated by the MEK–ERK1/2 signaling pathway.

### MATERIALS AND METHODS

### Reagents and Antibodies

Bovine serum albumin (BSA), recombinant human FGF10, Tert-Butyl hydroperoxide (TBHP), and U0126 (selective MKK1/2 inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Trypsin-EDTA (0.25%), and 4′, 6-Diamidino-2 phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against cleaved Caspase-3 (catalog number: 9661), cleaved Caspase-9 (catalog number: 9507), and phospho-ERK1/2 (catalog number: 9101) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-ERK1/2 antibody (catalog number: 82380) was purchased from Thermo Fisher Scientific (Sunnyvale, CA, USA). Antibodies against GRP78 (catalog number: ab21685), ATF-6 (catalog number: ab203119), ATF-4 (catalog number: ab23760), PDI (catalog number: ab154820), CHOP (catalog number: ab11419), XBP1 (catalog number: ab37152), and GAPDH (catalog number: ab9485) were purchased from Abcam, Inc. (Cambridge, MA, USA). The secondary antibodies were purchased from Abcam, Inc. (Cambridge, MA, USA) or Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Annexin V-FITC-PI Apoptosis Detection Kit was purchased from Becton Dickinson, Inc. (San Jose, CA, USA). High sensitivity ECL substrate kit, Hematoxylin and Eosin (H&E) staining kit, and Periodic Acid Schiff (PAS) staining kit were purchased from Abcam, Inc. (Cambridge, MA, USA). The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) Assay Kit was purchased form Abcam, Inc. (Cambridge, MA, USA).

### Renal I/R Injury Model and Assessment of Renal Function

To confirm the protective effect of FGF10 treatment on kidney after reperfusion, rat renal I/R injury model was established by surgical operation. Male Sprague–Dawley (SD) rats, eight weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed in a Specific-pathogen-free (SPF) facility. The experimental protocol was approved by the Institutional Animal Ethical and Use Committee of Wenzhou Medical University. The rat model of renal I/R injury was established as we described in our previous study (Tan et al., 2017). Briefly, SD rats were anesthetized with intraperitoneal (ip) injection of pentobarbital sodium (25 mg/kg) and placed on a thermostatic surgical table. A small incision was made through the medioventral line and exposed the right kidney. The right kidney was carefully liberated from the surrounding tissue, and nephrectomy was performed. The left kidney was exposed, and the renal artery was clamped using a nontraumatic vascular clamp. Renal blood flow was re-established after 45 min ischemia, and the muscle layer and skin layer were closed using a medical suture. For measurement of renal function, serum creatinine (Cr) was measured 1 day following renal ischemia by the hospital medicine biochemical laboratory (at The First Affiliated Hospital of Wenzhou Medical University). Kidneys were harvested and stored in cryogenic refrigerator for further experiments. Rats were randomly divided into three groups: (a) Sham group: Sham-operated rats with unconstricted renal artery; (b) I/R group: rats were subjected to 45 min of ischemia via renal artery followed by reperfusion; (c) I/R–FGF10 group: rats were treated with 0.5 mg/kg FGF10 (ip) 1 h before ischemia. FGF10 was dissolved in sterile saline.

### Cell Culture

The results of in vivo experiments in the present study have demonstrated that FGF10 could increase the phosphorylation of ERK1/2 in kidney tissues after reperfusion. To further clarify the role of MEK–ERK1/2 signaling pathway in the protective effect of FGF10, NRK-52E, a rat renal tubular epithelial cell line, was utilized in our present study. We verified the protective effect of FGF10 on damaged NRK-52E induced by TBHP. Furthermore, the participation role of MEK–ERK1/2 signaling pathway in the protective effect of FGF10 was clarified in the in vitro experiment. NRK-52E was purchased from the American Type Culture Collection (Manassas, USA) and cultured in DMEM supplemented with 10% FBS, antibiotics (100 units/ml penicillin, 100 mg/ml streptomycin) and incubated under 37°C, 95% air, and 5%CO2. To detect the effect of FGF10 on ER stress induced by TBHP, NRK-52E was cultured on 6-well plates with 2×105 cells per well and randomly divided into four groups: (a) Control group: NRK-52E was cultured in complete medium without any supplement; (b) TBHP group: NRK52E was cultured in complete medium, and then TBHP (200 mmol/L) was added for an additional 12 h; (c) TBHP + FGF10 group: NRK-52Ewas pretreated with recombinant FGF10 (100 ng/ml) for 2 h, and then TBHP (200 mmol/L) was added for an additional 12 h; (d) TBHP + FGF10 + U0126: NRK-52E was pretreated for 2 h with U0126 (20 mmol/L), and then cells were treated the same as TBHP + FGF10 group. The pretreatment compounds in the culture medium were not removed before successive treatment conditions. All experiments with NRK-52E were performed in triplicates.

### Western Blot Analysis

To assess the regulatory role of FGF10 on ER stress and apoptosis, the expression of relevant proteins was analyzed by western blot. For protein analysis of in vivo samples, total kidney tissues (contain both of cortex and medulla, but don't contain the renal fibrous capsule) were homogenized and total proteins were extracted using tissue lysis buffer. For protein analysis of in vitro samples, NRK-52E cultured in a petri dish was rinsed with PBS buffer three times; total proteins were extracted using cell lysis buffer. An equivalent of 100 mg protein of the in vivo sample (30 mg protein of the in vitro sample) was separated by Sodium Dodecyl Sulfate PolyAcrylamide and then transferred to a polyvinylidene fluoride (PVDF) membrane for immunoblot analysis. Primary antibodies against cleaved Caspase-3 (1:1,000), cleaved Caspase-9 (1:1,000), Bax (1:3,000), Bcl-2 (1:1,000), GRP78 (1:1,000), CHOP (1:5,000), XBP-1 (1:1,000), ATF-4 (1:1,000), ATF-6 (1:2,000), PDI (1:2,000), ERK1/2 (1:1,000), and phosphor-ERK1/2 (1:1,000) were used in the present study. GAPDH (1:2,500) was used as loading control. The signals were visualized with the ChemiDic™ XRS + Imaging System (Bio-Rad Laboratories). The band densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).

### Fluorescence Activated Cell Sorting Analysis

To assess the protective effect of FGF10 on NRK-52E against apoptosis induced by TBHP, apoptosis of NRK-52E in each group was quantified with Annexin V-FITC-PI Apoptosis Detection Kit following the manufacturing process instructions. Briefly, NRK-52E was cultured and randomly divided into four groups as described above. Cells were collected and washed twice with PBS and resuspended in binding buffer before the addition of Annexin V-FITC-PI. Cells were then gently vortex mixed and incubated for 15 min in the dark at room temperature before analysis using a BD FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, San Carlos, CA, USA).

### Immunohistochemistry and Immunofluorescence Staining

To observe the expression and location of ER stress and apoptosis relevant proteins in kidney tissues, immunohistochemistry and immunofluorescence staining were performed. The renal morphology was detected as we described in a previous study (Tan et al., 2017). Briefly, kidneys (both of cortex and medulla)

were excised and harvested 1 day after I/R injury. After being dehydrated in gradient ethanol, renal tissue was embedded in paraffin and cut into 5 mm sections. For immunohistochemistry staining, slides were incubated with antibodies against cleaved Capase-3 (1:300), CHOP (1:300), GRP-78 (1:500), and ATF6 (1:300) separately, and then incubated at 4°C overnight. After being incubated with primary antibodies and washed with PBS solution for three times, the slides were incubated with secondary antibodies for 1 h at room temperature, washed with PBS solution for three times, stained with Diaminobenzidine (DAB), and counterstained with hematoxylin. The slides were then subjected to gradient ethanol dehydration, dimethyl benzene transparent, and mounted with neutral resin cover slides. Images were captured using a Nikon ECLPSE 80i. For immunofluorescence staining, slides were incubated with primary antibodies against CHOP (1:300) and GRP-78 (1:500) incubated at 4°C overnight. After reacting with the primary antibodies, the slides were washed 3 times with PBS and then incubated with secondary antibodies (AlexaFluor 488, Abcam) for 1 h at room temperature. Images were captured using a laser confocal microscope (Nikon, Ti-E&A1 plus).

### Renal Histopathology Damage Assessment

To evaluate the renal histopathology damages, slides were stained with hematoxylin and eosin (H&E) and Periodic acid Schiff (PAS), respectively. Each image of the sections was examined under light microscope (Nikon ECLPSE 80i, Japan). Renal histopathology damage degree was evaluated based on intraluminal necrotic cells, cell swelling, interstitial congestion, edema, and protein casts. The following 5 point scoring system was utilized to assess renal damage: 0 point (normal renal morphology), 1 point (damage of kidney tissue ≤10%), 2 points (damage of kidney tissue 11–25%), 3 points (damage of kidney tissue 26–45%), 4 points (damage of kidney tissue 46– 75%), 5 points (damage of kidney tissue ≥76%). The pathologists who assessed the images were blinded to the allocation group.

### TUNEL Assay

The Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is a method for detecting DNA fragmentation. TUNEL has been widely used to identify and quantify cell death in tissues within the last two decades. To assess the protective effect of FGF10 against apoptosis, TUNEL staining and immunohistochemistry staining of cleaved Caspase-3 were used to detect the apoptosis in the kidney tissue after reperfusion. The experimental protocol was exactly the same as we described in our previous study. Kidneys were excised and harvested 1 day after I/R injury. After being dehydrated in gradient ethanol, renal tissue was embedded in paraffin and cut into 5 mm sections. For TUNEL assay, slides were handled with the TUNEL Apoptosis Assay KIT following manufacturing process instructions. The images were captured under a Laser confocal microscope (Nikon, Ti-E&A1 plus). The apoptosis index was analyzed based on five randomly selected images from each group.

### Statistical Analysis

SPSS 19.0 statistical software (Cary, NC, USA) was used for the analysis of rat survival rate after reperfusion. The statistical evaluation of the data was performed using one-way Analysis of Variance (ANOVA) when two groups were compared in this study. Data are expressed as the mean ± SEM of n independent experiments (n ≥ 5). \*P < 0.05, \*\*P < 0.01, and \*\*\*P < 0.001 were considered statistically significant.

### RESULTS

### FGF10 Protects Renal Function and Histological Integrity

We utilized a rat model of I/R injury to investigate the protective effect of FGF10 on AKI as depicted in Figure 1A. To evaluate the protective effect of FGF10, survival rate was analyzed for 30 days after reperfusion. As shown in Figure 1B, the 30-days survival rate was significantly improved in the I/R–FGF10 group (91.7%) compared with the I/R group (66.7%). Serum creatinine (Cr) levels were measured at 24 h post reperfusion to assess renal function. As expected, the level of serum Cr was increased nearly five folds in I/R rats (n = 5) compared to the Sham group (Figure 1C). However, the level of serum Cr in the I/R–FGF10 group was significantly decreased compared to the I/R rats (P < 0.05). Renal morphological changes were assessed by H&E staining; no obvious damage in the kidney of the Sham group was detected (Figure 1D-a), whereas the kidney of the I/R group showed typical pathological features of AKI. The arrows represent intraluminal necrotic cells, swelling of renal tubular cells, interstitial congestion, and edema. The asterisks represent protein casts in delated tubulars (Figure 1D-b). Administration of FGF10 significantly attenuated the extent of renal damages (Figure 1D-c) and largely preserved the integrity of renal morphology. Tubular injury score was analyzed based on H&E staining. As shown in Figure 1E, FGF10 treatment strikingly ameliorated the damage of the kidney tissue after I/R injury. There is no significant difference of the tubular injury between the I/R–FGF10 group and Sham group. PAS staining for glycogen deposition (purple plaques) further indicated that the integrity of brush border on the surface of the renal proximal epithelial cell was damaged in I/R rats. As shown in Figure 1F-b, the arrows point to the detachment of brush border from epithelial cell, and the asterisks represent tubular lumen narrowing caused by swelling of epithelial cells. The integrity of the brush border in the I/R–FGF10 group was significantly improved compared with the I/R group (Figure 1F-c). In addition, I/R rats displayed significantly increased glycogen accumulation in the glomerulus compared with that of the Sham rats, whereas FGF10 preadministration effectively reduced the deposition of glycogen in glomerulus after reperfusion (Figure 1F).

### FGF10 Prevents I/R-Induced Apoptosis of Renal Tubular Epithelial Cells

TUNEL staining was used to assess the apoptosis of renal tubular cells caused by I/R injury. As shown in Figure 2A, few TUNELpositive cells were observed in the kidney of the Sham group,

FIGURE 1 | FGF10 attenuates renal I/R injury. (A) Flow chart for animal procedures. (B) Survival rate. The survival rate of I/R–FGF10 group was significantly improved compared with the I/R group (n = 12). \*P < 0.05. (C) Serum creatinine levels of animals from Sham group, I/R group and I/R-FGF10 group. \*P < 0.05, \*\*P < 0.01. n = 5. (D) Histological evaluations of renal tissue stained with H&E staining. Panels are representative of five animals in each group. The arrows point to renal tubular swelling, interstitial congestion and glomerular basement membrane thickening. Asterisks represent protein casts in delated tubulars. Scale bars represent 50 mm. (E) Tubular injury score was analyzed based on H&E staining. \*\*\*P < 0.001. Results are representative of five rats in each group. (F) Brush border of renal proximal epithelial cell was evaluated with PAS staining (purple red). The arrows represent the abscission of brush border in proximal tubulars. Asterisks represent tubular lumen narrowing caused by swelling of epithelial cells. Panels are representative of five rats in each group. Scale bars represent 50 mm.

whereas the number of TUNEL-positive cells in the kidney of the I/R–FGF10 was dramatically increased. Importantly, the number of TUNEL-positive cells in the kidney of the I/R–FGF10 group was markedly reduced compared to the I/R group. Quantification analysis of the number of TUNEL-positive cells revealed that the average percentage of dead cells was 1.19% in the Sham group, 32% in the I/R group, and 4% in the I/R–FGF10 group, respectively (Figure 2B).

Caspase-3, also known as CPP32, is synthesized as an inactive proenzyme that is processed to an active form (cleaved Caspase-3) in cells undergoing apoptosis (Fernandes-Alnemri et al., 1994). Previous studies have demonstrated that Caspase-3 is the most important regulatory factor in the apoptotic cell both by death ligand (extrinsic) and mitochondrial (intrinsic) pathways (Salvesen, 2002). The Bcl-2 family, including Bax and Bcl-2, plays a crucial role in the mitochondrial apoptotic pathway (Havasi and Borkan, 2011). To further clarify the mechanism under the protective effect of FGF10 against renal I/R injury, we examined the activation of cleaved Caspase-3 by immunohistochemistry staining. As shown in Figure 3A, I/R injury increased the production of cleaved Caspase-3 as demonstrated by strong staining in the cytoplasm of renal tubular cells. The production of cleaved Caspase-3 was markedly decreased in the kidney tissue of the I/R–FGF10 group. Furthermore, several key proteins involved in the regulation of tubular cell apoptosis including cleaved Caspase-3, Bax, and Bcl2 were determined by western blot (Figure 3B). As shown in Figures 3C, D, the production of cleaved Caspase-3 and Bax in the kidney tissue was markedly increased after reperfusion, whereas administration of FGF10 inhibited the production of cleaved Caspase-3 and Bax. Together, these results indicated that FGF10 preadministration protects the kidneys via alleviating apoptosis of the renal tubular epithelial cells after reperfusion.

FIGURE 3 | FGF10 reduced the expression of proapoptotic proteins. (A) Immunohistochemistry staining of kidney tissues 1 day after reperfusion for the activation of Caspase-3. The expression of cleaved Caspase-3 was significantly increased in the cytoplasm of renal tubular cells after reperfusion, whereas FGF10 treatment reduced the expression of cleaved Caspase-3. Panels are representative of five rats in each group. Scale bars represent 50 mm. (B) Western blot analysis of apoptosis index expression. Total kidney tissues (contain both cortex and medulla, but don't contain renal fibrous capsule) were used for the analysis of protein expression in kidney. The expression levels of cleaved Caspase-3, Bax, and Bcl2 were detected. GAPDH was used as a loading control. (C, D) The column panels show the normalized optical density analysis. FGF10 significantly reduced the expression of cleaved Caspae-3 and Bax compared with I/R group. \*P < 0.05, \*\*P < 0.01.

### The Protective Effect of FGF10 Against Renal I/R Injury Is Associated With ER Stress

ER stress is a common feature of I/R injury and known to impact renal tubular cell survival. To investigate whether the protective effect of FGF10 on renal tubular cells is associated with the inhibition of excessive ER stress, we examined the expression of ER stress related proteins by immunohistochemistry staining. As shown in Figures 4A–C, the expression of CHOP was dramatically increased in the nucleus and cytoplasm of renal

tissues after 1 day of reperfusion. The expression of CHOP was significantly increased in the nucleus and cytoplasm of renal tubular epithelial cells after reperfusion, whereas FGF10 treatment reduced the expression of CHOP. (B) Immunohistochemistry staining for GRP78. FGF10 treatment reduced the expression of GRP78 in the cytoplasm of epithelial cells after reperfusion. (C) Immunohistochemistry staining for ATF-6. The expression of ATF-6 was significantly increased in the nucleus of renal tubular epithelial cells after reperfusion, whereas FGF10 treatment reduced the expression of ATF-6 compared to I/R alone. Panels are representative of five rats in each group. Scale bars represent 50 mm.

tubular epithelial cells after reperfusion, whereas FGF10 treatment reduced the expression of CHOP. FGF10 treatment reduced the expression of GRP78 in the cytoplasm of epithelial cells after reperfusion. The expression of ATF-6 was increased in the nucleus of renal tubular epithelial cells after reperfusion, FGF10 largely reduced the expression of ATF-6 compared to the I/R group. Results of immunohistochemistry staining confirmed that FGF10 could strikingly decreased the expression of ER stress relevant proteins induced by renal I/R injury. Western blotting was used to examine the expression of CHOP, GRP78, ATF-4, ATF6, PDI and XBP1, all of which are ER-stress effectors that, via regulation of the unfolded protein response, contribute to cellular homeostasis in kidney. As shown in Figure 5A and quantification analysis in Figures 5B–G, we observed elevated expression of these proteins in the kidney tissue of the I/R group, whereas pretreatment with FGF10 significantly down-regulated the expression of the proteins mentioned above. These results suggest that preadministration of FGF10 can effectively ameliorate I/R-induced maladaptive ER-stress response, which may contribute to mitigate tubular cell apoptosis. In addition, the apoptosis of renal tubular epithelial cells is the primary reason for AKI caused by I/R injury. We observed that cleaved Caspase-3, CHOP, GRP78, and ATF-6 are mainly expressed in the epithelial cells of the renal tubules (Figures 3A and 4). Based on the results of immunohistochemistry staining, we could infer that FGF10 reduces apoptosis of renal tubular epithelial cells via inhibiting the excessive ER stress.

### The Protective Effect of FGF10 Against Apoptosis Is Related to ERK1/2 Pathway

MAPK/ERK1/2 is a critical downstream pathway of FGF, which plays an important role in the regulation of variety of cellular processes including cell survival, proliferation, migration, and differentiation (Lunn et al., 2007). As mentioned above, FGF10 specifically binds to FGFR2b, which was distributed in the membrane of epithelial cells. To assess the effect of FGF10 on the activation of ERK1/2 pathway, we detected the phosphorylation of ERK1/2 in the kidney tissue of the Sham group, the I/R group, and the I/R–FGF10 group. As shown in Figures 5A, H, the phosphorylation of ERK1/2 was mildly increased in the kidney tissue of the I/R group compared to the Sham group. Preadministration of FGF10 led to a robust increase in the phosphorylation of ERK1/2 compared to the I/R group. The PI3K–Akt signal transduction pathway also plays an important role in the regulation of cell survival, proliferation, and migration (Wang et al., 2012). We also detected the phosphorylation of AKT in the kidney tissue of the Sham group, I/R group, and I/R–FGF10 group. As shown in Figures 5A, I, the phosphorylation of AKT was increased in the kidney tissue of the I/R group compared to the Sham group. However, there is no significant difference in the

\*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001, ns represents no significant difference.

phosphorylation of AKT in the kidney tissue between the I/R group and I/R–FGF10 group. Those results may imply that FGF10 protects against renal I/R injury through activating the ERK1/2 signaling pathway, not the PI3K-Akt signaling pathway. To further clarify the relationship between the ERK1/2 signaling pathway and the protective effect of FGF10, we treated NRK-52E cells with TBHP, a commonly ROS inducer which is much stable compared with hydrogen peroxide (H2O2) solution. The apoptosis of NRK52E cells was detected by flow cytometric analysis with Annexin V-FITC-PI Apoptosis Detection Kit. As shown in Figures 6A, B, FGF10 treatment significantly reduced the apoptosis rate of NER-52E caused by TBHP. However, U0126, a selective inhibitor of MEK1/2 that blocks the phosphorylation of ERK1/2 (Shukla et al., 2007), largely abolished the protective effect of FGF10 on NRK-52E cells. To further confirm the role of ERK1/ 2 signaling pathway in the protective effect against TBHP-induced apoptosis, the expression of cleaved Caspase-3, cleaved Caspase-9, Bax, and Bcl2 was detected by immunoblots. Caspase-9 is an initiator caspase which could further process the activation of other caspases, including Caspase-3, to start the caspase cascade leading to apoptosis. Under the action of the apoptotic signals, the release of Cytochrome c from the mitochondria and activation of Apoptotic protease activating factor 1 (APAF1) cleave pro-caspase 9 into the active form (Li et al., 1997). Our results indicated that FGF10 pretreatment effectively antagonized TBHP-induced Caspase-3 and Caspase-9 cleavages. More importantly, the effect of FGF10 was completely reversed in the presence of MEK inhibitor U0126. Consistently, the drastically increased production of Bax caused by TBHP also appeared to be restored with the treatment of FGF10. However, U0126 exposure partially reversed the effect of FGF10 (Figures 7A–D).

As shown in Figures 7A, E, FGF10 treatment significantly activated ERK1/2 phosphorylation compared to the TBHP group, which is consistent with what we observed in the kidney tissue. As expected, preaddition of U0126, a highly specific inhibitor of MEK, largely abolished the effect of FGF10 on the phosphorylation of ERK1/2 in NRK-52E cells. These results strongly suggest that ERK1/2 activation is a crucial mechanism in FGF10-mediated protection against cell apoptosis in both I/R injured kidney and TBHP injured NRK-52E cells.

proteins of NRK-52E were extracted using cell lysis buffer. NRK-52E was treated with different culture media and then the expression of cleaved Caspase-3, cleaved Caspase-9, Bax, Bcl-2, ERK1/2, and phosphor-ERK1/2 was detected by western blotting. (B–E) Histogram figures show the normalized optical density analysis. Results are representative of five rats in each group. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001.

### The Effect of FGF10 on the Regulation of ER Stress Is Related to the Activation of ERK1/2 Pathway

To clarify the relationship between the protective effect of FGF10 and ER stress, we examined the expression of CHOP and GRP78 by immunofluorescence staining in NRK-52E cells respectively (Figures 8A, B). We observed that FGF10 effectively attenuated ER stress relevant proteins induced by TBHP, which was inhibited by U0126 to a large extent. The expression levels of CHOP and GRP78 were also examined by western blotting. As

expression of CHOP and GRP78 in NRK-52E induced by TBHP, whereas U0126 largely eliminated the effect of FGF10. Panels are representative of five rats in each group. Scale bar represents 50 mm. (C) The expression of CHOP and GRP78 was detected by Western blotting. (D, E) Bar chart for quantification analysis for the expression of CHOP and GRP78. Results are representative of five rats in each group. GAPDH was used as a protein loading control. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001.

shown in Figures 8C, D, E, TBHP remarkably increased the expression of CHOP and GRP78 in NRK52E cells, indicating that oxidative stress triggered excessive ER stress in these cells. Similar to the results observed in vivo, FGF10 significantly reduced the expression of CHOP and GRP78 in NRK-52E cells treated with TBHP. The effect of FGF10 is partially abolished by U0126. Our results strongly suggested that activation of ERK1/2 signal pathway contributes to FGF10 mediated protection against maladaptive ER stress.

### DISCUSSION

AKI, mainly caused by renal I/R injury, remains a vexing health problem. Despite the current clinical advances in prevention and treatment, the morbidity and mortality of AKI in hospitalized patients remain very high (Basile et al., 2012; Winterberg and Lu, 2012). As a crucial mesenchymal–epithelial signaling growth factor in embryonic development, tissue repair, and regeneration, the role of FGF10 has been investigated in several disease conditions such as cerebral ischemia injury, pulmonary fibrosis, and wound healing (Li et al., 2016; Chen et al., 2017; Chao et al., 2017; El Agha et al., 2017). However, whether FGF10 is capable of delivering a protective effect on AKI in rat model of I/R injury is still unclear.

Currently, many studies reported the relationship between ERK1/2, ER stress, and apoptosis (Sun et al., 2015; Yao et al., 2017). Generally, ERK1/2 are activated upon phosphorylation by MEK1/2 and are considered to promote cell survival (Darling and Cook, 2014). ERK1/2 signaling inhibits apoptosis via activating the expression of prosurvival proteins (BCL-2, MCL-1, and BCL-XL) and repressing the expression of proapoptotic proteins (BAD, BIM, BMF, and PUMA). However, in some certain conditions such as starvation, ERK1/2 could also promote the expression of NOXA (phorbol-12-myristate-13 acetate-induced protein 1), a proapoptotic member of the BCL-2 family, to decide autophagy or apoptosis (Yao et al., 2017). ER stress could be triggered by a variety of extracellular stimuli and induces apoptosis. It has been reported that renal tubular cell apoptosis induced by I/R injury is associated with excessive ER stress. Excessive ER stress can activate apoptotic signaling pathways via CHOP, a master regulator of maladaptive ER stress-induced apoptosis (Rutkowski et al., 2006). The interaction of ERK1/2 signaling pathway and ER stress has been reported in many studies (Zhang et al., 2009; Darling and Cook, 2014). The activation of the ERK1/2 signaling pathway exhibits an antiapoptotic role during ER stress through regulating the IRE1 (inositol requiring enzyme 1) axis of the UPR (Darling and Cook, 2014). FGF10 is a member of the FGF family with multifunctional effect in the regulation of development, wound healing, and tissue regeneration. It has been proved that FGF10 can ameliorate cerebral I/R injury and spinal cord injury via inhibiting NF-kB-dependent inflammation and activating the PI3K/Akt signaling pathway (Li et al., 2016; Chen et al., 2017; Dong et al., 2019). In our previous study, we demonstrated that FGF10 can protect the kidney against apoptosis via the regulation of inflammatory response and autophagy (Tan et al., 2018). In our present study, the administration of FGF10 can not only reduced the expression of proapoptotic proteins, but also effectively alleviated ER stress kidneys after I/R injury. Therefore, FGF10 exhibits reliable capability in the protection against AKI caused by I/R injury via inhibiting maladaptive ER stress.

ER stress and UPR, which could be provoked by glucose depletion and hypoxia after renal I/R injury, have previously shown to play a pivotal role in the enhancement of protein folding ability (Belaidi et al., 2013; Wang et al., 2015). However, excessive and prolonged ER stress and UPR can elicit glomerular and tubular cell damage in patients with AKI and CKD (Bhatt et al., 2008; Inagi, 2009; Xu et al., 2016). Our present results indicated that the expression of ER stress relevant proteins including CHOP, GRP78, XBP-1, ATF-4, ATF-6, and PDI was significantly increased after reperfusion. Importantly, treatment of recombination FGF10 can reduce ER stress relevant proteins and thus inhibited renal tubular cell apoptosis caused by I/R injury. The present study suggested that the renoprotective effect of FGF10 is associated with the regulation of ER stress.

Mitogen-activated protein kinases (MAPKs) are among the most commonly activated signaling pathways associated with various renal injuries (Tian et al., 2000). ERK, an important member of MAPK family, is mainly activated by mitogenic stimuli such as growth factor and hormones. The ERK1/2 signaling pathway is particularly important in the regulation of cell survival, migration, differentiation, and proliferation in a variety of circumstances (Sun et al., 2015; Yao et al., 2017). The role of ERK1/2 in the restoration of renal structure and function is still controversial (Feliers and Kasinath, 2011; Zhang and Cai, 2016; Li et al., 2018). To confirm the role of ERK1/2 in the protective effect of FGF10 on AKI caused by renal I/R injury, we examined the expression of phospho-ERK1/2 in the kidney tissue after reperfusion as shown in Figure 5. Our experiments' results confirmed that FGF10 treatment increased the phosphorylation of ERK1/2 in the kidney tissues after reperfusion. As the effect of FGF10 in reducing apoptosis and inhibiting ER stress has been verified in the present study, we speculate that the protective effect of FGF10 in down-regulation of apoptosis may be related to the activation of ERK1/2 signaling pathway.

To further clarify the role of ERK1/2 signaling pathway in the regulation of FGF10 on ER stress after reperfusion, we then examined the protective effect of FGF10 against apoptosis with NRK-52E induced by TBHP. TBHP is a widely used oxidative stress inducer which can increase intracellular ROS production. Our present study demonstrated that FGF10 treatment can strikingly inhibit the apoptosis of NER-52E induced by TBHP. U0126, a specific inhibitor of MKK, abrogated the antiapoptosis effect of FGF10 via blocking the phosphorylation of ERK1/2. Moreover, U0126 also reversed the down-regulation effect of FGF10 on ER stress related proteins including GRP78 and CHOP. These results suggest that ERK1/2 signaling pathway is probably the downstream signals induced by FGF10 in the restoration of renal I/R injury. U0126 could suppress the activation of ERK1/2 and abolish the role of FGF10 in the regulation of ER stress on injured NRK-52E induced by TBHP.

As a multifunctional growth factor, FGF10 has been reported to play crucial roles in development and disease. However, the protective mechanism of FGF10 on AKI has not yet been clearly elucidated. In the present study, we confirmed that renal tubular epithelial cell apoptosis induced by hypoxia injury is related to the excessive activation of ER stress. Convincing experimental evidence has been provided both in vivo and in vitro that exogenously administered FGF10 could attenuate renal tubular epithelial cell apoptosis via inhibiting excessive ER stress. Through in vitro experiments, we also demonstrated that the protective effect of FGF10 is, at least partly, mediated by MEK– ERK1/2 signaling pathway. In conclusion, results of our present study have implications for understanding the pathophysiology of AKI caused by renal I/R injury and indicate the therapeutic potential of FGF10 in clinical applications. Future research should clarify the exact protective mechanisms of FGF10 in tissue repair and provide novel insights in the field of regenerative medicine.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ supplementary material.

### ETHICS STATEMENT

The animal study was reviewed and approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University, Wenzhou, China.

### AUTHOR CONTRIBUTIONS

XT, JX and J-SZ conceived and designed the experiments. XT, LY, and QT performed the animal operation. XT, LY, RY, and LX performed cell culture, apoptosis assay, FACS analysis, immunoblot, immunohistochemistry, and immunofluorescent staining. XT and LY analyzed the experiments data and prepared the figures. XT and J-SZ wrote and revised the manuscript. J-SZ and JX funded the project.

### REFERENCES


### ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (81500519, 81472601) and the Scientific Research Starting Foundation of Qingdao University (DC1900011202).

protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J. Biol. Chem. 269 (49), 30761–30764.


cardiomyocyte cell-cycle re-entry. Cardiovasc. Res. 104 (3), 432–442. doi: 10.1093/cvr/cvu232


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

Copyright © 2020 Tan, Yu, Yang, Tao, Xiang, Xiao and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Fibroblast Growth Factor 22 Inhibits ER Stress-Induced Apoptosis and Improves Recovery of Spinal Cord Injury

Sipin Zhu1† , Mengji Chen1,2† , Min Chen1,2† , Jiahui Ye1,2† , Yibo Ying1,2, Qiuji Wu1,2, Haicheng Dou<sup>1</sup> , Liyunian Bai 1,2, Fangmin Mao1\*, Wenfei Ni 1\* and Kehe Yu1\*

Wenzhou, China, <sup>2</sup> Second Medical College of Wenzhou Medical University, Wenzhou, China

<sup>1</sup> Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University,

### Edited by:

Zhouguang Wang, Albert Einstein College of Medicine, United States

#### Reviewed by:

Yingnyu Gao, Marshall University, United States Xiao Xiao, Merck, United States

#### \*Correspondence:

Fangmin Mao 13868816296@163.com Wenfei Ni niwenfei@126.com Kehe Yu kehefish@163.com

† These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 16 October 2019 Accepted: 07 January 2020 Published: 11 February 2020

#### Citation:

Zhu S, Chen M, Chen M, Ye J, , Ying Y, Wu Q, Dou H, Bai L, Mao F, Ni W and Yu K (2020) Fibroblast Growth Factor 22 Inhibits ER Stress-Induced Apoptosis and Improves Recovery of Spinal Cord Injury. Front. Pharmacol. 11:18. doi: 10.3389/fphar.2020.00018 Currently, inhibiting or reducing neuronal cell death is the main strategy to improve recovery of spinal cord injury (SCI). Therapies using nerve growth factors to treat SCI mainly focused on reducing the area damaged by postinjury degeneration to promote functional recovery. In this report, we investigated the mechanism of ER (endoplasmic reticulum) stress-induced apoptosis and the protective action of fibroblast growth factor 22 (FGF22) in vivo. Our results demonstrated that ER stress-induced apoptosis plays a significant role in injury of SCI model rats. FGF22 administration promoted recovery and increased neuron survival in the spinal cord lesions of model mice. The protective effect of FGF22 is related to decreased expression of CHOP (C/EBP-homologous protein), GRP78 (glucose-regulated protein 78), caspase-12, X-box binding protein 1 (XBP1), eukaryotic initiation factor 2a (Eif-2a) and Bad which are ER stress-induced apoptosis response proteins. Moreover, FGF22 administration also increased the number of neurons and the expression of growth-associated protein 43 (GAP43) which was related to axon regeneration. We also demonstrated that the protective effect of FGF22 effectively reduces neuronal apoptosis and promotes axonal regeneration. Our study first illustrated that the function of FGF22 is related to the inhibition of ER stress-induced cell death in SCI recovery via activation of downstream signals. This study also suggested a new tendency of FGF22 therapy development in central neural system injuries, which involved chronic ER stress-induced apoptosis.

Keywords: spinal cord injury, fibroblast growth factor 22, ER stress, apoptosis, nerve regeneration

### INTRODUCTION

Spinal cord injury (SCI) is a destructive event that usually leads to significant functional impairment for the patient (Angeli et al., 2018). It triggers very limited regeneration in humans, leading to irreversible damage that can result in permanent motor dysfunction (Kumamaru et al., 2018; Sofroniew, 2018). SCI pathology can be divided into two stages: primary injury caused by direct disruption of the spinal cord, and secondary injury including neuronal apoptosis, autophagy,

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vascular dysfunction, oxidative stress and inflammation, which lead to sustained and extensive tissue damage (Tohda and Kuboyama, 2011; Zhu et al., 2016; He et al., 2017a). SCI causes the injury loci to form a hypoxic microenvironment, which in turn causes neuronal death and dysfunction, ultimately limiting function recovery after SCI (Sabelstrom et al., 2013). Therefore, SCI treatment focuses on reducing neuronal death by inhibiting neuronal apoptosis.

The endoplasmic reticulum (ER) was initially identified as an intracellular organelle responsible for maintaining cellular homeostasis and resisting potential injury caused by misfolded proteins on the ER (Zhang et al., 2015; He et al., 2017b). During SCI, a large amount of protein misfolding caused by alteration of the microenvironment in the injured area leads to the unfolded protein response (UPR) (Hetz, 2012). The UPR activates three signal pathways that are regulated by activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), which increases the expression of apoptotic proteins and leads to neuronal death (Lee et al., 2014; Dou et al., 2018). Extensive neuronal death induced by apoptosis is the largest obstacle in recovery of spinal cord injury (Crowe et al., 1997).

The family of fibroblast growth factors and their receptors have been important regulators of presynaptic differentiation. FGF22 is a member of the FGF7 subfamily. It mainly activates FGF receptors FGFR1b and FGFR2b (Umemori et al., 2004). Studies have shown that FGF22 plays a significant part in SCI recovery as an endogenous regulator of synaptic plasticity and circuit remodeling in the adult nervous system (Jacobi et al., 2015). In this study, we injected different concentrations of FGF22 into the site of SCI in rats to observe the recovery of injured spinal cord. Here, we show that FGF22 can inhibit neuronal apoptosis induced by ER stress and promote SCI recovery.

## MATERIALS AND METHODS

### Cell Culture and Treatment

PC-12 cell lines obtained from the American Type Culture Collection (ATCC) were cultured in RPMI 1640 medium, which consisted of 10% fetal bovine serum (FBS), RPMI 1640 and 1% antibiotics. Cell incubation was performed in a humidified incubator at 37°C and 5% CO2. All cells were randomly divided into four groups which included the 4-PBA (4-phenylbutyric acid) group, TG (thapsigargin) group, control group, treatment group with 5 mg/ml FGF22, treatment group with 10 mg/ml FGF22 and treatment group with 15 mg/ ml FGF22.

### In Vitro Scratch Motility Assay

A linear scratch was applied to cell monolayers with the help of a 200 µl pipette tip to create a cell-free area; then, cells were washed twice with PBS. Wound analysis and photography were performed by an inverted phase-contrast microscope and the wound area was calculated by ImageJ software.

### Apoptosis Assay

The apoptotic rates of the PC-12 cells treated with TG, TG + 4- PBA, TG+5 mg/ml FGF22, TG + 10 mg/ml FGF22 and TG + 15 mg/ml FGF22 were measured by the PI/Annexin V-FITC kit (Invitrogen, Carlsbad, CA, USA) and then analyzed by FACS can flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) as the manual description.

### Western Blot Analysis

For protein analysis, PC12 cells were in RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS) containing the protease and phosphatase inhibitor. Cracked. The above extracts were quantified using bicinchoninic acid (BCA) reagent (Thermo, Rockford, IL, USA). 50 mg of protein was placed on the 11.5% gel and then transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% milk (Bio-Rad) in TBS containing 0.05% Tween 20 for 1 h and incubated with the following antibodies: CHOP (1: 300), GRP78 (1:300), caspase-12 (1:1000), and GAPDH (1:1000). The membrane was washed 3 times with TBS and treated with a horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The signals were visualized by the ChemiDoc ™ XRS+ imaging system (Bio-Rad), and the band density was quantified using the Multigauge Software (FUJIFILM Corporation, Tokyo, Japan) of the 2006 Scientific Laboratory. We analyzed the relative density of the bands using quantity one (version 4.5.2; Bio-Rad).

### Animal Model of Spinal Cord Injury

Eighty adult female SD rats (weighing 220–250 g at the start of the experiment) were purchased from the Animal Center of Chinese Academy of Sciences, Shanghai, China. The Animal Care and Use Committee of Wenzhou Medical College ratified the experiments, and the experiments were carried out in accordance with the guidelines of the Care and Use of Laboratory Animals from the National Institutes of Health. Animals were randomly divided into four groups, which included the SCI group, sham group, treatment group with 5 mg/ml FGF22 and treatment group with 10 mg/ml FGF22. The animals were fixed with a skin incision along the midline of the back. The 8th-to-10th thoracic spinal vertebrae were exposed. The model of acute spinal cord injury was established by striking T9 segments of the spinal cord with a 10-g hammer and a 25-mm-height free fall. The sham group rats underwent the operation similarly without injury by collision. Animal care and handling involved bladder massage to induce urination twice daily, once in the morning and again in the evening, until cefazolin sodium reconstructed reflex bladder function (50 mg/kg, i.p.).

### FGF22 Transplantation to Treat SCI

After building the SCI model, surgery was carried out immediately to microinject 5 mg/ml and 10 mg/ml of FGF22 into the rats of the two FGF22 treated groups. Through stereoscopic positioning instruments and microsyringes, FGF22 orthotopically arrived at the injured lesion. The sham group was perfused with saline at the same site. All animals were returned to cages for recovery. We provided each group with the same moderate diet at fixed times.

### Locomotion Recovery Assessment

To examine the locomotor function of rats after SCI, behavioral analyses were conducted by two well-trained investigators familiar with the score criteria but blinded to the experimental conditions.

Basso-Beattie-Bresnahan (BBB) is a scale with a total score of 22 points (scores 0–21) that logically and systematically follows functional recovery of hindlimbs from 0 points, reflecting complete paralysis of the lower limbs, to 21 points, reflecting normal locomotor function (Basso et al., 1995). The scale was formulated based on the natural progression of motor recovery in rats with thoracic spinal cord injury.

The inclined plane test was carried out by means of a testing apparatus (Rivlin and Tator, 1977). The maximum angle in which a rat's position could be maintained for 5 s without falling was recorded for each position, and then, the averages were taken to determine each rat's scores. Footprint analysis was performed by dipping the animal's hind paws in red dye and allowing them to crawl past a box of suitable size (1 m long and 7 cm wide) (de Medinaceli et al., 1982). Footprint scanning and digital image analysis were performed.

### H&E Staining and Nissl Staining

The rats were re-anesthetized with 1% pentobarbital (40–50 mg/ kg, i.p.). Thoracotomy was performed on the 60th day after injection. Rats were perfused with 0.9% NaCl, followed by 500 ml paraformaldehyde phosphate buffer solution injected into the heart to harden the tissue for complete removal. Spinal cord excision at the 8th-to-10th thoracic spinal vertebral level around the injury was performed. Spinal cords were fixed overnight in cold 4% paraformaldehyde and embedded with paraffin. For histopathological examination, transverse paraffin sections (10 mm thick) were subjected to hematoxylin and eosin (H&E) staining. For Nissl staining, sections were incubated in 1% cresyl violet. Two stained sections were examined and scanned under a light microscope.

### Immunohistochemistry

Transverse paraffin sections were incubated in 80% carbinol and 3% H2O2 for 30 min, and then, the sections were transferred to blocking solution at room temperature for 1 h. Subsequently, the sections were incubated at 4°C overnight with the following primary antibodies: GRP78 (1:200), CHOP (1:200), caspase-12 (1:4000), XBP1 (1:400), Eif-2a (1:100) and Bad (1:200). After washing with PBS three times, sections were incubated with horseradish peroxidase-conjugated secondary antibodies at 37°C for 2h. We used 3,3-diaminobenzidine (DAB) to stop the reaction. Positive neuron numbers and optical densities of CHOP, GRP78, XBP1, Eif-2a, Bad and caspase-12 were counted in six randomly selected fields per sample. The results were imprinted using Nikon ECLPSE 80i (Nikon, Tokyo, Japan).

### Video Images Locomotor Function

Forty-two adult female SD rats (weighting 220–250 g) are divided into six groups, sham group, SCI group, TG group, 4- PBA group, 5 mg/ml FGF22 group and 10 mg/ml FGF22 group. Using a camera (Leica), each group rats were photographed while walking through a 1-m-long glass runway with markers on the hindlimbs to estimate hip, knee, ankle, and foot position. The following parameters were used to evaluate locomotion: 1) weight support (height; hip height minus trunk width, equal to the torso gap on the ground), 2) leg extensor spasms (quantified as the time the foot is overstretched and dragged, relative to the foot cycle The duration is on the back surface), 3) the number of footsteps (the number of footsteps calculated relative to the number of forefoot steps) and 4) the posture of the foot (measurement of the foot offset behind the hip at the beginning of the ankle). The walking step rhythm is defined by the front leg (front leg steps/sec).

### Immunofluorescence Staining

Sections were incubated with 10% normal bovine serum at 37°C for 1 h in PBS containing 0.1% Triton X-100 and then incubated with suitable primary antibodies overnight in the same buffer solution at 4°C. Nuclei were stained with DAPI (0.25 mg/ml) dye. To detect neurons and GAP43, we used anti-NeuN (1:500, Millipore) and anti-GAP43 (1:50) primary antibodies, respectively. After incubation with primary antibody, sections were triple washed with PBS at room temperature, followed by incubation with secondary antibody (1:500) at 37°C for 1 h. Then, sections were washed with PBS containing 0.1% Triton X-100 4×10 min and then washed with PBS 3×5 min. All images were captured under a Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan).

### Statistical Analysis

Statistically, data were expressed as the mean ± SEM. For two experimental groups, the Student's t test was used to determine statistical significance. Values of P ≤ 0.05 were deemed significant. When there were more than two experimental groups, one-way analysis of variance (ANOVA) and Dunnett's post hoc test were used to statistically evaluate data, and P ≤ 0.05 was considered statistically significant.

### RESULTS

### FGF22 by Inhibiting the Endoplasmic Reticulum Stress Increases the Migration and Repair Capability of PC-12 Cells

PC12 cells were derived from murine adrenal medullary pheochromocytoma, and were similar to normal nerve cells in terms of cell morphology, physiology, biochemistry and other functions (Shafer and Atchison, 1991). Wound healing assay results were shown for the control group, TG group, 4-PBA group, 5 mg/ml FGF22 group, 10 mg/ml FGF22 group and 15 mg/ml

FGF22 group 12 h and 24 h after scratching (Figure 1A). Compared to the control group, the TG group displayed a larger cell-free area, and compared with the TG group, the 10 mg/ml FGF22 treatment group displayed an obviously reductive wound area and faster migration. Although the 4-PBA group, the 5 mg/ml FGF22 group and the 15 mg/ml FGF22 group had improved cell migration, the 10 mg/ml FGF22 treatment group was even more improved (Figure 1B). The effect of the 15 <sup>m</sup>g/ml FGF22 group was not as good as that of the 10 mg/ml FGF22 group, which may be due to the fact that FGF22 was the biological macromolecule. If the concentration was too high, the cells will dehydrate and the cell migration capacity will be reduced (Yurinskaya et al., 2005).

### FGF22 Inhibit ER Stress-Induced Cell Apoptosis of PC-12 Cells

To further confirm the role of FGF22 in the ER stress induced apoptosis in vitro. The apoptosis model was replicated by ER stress specific activator TG was using to treat PC12 cells. Cell apoptosis rates were analyzed, and we found that FGF22 10 ug/ml significantly reduced TG-induced apoptosis rate in PC12 cells, compared with the TG, TG + 4-PBA, FGF22 5 mg/ml and FGF22 <sup>15</sup> <sup>m</sup>g/ml groups (Figures 2A, B). Furthermore, the ER stressrelated protein levels were detected by western blot. We observed that the levels of CHOP, GRP78, and cleaved caspase-12 protein significantly decreased in FGF22 10 ug/ml group than other groups (<sup>P</sup> < 0.01) (Figures 2C–F).

### Maximizing Functional Recovery After SCI With a Higher Concentration of FGF22

FGF22 treatment reduced spinal cord injury and neuron death and improved motor recovery after SCI. The FGF22 injection groups showed significantly higher BBB scores than the SCI group after the operation, indicating that FGF22 promoted the recovery of nerve function. The results of the inclined plane test were similar. The 10 mg/ml FGF22 group showed the most complete anatomical appearance of SCI (Figure 3A). The mean BBB scores and angle of incline scores assessing locomotor skills over time were higher in the 10 mg/ml FGF22 group than in the other three groups (Figures 3B, C). Rats

injected with 10 mg/ml FGF22 also showed a significant improvement in footprint analysis and at day 60 (Figure 3D).

### FGF22 Increases Neuron Survival and Improves Tissue Density in SCI

Transverse and longitudinal H&E staining was performed on spinal cord samples in the sham group, SCI group, 5 mg/ml FGF22 group and 10 mg/ml FGF22 group after 60 days contusion (Figures 4A, B). Compared with the sham group, the SCI group showed progressive destruction of the dorsal white matter and central gray matter tissue. Compared with the SCI group, the 10 mg/ml FGF22 treatment group showed obvious protective impacts, such as less necrosis and karyopyknosis. Although the 5 mg/ml FGF22 group showed improvements in spinal cord sections, 10 mg/ml FGF22 treatment provided more amelioration (Figures 4A, B). These results further strengthen the neuroprotective efficacy of 10 mg/ml FGF22 for motor neurons in this SCI rat model.

### FGF22 Treatment Inhibits ER Stress-Induced Apoptosis and Improves Recovery of SCI

To determine whether FGF22 promoted SCI recovery via inhibiting ER stress-induced apoptosis, we measured the expression of GRP78, caspase-12 and CHOP, which are related to ER stress, by immunofluorescence staining. As shown in Figure 5, caspase-12, GRP78 and CHOP expression were increased in SCI and remarkably inhibited by FGF22. Moreover, the 10 mg/ml FGF22 group showed fewer positive points than the 5 mg/ml FGF22 group. This indicates that, within a certain range, higher concentrations of FGF22 can better inhibit apoptosis and promote recovery of SCI (Figures 5A–D).

After SCI, the lateral intumescent spinal cord anterior horn displayed a decreased number of large and medium-sized neurons compared with the sham group, the survival rate decreased, and the cell outline was not clear, as seen by fuzzy Nissl staining. Nissl staining of crosscutting showed that the 10 mg/ml FGF22 group recovered well and the spinal cord anterior horn neurons of Nissl increased compared to the SCI group, providing further evidence that spinal cord function restoration and the treatment effect were more pronounced in the 10 mg/ml FGF22 group than in the 5 <sup>m</sup>g/ml FGF22 group (Figures 5A, E).

### Regulation of Upstream and Downstream Signals Is Important for the Protection of FGF22

Eif-2a was an upstream factor of CHOP, which promoted JNK signaling and inhibited AKT signaling to promote apoptosis. XBP1 promoted apoptosis by activating JNK signaling (Li et al., 2019). Bad was the downstream factor of CHOP, CHOP-

ERO1a-caspase-dependent apoptosis signaling pathway activates mitochondrial-mediated apoptosis signaling pathway by up-regulating BAD and caspase-3 and down-regulating BCL-2/BAX ratio (Chen et al., 2019). To determine that the regulation of upstream and downstream signals was important for protecting FGF22, we measured the expression of Bad, XBP1, and Eif-2a associated with endoplasmic reticulum stress by immunohistochemical staining. As shown in Figure 6, the expression of Bad, XBP1 and Eif-2a increased in SCI and were significantly suppressed by FGF22. In addition, the 10 mg/ml FGF22 group had fewer positive spots than the 5 mg/ml FGF22 group. This indicates that within a certain range, 10 mg/ml FGF22 can better inhibited apoptosis and promoted the recovery of SCI (Figures 6A–D).

### Recovery of Hindlimb Function in Rats Treated With FGF22 Inhibiting ER Stress-Induced Apoptosis

TG was an endoplasmic reticulum stress agonist and 4-PBA was an endoplasmic reticulum stress inhibitor (Rellmann et al., 2019). Adding these two groups as the comparison can showed that inhibition of endoplasmic reticulum stress can promoted the recovery of SCI. The FGF22 groups showed lower index of foot error and higher index of height and plantar steps which indicated preferable recoveries (Figure 7A). The 4-PBA group also showed better functional recovery, this illustrated that inhibiting ER stress promoted the recovery after SCI. It can also be obtained from video data that 10 mg/ml FGF22 group showed significantly recovery of hindlimb function of rats after SCI. It had least foot error, highest index of height and plantar steps than the other two groups (Figures 7B–D). Meanwhile, the TG treated group showed no obvious improvement. This suggested that ER stress has a negative effect on functional recovery after SCI. Inhibiting ER stress-induced apoptosis effectively promote the hindlimb function of SCI rats.

### FGF22 Treatment Promotes Neuronal Survival and Nerve Regeneration

To determine the effect of FGF22 on apoptosis inhibition, NeuN was used to detect neuronal numbers. The neurons in the SCI group were largely lost, while the FGF22 treatment groups showed increases in the number of neurons, which proved that apoptosis was effectively inhibited. The higher concentration of FGF22 had more obvious protective effects on neurons. The experimental results showed an obvious enhancement of positive green fluorescence signal in the 10 mg/ml FGF22 administration group compared with the 5 mg/ml FGF22 group and SCI group (Figures 8A, C).

Moreover, we used GAP43 to detect the effect of FGF22 on axons. After SCI, axons break and are difficult to regenerate. The number of axons is significantly increased with the injection of FGF22. The experimental results showed an obvious enhancement of positive green fluorescence signal in the axons in the 10 mg/ml FGF22 administration group compared with the <sup>5</sup> <sup>m</sup>g/ml FGF22 group and SCI group (Figures 8B, D). This indicates that FGF22 can promote axonal regeneration after spinal cord injury, restore the nerve conduction pathway and promote functional recovery.

### DISCUSSION

After SCI, the injury site forms a microenvironment of ischemia, hypoxia and inflammatory infiltration (Abbott et al., 2010). After the neurons are stimulated by the unbalanced microenvironment, protein folding errors, unfolded protein aggregation, and Ca2+ balance disorders occur in the ER (Soboloff and Berger, 2002; Montague et al., 2014). This state is called endoplasmic reticulum stress. ER stress can trigger a series of physiological changes, and the misfolded proteins accumulated in the endoplasmic reticulum are

FIGURE 5 | FGF22 inhibited ER stress-induced apoptosis and improves the recovery of SCI. (A) Immunohistochemistry for GRP78, CHOP, and caspase-12 in the sham, SCI, 5 mg/ml FGF22 and 10 mg/ml FGF22 groups. (B) Nissl staining of crosscutting for the sham, SCI, 5 mg/ml FGF22 and 10 mg/ml FGF22 groups. (C–E) Analysis of positive cells in immunohistochemistry and Nissl staining of crosscutting. "\*" and "\*\*" represent P < 0.05 or P < 0.01 versus the sham group or SCI group, " # " and " ##" represent P < 0.05 or P < 0.01 comparing the 10 mg/ml FGF22 group to the 5 mg/ml FGF22 group, indicating statistical significance. Data are the mean values ± SEM, n = 6.

sham group or SCI group, " ##" P < 0.01 indicating statistically significant. Data are the mean values ± SEM, n = 6.

processed, which is beneficial to the recovery of cell function (Lin et al., 2005). This process is called the unfolded protein response (UPR) and is a cytoprotective mechanism that restores ER homeostasis under cell pressure (Grootjans et al., 2016). However, if the UPR is beyond the control of protein folding and uncompensated, it will activate the apoptotic signaling pathway (Ohri et al., 2011). Unfolded proteins are stored in the ER and induce activation of PERK/ATF6 signaling pathways. The activation of this pathway results in upregulated expression of chaperones including GRP78, followed by activation of caspase-12 and other apoptotic proteins, such as CHOP, to induce cell apoptosis (Nakagawa et al., 2000; Li et al., 2016).

When ER stress occurs, ER oligomerization occurs, and phosphorylation leads to phosphorylation of Eif-2a, which forms the p-eIF2a pathway, inhibits and inhibits mRNA transcription, and reduces protein accumulation (Sozbilen et al., 2018). In addition to Eif-2a, XBP1 is also activated in the transcription of the ER chaperone gene (Wang et al., 2013). The following is the binding of the general transcription factor nuclear factor Y to the CCAAT part of the ERSE, which leads to the activation of ER chaperone gene transcription (Ubeda and Habener, 2000; Shifman et al., 2008).

Previous studies have indicated that SCI causes a large number of neuronal deaths, axonal rupture, and disruption of nerve conduction pathways (Zhou et al., 2017; Anderson et al., 2018). Among them, the loss of neurons has the most direct relationship with motor dysfunction (Liu et al., 2015). There is increasing evidence indicating that ER-stress-activated pathways may play a significant role in promoting cell death caused by apoptosis in CNS injury diseases (Byrnes et al., 2007; Ogawa et al., 2007). Therefore, a variety of mechanisms, such as disturbed ionic oxidative stress, homeostasis, ER stress, and inflammatory response, participate in the progression of the second pathological process of SCI (Rami and Kogel, 2008). Since neurons are difficult to regenerate, the best solution for neuronal loss is to protect neurons and reduce their apoptosis. Therefore, inhibiting this process to protect neurons from apoptosis might be an ideal therapeutic strategy for SCI.

Many therapeutic interventions utilizing growth factors, such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF), have been confirmed to promote functional recovery after SCI (Rathbone et al., 1999; Zhu et al., 2016; Anderson et al., 2018). However, these factors are usually applied to promote

FIGURE 7 | FGF22 improved hindlimb function SCI rats via the inhibition of ER stress-induced apoptosis. (A) Video image sequence of rat walking posture of the sham group, SCI group, TG group, 4-PBA group, 5 mg/ml FGF22 group and 10 mg/ml FGF22 group in spinal cord injury, at 14 days after SCI. Lines and dots were used to mark the ankle, knee and hip joint, and foot movement was shown by arrows, scale bar = 500 mm. (B) Body height for each rat of the six groups. "\*" and "\*\*" represent P < 0.05 or P < 0.01 versus the SCI group, " # " represent P < 0.05. Data are the mean values ± SEM, n = 6. (C) Number of successful hind-limb steps for each rat of the six groups. "\*" and "\*\*" represent P < 0.05 or P < 0.01 versus the SCI group, " # " represent P < 0.05. Data are the mean values ± SEM, n = 6. (D) Foot-placement error for each rat of the six groups. "\*" and "\*\*" represent P < 0.05 or P < 0.01 versus the SCI group, " # " represent P < 0.05. Data are the mean values ± SEM, n = 6.

angiogenesis, neuronal proliferation and differentiation but are rarely used to inhibit neuronal apoptosis.

In this research, we demonstrated that ER stress-induced apoptosis participates in the response to SCI and the levels of proteins activated by apoptosis, including CHOP, GRP78, and caspase-12, were significantly increased by SCI. As intermediate signaling molecules of the ER stress pathway, these proteins mediate apoptosis through various pathways such as the IRE-1- CHOP pathway, ATF6-CHOP pathway, and PERK-elF2a-ATF4-CHOP pathway, which affect the expression and function of downstream proteins.

As an important promoter of circuit remodeling in the injured spinal cord, FGF22 treatment inhibited the expression of protein activated by ER stress and improved motor function and SCI recovery (Singh et al., 2012). Previous studies also showed that genetic destruction of the FGF22 signal not only reduces the number of new axons formed in the injured loci but also changes the molecular organization of the forming axons (Terauchi et al., 2010). Therefore, treatment of injured spinal cord with FGF22 can effectively promote axonal regeneration and functional recovery (Huang et al., 2017). The experimental results showed that apoptosis signaling molecules in the FGF22 treatment group were inhibited, which indicated that FGF22 can effectively inhibit endoplasmic reticulum stress, reduce neuronal apoptosis and play a crucial role in neuronal protection.

In conclusion, our research demonstrated that FGF22 plays a role in protecting neurons of SCI rats, promoting functional recovery after SCI by reducing apoptosis induced by ER stress and promoting axon regeneration [46]. To further prove that FGF22 indeed functions through the ER stress pathways to promote SCI recovery, we will use Chop knockout SD rats and further verify our experimental results.

### REFERENCES


### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ supplementary material.

### ETHICS STATEMENT

The animal study was reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical College.

### AUTHOR CONTRIBUTIONS

SZ, MiC, MeC, and JY coordinated and carried out most of the experiments and data analysis and participated in drafting the manuscript. MeC to participate in the data. YY, QW, HD, and LB provided technical assistance,including drawing and tabulating. SZ, MiC, and JY carried out data analysis and revised the manuscript. FM, WN, and KY supervised the project and experimental design and provided financial support. SZ and MiC wrote the main part of the paper. All authors read and approved the final manuscript.

### FUNDING

This work was partly supported by a research grant from the National Natural Science Foundation of China (81802235), Zhejiang Experimental Animal Science and Technology Project of China (2018C37112), Zhejiang Provincial Natural Science Foundation of China (LY17H060009, Y17H060051) and Wenzhou Basic Science Research Plan Project (Y20180033), which in part supported SZ as a visiting scholar at UWA. SZ made mutual collaborative visits for the purpose of this study.


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Zhu, Chen, Chen, Ye, Ying, Wu, Dou, Bai, Mao, Ni and Yu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Protective Effect of Basic Fibroblast Growth Factor on Diabetic Nephropathy Through Remodeling Metabolic Phenotype and Suppressing Oxidative Stress in Mice

Tingting Wei 1,2, Qi Shu<sup>1</sup> , Jie Ning<sup>1</sup> , Shuaijie Wang<sup>1</sup> , Chen Li <sup>1</sup> , Liangcai Zhao<sup>1</sup> , Hong Zheng1\* and Hongchang Gao1\*

#### <sup>1</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Laboratory Animal Centre, Wenzhou Medical University, Wenzhou, China

#### Edited by:

Jin-San Zhang, Mayo Clinic, United States

#### Reviewed by:

Yi Tan, University of Louisville Physicians, United States Sandra Donnini, University of Siena, Italy

\*Correspondence:

Hong Zheng 123zhenghong321@163.com Hongchang Gao gaohc27@wmu.edu.cn

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 08 November 2019 Accepted: 22 January 2020 Published: 21 February 2020

#### Citation:

Wei T, Shu Q, Ning J, Wang S, Li C, Zhao L, Zheng H and Gao H (2020) The Protective Effect of Basic Fibroblast Growth Factor on Diabetic Nephropathy Through Remodeling Metabolic Phenotype and Suppressing Oxidative Stress in Mice. Front. Pharmacol. 11:66. doi: 10.3389/fphar.2020.00066 Diabetic nephropathy is a common complication in diabetes, but still lack of effective therapeutic strategies. This study aimed to investigate the therapeutic effect of basic fibroblast growth factor (bFGF) in db/db mice with diabetic nephropathy and explore its possible metabolic mechanisms using a nuclear magnetic resonance-based metabolomic approach. We found that bFGF treatment significantly alleviate urinary albumin to creatinine ratio and renal fibrosis in db/db mice, suggesting a potential renal protective effect. Metabolomics results reveal that bFGF remodeled metabolic phenotypes of the kidney and urine in db/db mice, mainly involving energy metabolism, methylamine metabolism, osmoregulation, and oxidative stress. Furthermore, the results show that bFGF-induced reductions of oxidative stress and apoptosis in db/db mice might be mediated by NOX-ROS-Nrf2 signaling. Therefore, our study suggests that the protective effect of bFGF on diabetic nephropathy could be mediated by remodeling metabolic phenotype and suppressing oxidative stress.

Keywords: bFGF, diabetes, metabolomics, nephropathy, taurine, oxidative stress

## INTRODUCTION

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes and has become the leading cause of end-stage renal failure and death of both type I and II diabetic patients (Yu and Bonventre, 2018; Wang et al., 2019). The progression of DN involve several stages, such as glomerular hypertrophy, proteinuria, glomerulosclerosis, interstitial fibrosis, and renal failure (Li et al., 2019). Several potential mechanisms underlying DN development have been reported, including oxidative stress (Barman et al., 2018), inflammation (Olatunji et al., 2018), advanced glycation end products, and polyol pathway (Pan et al., 2010). However, there is still a lack of effective treatments for DN, so elucidating the mechanisms and discovering new drugs of DN treatment are urgently required.

Basic fibroblast growth factor (bFGF), a member of the growth factor family, has been reported to reduce the functional and morphological damages in chronic kidney disease and induce the re-expression of nephrogenic/ angiogenic factors (Villanueva et al., 2014). bFGF treatment can effectively reduce serum glucose and lipid levels in STZinduced diabetic rats and prevent DN through inhibition of inflammation (Lin et al., 2016; Sheng et al., 2018). Additionally, Tan et al. reported that bFGF protects against renal ischemia reperfusion injury by attenuating mitochondrial damage and proinflammatory signaling (Tan et al., 2017). Of note, a recent review revealed that the FGF family possesses multifarious roles in metabolic homeostasis (Li, 2019). Yet, the metabolic mechanisms of bFGF on diabetic kidney diseases remain unclear.

Metabolomics is an omics approach that aims to analyze a comprehensive set of low-molecular weight metabolites in biological samples under pathophysiological conditions (Nagana Gowda and Raftery, 2017). It has been extensively applied for identifying potential biomarkers and exploring pathogenesis of diseases (Nicholson et al., 1999; Lu et al., 2013; Chen et al., 2014). Nuclear magnetic resonance (NMR) spectroscopy is a common analytical technique used in metabolomics studies owing to its advantages, such as simple sample preparation, rapid analysis, and high reproducibility. In our previous studies, we have reported several defective metabolic pathways in the kidney of diabetic mice and rats, such as TCA cycle, glycolysis, methylamine pathway, fatty acids b-oxidation, ketogenesis, and glycogenic amino acid pathway (Guan et al., 2013; Wei et al., 2015). Moreover, Zhao et al. have found that abnormal energy metabolism in the kidney of rats was associated with the pathogenic process of DN (Zhao et al., 2011).

In the present study, we analyzed metabolic profiles of the kidney and urine in type 2 diabetic db/db mice with DN after bFGF treatment by using a <sup>1</sup> H NMR-based metabolomic approach. The purposes of this study are (1) to examine the therapeutic effect of bFGF on DN, and (2) to explore its potential metabolic mechanisms.

### MATERIALS AND METHODS

### Animals

Eight-week-old male db/db (C57BLKS/J-leprdb/leprdb) mice and age-matched wild-type (wt) mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Mice were housed in specific pathogen-free (SPF) colony under a fully controlled condition (room temperature, 22 ± 2°C; humidity, 50–60%; light/dark, 12 h/12 h) at the Laboratory Animal Center of Wenzhou Medical University (WMU, Wenzhou, China). All mice were given free access to standard rat chow and tap water. The present study was conducted on the basis of the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of WMU.

### bFGF Treatment

All mice were acclimatized for 1 week and then randomly divided into bFGF-treated and control groups at 10 weeks of age. For bFGF group, db/db mice were intraperitoneally (i.p.) injected with bFGF at a dose of 0.5 mg/kg body weight every other day for 10 weeks (Figure 1A). The dose of bFGF treatment was selected according to the previous publication (Tan et al., 2017). Meanwhile, the wt mice and db/db mice in the control group received 0.9% saline using the same schedule.

### Urine and Kidney Sample Collection

Urine samples were individually collected from mice before sacrifice in metabolic cages for 12 h. The urine samples were added with 0.1 ml of 1% sodium azide solution for avoiding bacterial contamination and then centrifuged at 3,000g at 4°C for 10 min. The supernatant was transferred into a new tube and stored at -80°C until analysis. Mice were sacrificed by cervical dislocation at 20 weeks of age, and their renal tissues were isolated, immediately snap-frozen in liquid nitrogen, and kept at -80°C until use.

### Biochemical Analysis and Histopathological Examination

In this study, blood glucose level was measured using the Precision G Blood Glucose Testing System (Abbott Laboratories, Abbott Park, IL). Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed on mice after 10 weeks of bFGF treatment (Figure 1A). After a 12 h fast, mice were orally administered with 50% glucose solution at a dose of 2 g/kg body weight. For ITT, mice were i.p. injected with insulin solution at a dose of 0.35 U/kg body weight after a 4 h fast. Blood glucose levels were measured from the tail vein at time 0, 30, 60, 90, 120, and 150 min. The levels of urea nitrogen (UN), uric acid (UA), urinary albumin to creatinine ratio (UACR) in the urine was determined using an automatic biochemistry analyzer (Mindray BS-300).

For pathologic examination, the kidney tissues were harvested, fixed overnight in 4% paraformaldehyde, and then embedded in paraffin. After deparaffinization and rehydration, the paraffin sections (5 mm) were stained with hematoxylin and eosin (HE) for routine histopathological observations. Periodic acid-Schiff (PAS) and Masson's trichrome staining were used to determine collagen deposition and fibrosis, respectively.

### RNA Extraction, cDNA Synthesis, and Quantitative RT-PCR

Total RNA was extracted from renal tissues with Trizol reagent (Invitrogen, Carlsbad, CA). The purity of RNA was measured by a nanodrop spectrometry (Thermo Fisher Scientific, Beverly, MA) and expressed as the ratio of OD values at 260/280 nm. An OD 260/280 ratio greater than 1.80 represents high RNA purity. Then, RNA was used to synthesize the first-strand cDNAs by the Prime Script™ RT Reagent Kit (TaKaRa, Kusatsu, Japan). The quantification process was carried out in a 10 ml final

0.001 versus wildtype (wt) mice; \*P < 0.05 and \*\*\*P < 0.001 versus db/db mice.

reaction volume using a SYBR Green PCR Master Mix (Bio-Rad, CA, USA). In this study, GAPDH served as an endogenous control, and the primers were synthesized by Sunny Biotechnology (Sunny, Shanghai, China).

### Western Blot Analysis

The renal tissues (30–40 mg) were lysed with RIPA buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, MA). Total protein concentration was measured using the BCA protein assay kit (Bio-Rad, CA, USA). After normalization, equal amounts of proteins were separated by 10% SDS–PAGE and transferred to PVDF membranes (0.45 mm, Millipore, Germany). The membranes were blocked with 5% nonfat milk in TBST for 2 h and incubated overnight with primary antibodies at 4°C. After three washes with TBST, the membranes were incubated with secondary antibodies (Thermo Fisher Scientific, 1:10,000) at room temperature for 1 h. Finally, the blots were washed with TBST three times and incubated using the EasySee western Blot Kit (Transgen Biotech, China) to visualize the immunoreactive bands.

### Sample Preparation and NMR Measurement

1 H NMR spectra were acquired at 25°C on a Bruker AVANCE III 600 MHz NMR spectrometer equipped with a triple resonance probe and a z-axis pulsed field gradient (Bruker BioSpin, Rheinstetten, Germany). Prior to NMR analysis, urine samples were thawed, and 200 ml aliquots of the samples were mixed with 50 ml D2O containing sodium trimethylsilyl propionate-d4 (TSP, 0.36 mg/ml) and 300 ml of phosphate buffer (0.2 M Na2HPO4/ NaH2PO4, pH 7.4) to minimize pH variations (Xiao et al., 2009). The mixtures were centrifuged and then 500 ml of the supernatant was transferred to 5-mm NMR tubes for metabolomics analysis. One-dimensional NOESY pulse sequence with water signal pre-saturation was performed to acquire NMR spectra of urine samples. The main acquisition parameters were set as follows: spectral width = 12,000 Hz; data points = 256 K; relaxation delay = 4 s; acquisition time = 2.66 s per scan.

The frozen renal tissues were weighed into a centrifuge tube. Then, ice-cold methanol (4 ml/g) and distilled water (0.85 ml/g) was added into the tube, homogenized at 4°C after thawing and mixed by vortex for 15 s. Subsequently, ice-cold chloroform (2 ml/g) and distilled water (2 ml/g) was added into the tube and mixed again for 15 s. The mixture were kept on ice for 15 min and centrifuged at 10,000g for 15 min at 4°C. The supernatant was extracted into a new tube and lyophilized for about 24 h. The lyophilized extract was reconstituted in 500 ml D2O containing TSP and transferred to 5 mm NMR tubes for analysis. A onedimensional ZGPR pulse sequence with water signal presaturation was used to acquire NMR data. In addition, the main acquisition parameters were set as follows: data points = 256 K; spectral width = 12,000 Hz; relaxation delay = 4 s; acquisition time = 2.66 s per scan.

### Multivariate Data Analysis and Statistical Analysis

All NMR spectra were manually corrected for phase/baseline and referenced to TSP peak at 0 ppm using TopSpin 3.0 software (Bruker BioSpin, Rheinstetten, Germany). Then, the "icoshift" procedure was employed to align NMR spectra under the MATLAB environment (R2012a, The MathWorks Inc., Natick, MA, USA) (Savorani et al., 2010). The spectral region from 0.0 to 9.0 ppm excluding the residual water signals (4.65–5.05 ppm for kidney extract; 4.78–4.83 ppm for urine) were subdivided and integrated to binning data with a size of 0.01 ppm for further multivariate analysis.

To discriminate metabolic patterns between different groups, partial least squares-discriminant analysis (PLS-DA) was performed using Pareto-scaled NMR data in SIMCA 12.0 software (Umetrics, Umeå, Sweden). Leave one-out cross validation and permutation tests (200 cycles) were used to examine the performance of the model. PLS-DA loading plots were used to identify the important metabolites for the separation of groups. The significance of metabolites in PLS-DA was assessed using the absolute value of the correlation coefficient, |r|, and high |r| value was considered important.

The differences in metabolite levels between two groups were analyzed using independent-samples t-tests with SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). Additionally, the differences in OGTT and ITT between two groups were assessed with repeated measure ANOVA in SPSS 13.0 software. A statistically significant difference was considered when P value <0.05.

### RESULTS

### The Protective Effect of bFGF on Diabetic Nephropathy in db/db Mice

In this study, as expected, db/db mice at 10 weeks of age exhibited a higher level of blood glucose than age-matched wild-type (wt) mice (Figure 1B). Of note, we found that blood glucose level was significantly reduced in db/db mice after 5 and 10 weeks of bFGF treatment, as shown in Figure 1B. Furthermore, OGTT demonstrated that administration of bFGF significantly improved blood glucose clearance in db/db mice (Figures 1C, D). As can be seen from insulin tolerance test, insulin sensitivity was also enhanced in db/db mice with bFGF treatment (Figures 1E, F). To evaluate renal function of mice, we measured UACR, which is a clinical marker of kidney damage and dysfunction (Teimoury et al., 2014). The result shows that the UACR level was significantly increased in db/db mice compared with age-matched wt mice (Figure 1G), whereas bFGF treatment significantly decreased its level in db/db mice. Additionally, uric acid and urea nitrogen are another two hallmarks of renal injury (Barutta et al., 2010). We found that the UA level was significantly reduced in db/db mice relative to age-matched wt mice (Figure 1H). However, interestingly, its level was significantly increased after bFGF treatment and reached to the normal level. In addition, db/db mice had a higher UN level than age-matched wt mice, but this

increase can be recovered after bFGF treatment (Figure 1I). Figure 2A shows that db/db mice displayed notable glomerular hypertrophy and mesangial matrix expansion as compared with age-matched wt mice, while these symptoms were markedly alleviated after bFGF treatment. In addition, Masson trichrome staining revealed a significant increase in renal fibrosis in db/db mice, but this change cannot be observed in db/db mice treated with bFGF. Consistent with our histological findings, the relative mRNA expression levels of profibrotic molecules transforming growth factor (TGF) b1 and type IV collagen were significantly increased in the kidney of db/db mice relative to age-matched wt mice; however, of note, these two indicators were recovered to the normal level after bFGF treatment (Figure 2B). Moreover, bFGF treatment induced parallel changes in the protein expression of these two profibrotic markers in renal tissues (Figures 2C, D). Taken together, these findings revealed that bFGF had a potential protective effect on diabetic nephropathy in db/db mice.

### Changes of Metabolic Phenotypes in the Kidney and Urine in db/db Mice Treated With bFGF

Typical <sup>1</sup> H NMR spectra obtained from the kidney and urine samples of wt mice are illustrated in Figures 3A, B, respectively. In total, 43 metabolites were identified from NMR-based metabolome, mainly including energy metabolism-related metabolites (succinate, 2 oxoglutarate, citrate, creatine, creatinine, lactate, glucose, fumarate, ATP, AMP, and NAD+), amino acids (leucine, isoleucine, valine, alanine, glutamate, aspartate, taurine, glycine, tyrosine, and phenylalanine), methylamine metabolites (methylamine, dimethylamine, trimethylamine, choline, and TMAO), ketobodies (3-hydroxyisovalerate, 3-hydroxybutyrate, and 2-hydroxybutyrate), nucleotide metabolism-related metabolites (uracil, uridine, GTP, and allantion), short-chain fatty acids (formate, acetate, and propionate), and others (GPC, myo-inositol, inosine, niacinamide, 1-methylnicotinamide, hippurate, and 3-indoxylsulfate).

Furthermore, PLS-DA was used to examine changes in metabolic patterns among wt, db/db, and bFGF groups and identify key metabolites. The PLS-DA score plot showed a clear separation among these three groups (Figure 4A), which is validated by a permutation test (Figure 4B). According to its loading plot, a series of metabolites that contribute to this separation were identified, such as lactate, succinate, ATP,

AMP, glutamate, taurine, glycine, tyrosine, uracil, GTP, TMAO, inosine, and niacinamide, as shown in Figure 4C. However, of note, taurine showed the most significant contribution. In addition, PLS-DA score plot based on urine metabolome also showed a clear difference among these three groups (Figure 5A) which is validated by a permutation test (Figure 5B). The corresponding loading plot identified a series of important metabolites, including acetate, succinate, 2-oxoglutarate, citrate, creatine, creatinine, fumarate, hydroxybutyrate, DMA, propionate, allantion, hippurate, and 3-indoxylsulfate (Figure 5C).

### Metabolic Changes in the Kidney and Urine of db/db Mice Treated With bFGF

Figure 6 shows the metabolic pathway changes in db/db mice after bFGF treatment. We found that the levels of creatinine, citrate, ATP, fumarate, TMAO, DMA, and myo-inositol were significantly increased in db/db mice compared with agematched wt mice, but these increased trends were significantly reduced after bFGF treatment (Figure 6). Moreover, db/db mice had significantly lower creatine and taurine levels than agematched wt mice, while bFGF treatment significantly increased their levels, as shown in Figure 6. Relative to age-matched wt mice, we also found that db/db mice had significantly decreased levels of alanine, uracil, acetate, glycine, methylamine, TMA, choline, and glutamate as well as increased levels of fumarate and AMP in the kidney, but no significant alterations were observed after bFGF treatment. Together, these metabolites mainly involved energy metabolism, amino acid metabolism, and methylamine metabolism.

### bFGF Suppress Oxidative Stress in the Kidney of db/db Mice

To investigate whether bFGF can suppress oxidative stress, several relevant markers were detected in the kidney of db/db mice, as shown in Figure 7. As compared with age-matched wt mice, db/db mice had a significantly increased MDA level (Figure 7A) and a significantly decreased SOD level (Figure 7B). However, of note, these two markers were recovered to the normal level after bFGF treatment. In addition, the relative mRNA expression levels of antioxidant biomarkers, including nuclear factor erythroid-2 related factor 2 (Nrf2), NAD(P)H dehydrogenase quinone 1 (NQO1), and superoxide dismutase-2 (SOD2), were significantly down-regulated in the kidney of db/ db mice, while the oxidative marker levels, such as NADPH oxidases 2 and 4 (NOX2 and NOX4), were significantly

increased, as shown in Figure 7C. However, interestingly, these changes were recovered to the normal level after the treatment of bFGF. These results were further validated at the protein level using western blot analysis, where we found that bFGF treatment corrected the decreased levels of Nrf2, NQO1, and SOD2 as well as the increased levels of NOX2 and NOX4 in the kidney of db/db mice (Figures 7D, E). Additionally, we also observed that db/db mice had significantly increased levels of pro-apoptotic proteins (caspase-3, cleaved caspase-3, and Bax) and decreased level of apoptotic protein (Bcl-2) in the kidney relative to age-matched wt mice (Figure 7F). Of note, the recovery of these protein marker levels was obtained after bFGF treatment. Furthermore, we quantified the ratios of cleaved caspase-3/caspase-3 and Bax/ Bcl-2, which have been commonly used to assess cell apoptosis. The results showed that these two ratios were significantly increased in the kidney of db/db mice relative to age-matched wt mice (Figures 7G, H), suggesting an enhanced cell apoptosis. Yet, bFGF treatment can significantly reduce renal cell apoptosis in db/db mice. Figure 8 illustrates that the protective effect of bFGF on diabetic nephropathy may be mediated by correcting metabolic disorders and suppressing oxidative stress in db/ db mice.

### DISCUSSION

Diabetic nephropathy is a common diabetic complication and seriously affect human health worldwide (Yang, 2019), but so far no effective treatment strategies can be available. In the present study, our results reveal that bFGF could be a promising drug for the treatment of diabetic nephropathy, as indicated by reductions in urinary albumin to creatinine ratio and renal fibrosis. To explore its potential therapeutic mechanisms, we performed an NMR-based metabolomics study to analyze metabolic changes in the kidney and urine of db/db mice after bFGF treatment. The results show that bFGF treatment altered metabolic phenotypes in the kidney and urine of db/db mice, mainly involving energy metabolism, methylamine metabolism, osmoregulation, and oxidative stress.

Kidney is an organ with abundant mitochondria and high energy demand, followed by heart in the body (Forbes, 2016). Therefore, diabetic nephropathy is usually accompanied by abnormal energy metabolism (Zhong et al., 2012; Wei et al., 2015; Tanaka et al., 2018). In the present study, relative to agematched wt mice, we found a disordered energy metabolism in db/db mice, as indicated by significantly increased levels of

tricarboxylic acid (TCA) cycle intermediates, such as citrate, fumarate, ATP, and AMP. Interestingly, abnormal energy metabolism can be recovered by bFGF treatment. Mitochondrial damage was considered as the hallmark of kidney injury (Galvan et al., 2017), and the TCA intermediates were identified as the potential biomarkers for impairment of mitochondrial function in the kidney of diabetic animals (Zhao et al., 2011; Jiang et al., 2019). We thereby hypothesize that bFGF has a protective effect on renal mitochondrial function. Creatinine, as an indicator of renal function, is formed exclusively from creatine in the body, and creatine is a nitrogen-containing organic acid that provides energy for maintaining normal cellular physiology (Akira et al., 2012). In this study, we found a significantly higher metabolic rate from creatine to creatinine in db/db mice compared with age-matched wt mice, but notably this abnormal metabolism can be recovered in db/db mice after bFGF treatment. Taken together, our results reveal that the protective effect of bFGF on diabetic nephropathy in db/db mice may be associated with energy metabolism regulation. In addition, amino acid metabolism is necessary to protein synthesis for cell growth and also supports for energy metabolism. In this study, we found a significant reduction of amino acid metabolism in the kidney of db/db mice relative to age-matched wt mice, as indicated by decreases in alanine, glycine, and glutamate. Yet, their levels were not restored after

the treatment of bFGF, which may imply that the protective effect of bFGF on diabetic nephropathy could be not achieved through amino acid metabolism.

Methylamine metabolites are produced from the degradation of dietary choline to TMA, TMAO, and DMA by the gut microflora (Messenger et al., 2013). In this study, db/db mice had significantly higher DMA and TMAO levels than agematched wt mice, whereas their levels were significantly decreased by bFGF treatment, indicating a recovery of methylamine metabolism. TMAO has been reported to be closely associated with decreasing renal function (Mueller et al., 2015; Tang et al., 2015; Missailidis et al., 2016). Tang et al. also found that chronic dietary TMAO exposure directly resulted in progressive renal fibrosis and dysfunction in animal models. Thus, our results suggest that methylamine metabolism may contribute to the reno-protective effect of bFGF in db/ db mice.

Myo-inositol as an osmolyte plays an important role in normal cellular physiology (Carlomagno et al., 2012). Previous studies have reported that myo-inositol could be an indicator for tubular dysfunction and renal cell stress under hyperglycemia, and has a key role in the etiology of diabetes mellitus, particularly for diabetic nephropathy (Prabhu et al., 2005; Kanwar et al., 2008). Gil et al. reported that in the level of urinary myo-inositol excretion indicated severity of chronic kidney disease (Gil et al.,

2018). In the current study, we found a significantly increased myo-inositol level in the kidney of db/db mice relative to agematched wt mice; however, this abnormal increase can be significantly reduced after bFGF treatment. This finding suggests that the protective effect of bFGF on diabetic nephropathy in db/db mice may be implicated in myo-inositolmediated mechanisms such as osmoregulation.

It is worth noting that taurine was identified to have the greatest contribution to bFGF-induced metabolic changes. Taurine is one of abundant free amino acids in mammals' tissues and implicated in oxidative damage, endoplasmic reticulum stress, and apoptosis (Timbrell et al., 1995; Jong et al., 2017). Hence, taurine has been reported to protect against kidney injure (Adedara et al., 2017; Stacchiotti et al., 2018; Adedara et al., 2019). In the present study, db/db mice had a significantly lower level of renal taurine than age-matched wt mice, but its level was significantly increased after bFGF treatment. Our results also reveal that bFGF treatment corrected the decreased level of SOD, an antioxidant marker, and the increased level of MDA, a marker of lipid peroxidation, in the kidney of db/db mice. Additionally, significant reductions in apoptosis indicators were also detected in the kidney of db/db mice treated with bFGF. These findings suggest that the protective effect of bFGF on diabetic nephropathy may be also associated with taurine-mediated oxidative stress and apoptosis. Oxidative stress can increase ROS production and impair antioxidant capacity because of the generation of ROS exceeding the capacity of antioxidant defense system (Wilcox, 2005). Moreover, oxidative stress is believed to play an important role in cell apoptosis (Buttke and Sandstrom, 1994; Chandra et al., 2000) and also associated with diabetic complications (Ruiz et al., 2013; Yang et al., 2015). Nrf2 is a critical transcription factor to maintain redox homeostasis and regulates the expression of antioxidative stress genes including NQO1 and SODs (Dodson et al., 2019). It has been reported that hyperglycemia can induce oxidative stress and increase kidney injure via Nrf2 signaling (Wu et al., 2018). However, interestingly, our results show that bFGF treatment corrected the decreased levels of Nrf2, NQO1, and SOD2 in the kidney of db/db mice. In addition, NADPH oxidases (NOX2 and NOX4)

are a family of enzymes that catalyze the generation of ROS, and have been linked with the development of kidney disease (Holterman et al., 2015). In this study, we found that the levels of NOX2 and NOX4 were significantly increased in the kidney of db/db mice relative to age-matched wt mice, but bFGF treatment recovered their levels to the normal level. Therefore, these findings reveal that bFGF may ameliorate oxidative stress and apoptosis during the development of diabetic nephropathy in db/ db mice via NOX-ROS-Nrf2 signaling.

## CONCLUSION

In this study, bFGF treatment can effectively reduce urinary albumin to creatinine ratio and renal fibrosis as well as remodel metabolic phenotype in db/db mice. Metabolomics analysis identified that taurine, as an antioxidative metabolite, had the greatest contribution to bFGF-induced metabolic changes. Additionally, we found that bFGF treatment alleviated oxidative stress and apoptosis in the kidney of db/db mice though NOX-ROS-Nrf2 signaling. However, several limitations or further works should be considered: (1) The biological activities of bFGF are mediated by FGF receptors, so changes in bFGF and its receptors need to be further analyzed in the kidney; (2) A multi-analytical platform is recommended to detect more detailed metabolic pathway changes; (3) A multiomics analysis will advance a better understanding of the therapeutic effect of bFGF on diabetic nephropathy and its potential mechanisms. Further translational studies are encouraged to modify bFGF structure for excluding its potent mitogenic and angiogenic activities, and facilitate its clinical application for the treatment of diabetic nephropathy in humans.

## DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ supplementary material.

## ETHICS STATEMENT

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University.

## AUTHOR CONTRIBUTIONS

HG and HZ contributed to experimental design. TW, JN, and SW contributed to animal experiments and metabolomics analysis. TW, QS, CL, and LZ contributed to molecular biology experiments and data analysis. HZ and TW contributed to result interpretation and writing. All authors have read, revised, and approved the final manuscript.

### FUNDING

This work was supported by the National Natural Science Foundation of China (nos. 81600653, 21605115, and 21974096).

## REFERENCES


in chronic kidney disease. Am. J. Physiol. Renal Physiol. 306 (4), F430–F441. doi: 10.1152/ajprenal.00720.2012


formulation approach to preclinical study. Drug Deliv. 26 (1), 849–859. doi: 10.1080/10717544.2019.1642417


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Wei, Shu, Ning, Wang, Li, Zhao, Zheng and Gao. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Progress in Research on the Role of FGF in the Formation and Treatment of Corneal Neovascularization

Mengji Chen, Licheng Bao, Mengying Zhao, Jiarong Cao and Haihua Zheng\*

Department of Ophthalmology, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China

Corneal neovascularization (CNV) is a sight-threatening disease usually associated with inflammatory, infectious, degenerative, and traumatic disorders of the ocular surface. Fibroblast growth factor (FGF) family members play an important role in angiogenesis to induce corneal neovascularization, which significantly affects the differentiation, proliferation, metastasis, and chemotaxis of vascular endothelial cells. Both acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) demonstrate positive staining in capillaries and induce corneal stromal cells. The anabolism of endothelial cells is induced by bFGF in corneal neovascularization. FGFs exert their effects via specific binding to cell surface-expressed specific receptors. We believe that both anti-FGF antibodies and anti-FGF receptor antibodies represent new directions for the treatment of CNV. Similar to anti-vascular endothelial growth factor antibodies, subconjunctival injection and eye drops can be considered effective forms of drug delivery.

#### Edited by:

Saverio Bellusci, University of Giessen, Germany

### Reviewed by:

Elie El Agha, University of Giessen, Germany Tingting Yuan, University of Alabama at Birmingham, United States

#### \*Correspondence:

Haihua Zheng eyezhh@126.com

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 23 October 2019 Accepted: 28 January 2020 Published: 25 February 2020

#### Citation:

Chen M, Bao L, Zhao M, Cao J and Zheng H (2020) Progress in Research on the Role of FGF in the Formation and Treatment of Corneal Neovascularization. Front. Pharmacol. 11:111. doi: 10.3389/fphar.2020.00111 Keywords: corneal neovascularization, fibroblast growth factor, development, treatment, drug delivery

### CORNEAL NEOVASCULARIZATION

The incidence rate of corneal neovascularization (CNV), a sight-threatening condition, is approximately 1.4 million patients per year (Bonini, 2016). It is usually associated with inflammatory, traumatic, or infectious disorders of the ocular surface. Corneal neovascularization can cause vision damage and even blindness, the rate of which is as high as 57.4% (Chang et al., 2001). The cornea needs to be transparent to allow the passage of light into the retina. When CNV occurs, abnormal blood vessels directly block light, indirectly diffract light as canals for inflammatory cells, and damage the structure of the cornea by depositing lipids and proteins into the corneal stroma. Various mechanisms of corneal neovascularization have been studied. A balance exists between angiogenic factors (such as fibroblast growth factor and vascular endothelial growth factor) and anti-angiogenic factors (such as angiostatin, endostatin, and pigment epitheliumderived factor) in the cornea (Senturk et al., 2016). An imbalance leads to CNV, which can be divided into superficial neovascularization and deep stromal neovascularization. In the normal cornea, heparan sulfate prevents the release of potent angiogenic cytokines, such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). However, under stimulation, cytokines may be released, causing an imbalance (Gurung et al., 2018).

#### Chen et al. FGF in the Development and Treatment of Corneal Neovascularization

### FGF

### FGF

Fibroblast growth factor participates in cell proliferation, migration, and tissue repair in adults (Itoh and Ornitz, 2004). FGFs can stimulate many cell types, including endothelial cells and nerve cells. The FGF family comprises 22 members (FGF1- 23, except FGF15 because mouse FGF15 is the orthologue of human and chick FGF19) (Beenken and Mohammadi, 2009). Many of these FGFs, particularly FGF-1 (acidic FGF) and FGF-2 (basic FGF), have been shown to influence angiogenesis in several tissues in vivo (Table 1) by acting on endothelial cells. Different factors affect FGFs in different tissues. FGFs are produced by endothelial cells and are stored in the extracellular matrix. They show a high affinity for heparin (Klagsbrun, 1990).

FGFs activate transmembrane tyrosine kinases and their coupled intracellular signalling pathways to stimulate biological activities and transduce external signals through the PI3K, MAPK, and phospholipase Cg (PLCg) pathways (Klagsbrun, 1990; Zhou et al., 2009; Francavilla et al., 2013; Huang et al., 2016). A dual-receptor system comprising a family of four receptor tyrosine kinases (FGFRs) and heparan sulfate proteoglycans (HSPGs) mediates the stimulation of cellular metabolism by FGFs. Thus, many of the FGFR isoforms bind several FGFs. HSPG receptors may provide additional specificity (Klagsbrun, 1990). ERK1/2, JNK1/2, and p38 alpha/beta are three main MAPK subgroups (Zhang et al., 2009). Phospholipase C can receive the signal from FGFRs and then activate PLCg1 to induce cell proliferation and migration in vascular smooth muscle cells by cleaving the phospholipid phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-triphosphate (Michel, 1998). Studies have suggested that heparan may modulate the activity of FGFs (Ornitz, 2000). Additionally, studies have suggested that membrane-type 1 matrix metalloproteinase (MT1-MMP) plays

TABLE 1 | Fibroblast growth factors (FGFs) associated with angiogenesis.


an important role in modulating FGF-mediated signal pathways (Zhou et al., 2000). It controls FGF signalling by reducing the amount of FGF bound to the cell surface, increasing the proteolysis of FGFs and the downregulation of FGFR-1 and -4 (Tassone et al., 2015).

### FGFs in the Eye

mRNAs encoding basic FGF are produced by corneal epithelial cells, stromal fibroblasts, and corneal endothelial cells (Oladipupo et al., 2014). FGF-2 may have different functions in the three primary cell types of the cornea. Studies have shown that in corneal epithelial cells, FGF-2 stimulates the proliferation of corneal epithelial cells and increases the rate of epithelial wound healing through autocrine and paracrine effects (Kanayama et al., 2007). Rajesh et al. showed that FGF-2 promotes corneal stromal wound healing by increasing cellular proliferation (stimulated by TGF-beta via paracrine effects) (Moioli et al., 2006) and cellular motility (significant enhancement of stromal fibroblast motility by 100 ng/ml FGF-2 in animal experiments) (Rao et al., 1992). Cdc42 activation, Rho inactivation, and the phosphatidylinositol 3-kinase pathway in corneal endothelial cells (CECs) are essential for FGF-2 induced wound healing (Lee and Kay, 2006), and endothelial mesenchymal transformation can be mediated by FGFs in CECs. Additionally, FGFs can mediate the proliferation and regeneration of the lens (FGF-1 and FGF-2) (Zhu et al., 2012) and retinal cells (FGF-2, FGF-5, and FGF-9) through different signalling pathways (Gong et al., 2014). FGF-9, FGF-21, and FGF-23 are also expressed in choroidal endothelial cells and affect choroid plexus epithelial cell behaviour (Loren et al., 2009; de Oliveira Dias et al., 2011).

FGFs also play an important role in early mammalian eye development. The neuroepithelium of the optic vesicle separates into NR and RPE domains in a manner mediated by extrinsic factors that emanate from the surface ectoderm, for which fibroblast growth factors are prime candidates (Bassnett and Sikic, 2017).

### FGFS AND RELATED CORNEAL DISEASES

Many FGFs affect angiogenesis, but FGF-2 is the most common isoform to be detected in the eye and to cause CNV in different eye diseases. One of these diseases is ocular chemical burn. We focused on alkali burns because of their severity. CNV can be caused by severe corneal alkali burn (Nominato et al., 2018), in which deep corneal stroma or full-thickness corneal injury is involved. In this situation, angiogenic factors play an important role in CNV. For example, experimental data have shown that on the second day after alkaline burn, b-FGF is obviously expressed in the corneal epithelium, substantia propria layer, and endothelium (Xiao et al., 2012). Additionally, studies have shown that the upregulation of related genes (microRNA-296) after alkali burn is positively correlated with the expression of related FGF isoforms (FGF-23). FGF-23 may influence corneal inflammatory responses by participating in cytokine-cytokine receptor interaction pathways (Hayashi et al., 1996).

Keratitis is another important cause of corneal neovascularisation (Soiberman et al., 2017). In the initial stage of infection with herpes simplex virus type 1 (HSV-1), both virus and immune cells are present in the cornea. Meanwhile, various cytokines and growth factors (FGF-2 and Ang-2), which also permeate the cornea, lead to further inflammation. The origin of FGF-2 may not fibroblasts (keratocytes), epithelium, endothelium, blood endothelial cells, and lymphatic endothelial cells rather than leukocytes (Xu et al., 2019). In the late stage, immune cells and growth factors continue to be effective while the virus is removed from the cornea. Furthermore, FGF-2 mediates the expression of other cytokines (such as VEGF-A, IL-6, and Ang-2), which are crucial for HSV-1-induced corneal neovascularization (Klagsbrun, 1990).

Patients who undergo corneal transplantation can develop corneal neovascularization. Particularly, after high-risk keratoplasty, intense CNV outgrowth is a common phenomenon in the early postoperative period (Kelly et al., 2011). Corneal neovascularization can lead to an increased risk of graft rejection caused by an imbalance between angiogenic factors and anti-angiogenic factors. Additionally, FGF is an important angiogenic factor. Presently, no study has shown how FGFs cause CNV after corneal transplantation and which FGF isoform is involved, but this phenomenon warrants further research.

### ROLE OF FGFS IN CNV FORMATION

Angiogenesis is the process by which new blood vessels grow by sprouting from established blood vessels (Carmeliet and Jain, 2011). In the cornea, these existing blood vessels can be part of the vascular plexus around the limbus of the anterior ciliary artery. Corneal angiogenesis occurs because of the release of proangiogenic factors, such as FGF-2, VEGF, and several other chemokines, from hypoxic or inflammatory cells. The binding of angiogenic factors to corresponding receptors on vascular endothelial cells leads to many events, including the following: (a) injury to endothelial cell junctions through the activation of non-receptor Src family kinases and increased expression of integrins; (b) the promotion of endothelial cell proliferation by mitogen-activated protein kinase and phosphoinositide 3' kinase; (c) the secretion of metalloproteinases (MMPs) by endothelial cells to promote basal membrane disruption and pericyte detachment; and (d) blood vessel destabilization caused by the release of angiopoietin 2 (ANG 2) from endothelial cell granules (Figure 1). Additionally, murine tissue inhibitor of metalloproteinase-4 (TIMP-4) expression in the cornea may play a role in regulating extracellular matrix remodelling associated with corneal wound healing and angiogenesis; however, the mechanism is unclear.

Vascular endothelial cells are stimulated directly by FGFs from angiogenic tissues. FGF-producing cells also release FGFs through autocrine or nuclear actions (Figure 1), and secondary angiogenic factors then act on the vasculature. The vascular endothelium can induce the secretion of secondary regulatory molecules in the same way, and such secretion can also be influenced by other angiogenic factors, such as VEGF. The specific role of FGFs depends on the environment in which they are located (Beenken and Mohammadi, 2009).

### PROGRESS IN FGF RESEARCH FOR CNV TREATMENT

### Current Effective Treatments for CNV

Presently, laser treatment, drug treatment, and surgical treatment are available to treat corneal neovascularization. Drugs used for treatment include glucocorticoids, immunosuppressive agents, and various vascular growth factor inhibitors. While the topical administration of steroidal anti-inflammatory drugs is the firstline treatment for corneal neovascularization, many side effects

autocrine or nuclear actions during events such as trauma and hypoxia. (E) Limbus capillaries: activation of Src family kinases.

occur during long-term application (Mukwaya et al., 2019). Laser treatment is less effective for the dense mesh type of CNV. Corneal transplant surgery may also cause various complications, including HSV-1 infection of the cornea.

A novel therapy targeting angiogenic cytokines may have therapeutic potential for clinical use in the future, and it has been shown to inhibit corneal vascularization effectively in animal models (Klagsbrun, 1990). Repeated subconjunctival injections of Avastin® (the trade name of bevacizumab; 1.25 mg/0.05 ml) and topical cyclosporin-A drops appear to be safe and effective in treating aggressive corneal vascularization (Akpek et al., 2004; Stevenson et al., 2012). Subconjunctival bevacizumab injections were shown to be effective and safe in reducing corneal neovascularization within the first 4 months (Jacobs et al., 2009). Recent research has shown that bevacizumab can also be administered by corneal intrastromal injection for the inhibition of intrastromal vascularization after deep anterior lamellar keratoplasty (Sun et al., 2019). Additionally, there are few corneal epithelial side effects when bevacizumab eye drops are used to treat corneal neovascularization (Baradaran-Rafii et al., 2019). Anti-angiogenic effects and anti-fibrotic effects for the maintenance of corneal transparency have also been observed after the use of bevacizumab eye drops (25 mg/ml) for corneal burn (Koenig et al., 2012). However, only a few related studies using eye drops exist, and further verification is needed.

### Comparison of Anti-FGF and Anti-VEGF

Anti-VEGF drugs include recombinant full-length VEGF monoclonal antibodies (Avastin), recombinants of VEGF subtype monoclonal antibody fragments (Lucentis), and RNA aptamers (Macugen). The mechanism of these drugs involves binding to VEGF and inhibiting the specific binding of VEGF to its receptor. Recent observations have shown that the soluble pattern recognition receptor long-pentraxin-3 binds FGF2 with high affinity and specificity and thus acts as an FGF2 antagonist (Presta et al., 2018). The novel RNA aptamer (a short singlestranded nucleic acid molecule) APT-F2, which is specific for human FGF2, was also recently discovered as an anti-FGF drug (Jin et al., 2016), but it has not been applied in anti-angiogenic studies. More types of anti-FGF drugs are yet to be discovered. The mechanism of anti-FGF drugs is similar to that of anti-VEGF drugs.

Studies have demonstrated that bFGF expression induces VEGF, MT1-MMP, and CD31 in the experimental mouse cornea (Nor et al., 2001; Mimura et al., 2011). Therefore, FGF treatment may be more comprehensive. Researchers believe that angiogenesis induced by basic fibroblast growth factor (bFGF) is immune to anti-VEGF/VEGFR (vascular endothelial growth factor/receptor) therapy (Koolwijk et al., 1996). Furthermore, patients may develop tolerance to anti-VEGF drugs. Experimental evidence suggests that FGF2-targeted drugs might provide cooperative effects with anti-VEGF monoclonal antibodies for the treatment of angiogenesis-related diseases. Another important advantage of anti-FGF drugs is that they may be beneficial for the recovery of vision. Researchers established a model of anti-FGF-2 antibody-treated mouse corneal neovascularization and then evaluated the corneal sensitivity and visual acuity of these mice to assess their functional vision. Time-course experiments revealed partial recovery of visual acuity in mice treated with an anti-FGF-2 antibody compared with control mice (Zheng et al., 2001). This finding supports anti-VEGF therapy for corneal neovascularization, but further experimental confirmation is needed.

### FGF as a Drug Source and the Administration Route

Several anti-FGF2 monoclonal antibodies (mAbs) that can neutralize the activities of FGF2 in vitro and in vivo have been identified (Jin et al., 2016). However, to our knowledge, no anti-FGF2 mAb has entered clinical trials. Because of the similarity between VEGF and FGF antagonists, we can presume the likely effect of FGF antagonists in treating CNV based on the effects of anti-VEGF drugs.

A previous study demonstrated that the subconjunctival injection of anti-VEGF drugs (1.25 mg/0.05 ml bevacizumab) significantly reduces inflammation and fibroblast activity to inhibit corneal neovascularization in the cornea of a rat model of alkali burn. Rats in the test group were treated with a subconjunctival injection (Kim et al., 2013). Another researcher advocated combining subconjunctival (1.25 mg/0.05 ml) and intracorneal injection (1.25 mg/0.05 ml). They confirmed that combining subconjunctival and intracorneal injection did not damage corneal endothelial cells (Lichtinger et al., 2014). No clinical case featuring the administration of anti-FGF drugs through subconjunctival and intracorneal routes for the treatment of CNV exists, but we can refer to these modes of administration. Therefore, the drug concentration and side effects remain unclear, and further research is needed.

Recently, research on new eye drops has progressed. Considering the effects of anti-VEGF drugs, in the future, Avastin eye drops may be shown to exhibit the same anti-VEGF effects with fewer complications than intravitreal use (Kim et al., 2013). Bevacizumab eye drops seem to inhibit corneal neovascularization without inducing obvious corneal epithelial side effects. Bevacizumab eye drops are prepared by adding 0.9% physiological saline to the drug at a concentration of 5 mg/ml. The minimum storage temperature of the eye drops is – 20°C (before opening), the maximum storage temperature is 4°C (after opening), and the maximum storage time is 14 days; after opening, they needs to be used within 1 day (Bock et al., 2008). Therefore, we believe that preparations of anti-FGF drugs as eye drops can be applied for the clinical treatment of corneal neovascularization. The effects of the FGF antagonist tecogalan sodium against corneal neovascularization were tested in animal experiments, and tecogalan sodium demonstrated dosedependent antiangiogenic activity. The inhibitory effect of 100 ng terconazole sodium was weakly, and the effect of 250 ng was marked, indicating that corneal neovascularization induced by bFGF can be inhibited by the topical instillation of tecogalan sodium (Murata et al., 1995). However, no data regarding the recommended dose of other anti-FGF drugs for the treatment of CNV are available. It is possible that we need to start with a low dose and simultaneously test the corneal toxicity of the drug.

### SUMMARY

Corneal neovascularization is a pathological change in the cornea that blocks light, leads to inflammation and edema, and causes corneal scarring in severe cases. Ultimately, it leads to a serious decline in vision. When the balance between angiogenic and antiangiogenic factors shifts towards the former, corneal neovascularization occurs. FGF, particularly bFGF, plays a very important role in corneal neovascularization due to various factors. However, its exact role and mechanism require further research. Therefore, anti-FGF drugs can be used as new candidates for treating corneal neovascularization. Presently, steroidal anti-inflammatory drugs are the first-line therapy for corneal neovascularization. New types of drugs with fewer side effects need to be developed; anti-VEGF drugs are one of the candidates. Compared with anti-VEGF drugs, anti-FGF drugs have advantages. The discovery of additional FGF antagonists and the route of administration of anti-FGF drugs will become a new research direction. The authors believe that the

### REFERENCES


subconjunctival and corneal stroma injection of anti-FGF drugs and anti-FGF eye drops will provide new strategies to treat corneal neovascularization.

### AUTHOR CONTRIBUTIONS

MC and HZ participated in drafting the manuscript. LB, MZ, and JC provided technical assistance. MC and HZ revised the manuscript. HZ supervised the project and provided financial support. MC and HZ wrote the main part of the paper. All of the authors read and approved the final manuscript.

### ACKNOWLEDGMENTS

This study was supported by all members of the Ophthalmology Department of the Second Affiliated Hospital of Wenzhou Medical University and by the Zhejiang Natural Science Foundation (LY18H120009). The authors also thank Professor Qianying Gao from the State Key Laboratory of Sun Yatsen University.


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Chen, Bao, Zhao, Cao and Zheng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# FGF21 Attenuated LPS-Induced Depressive-Like Behavior via Inhibiting the Inflammatory Pathway

Xue Wang1,2† , Liyun Zhu1† , Jian Hu<sup>1</sup> , Ruili Guo<sup>1</sup> , Shasha Ye<sup>1</sup> , Fei Liu<sup>1</sup> , Dongxue Wang<sup>1</sup> , Yeli Zhao1,2, Aiping Hu1 , Xiaojie Wang1,2, Kaiming Guo1\* and Li Lin1,2\*

<sup>1</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Engineering Laboratory of Zhejiang Province for Pharmaceutical Development of Growth Factors, Biomedical Collaborative Innovation Center of Wenzhou, Wenzhou, China

#### Edited by:

Jin-San Zhang, Mayo Clinic, United States

### Reviewed by:

Ying Peng, Chinese Academy of Medical Sciences and Peking Union Medical College, China Yun Seon Song, Sookmyung Women's University, South Korea

#### \*Correspondence:

Kaiming Guo gkm6662@163.com Li Lin linliwz@163.com

† These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 23 October 2019 Accepted: 05 February 2020 Published: 28 February 2020

#### Citation:

Wang X, Zhu L, Hu J, Guo R, Ye S, Liu F, Wang D, Zhao Y, Hu A, Wang X, Guo K and Lin L (2020) FGF21 Attenuated LPS-Induced Depressive-Like Behavior via Inhibiting the Inflammatory Pathway. Front. Pharmacol. 11:154. doi: 10.3389/fphar.2020.00154 Major depressive disorder is a serious neuropsychiatric disorder with high rates of recurrence and mortality. Many studies have supported that inflammatory processes play a central role in the etiology of depression. Fibroblast growth factor 21 (FGF21), a member of the fibroblast growth factors (FGFs) family, regulates a variety of pharmacological activities, including energy metabolism, glucose and lipid metabolism, and insulin sensitivity. In addition, recent studies showed that the administration of FGF21, a regulator of metabolic function, had therapeutic effects on mood stabilizers, indicating that FGF21 could be a common regulator of the mood response. However, few studies have highlighted the antidepressant effects of FGF21 on lipopolysaccharide (LPS) induced mice, and the anti-inflammatory mechanism of FGF21 in depression has not yet been elucidated. The purpose of the current study was to determine the antidepressant effects of recombinant human FGF21 (rhFGF21). The effects of rhFGF21 on depression-like behaviors and the inflammatory signaling pathway were investigated in both an LPS-induced mouse model and primary microglia in vitro. The current study demonstrated that LPS induced depressive-like behaviors, upregulated proinflammatory cytokines, and activated microglia in the mouse hippocampus and activated the inflammatory response in primary microglia, while pretreatment with rhFGF21 markedly improved depression-like behavior deficits, as shown by an increase in the total distance traveled and number of standing numbers in the open field test (OFT) and a decrease in the duration of immobility in the tail suspension test (TST) and forced swimming test (FST). Furthermore, rhFGF21 obviously suppressed expression levels of the proinflammatory cytokines interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) and inhibited microglial activation and the nuclear factor-kB (NF-kB) signing pathway. Moreover, coadministration of rhFGF21 with the fibroblast growth factor receptor 1 (FGFR1) inhibitor PD173074 significantly reversed these protective effects, indicating that the antidepressant effects of rhFGF21 occur through FGFR1 activation. Taken together, the results of the current study demonstrated for the first time that exogenous rhFGF21 ameliorated LPS-induced depressive-like behavior by inhibiting microglial expression of proinflammatory cytokines through NF-kB suppression. This new discovery suggests rhFGF21 as a new therapeutic candidate for depression treatment.

Keywords: major depressive disorder, depressive-like behavior, FGF21, inflammation, NF-kB signaling pathway

### INTRODUCTION

Major depression disorder (MDD), a mood disorder characterized by the symptoms of a persistent feeling of sadness, loss of interest, and worthlessness, affects approximately 300 million people worldwide (Zhang et al., 2019). A total of 0.8 million patients with depression commit suicide annually (Lee et al., 2019). There are some clinical antidepressants on the market, such as norepinephrine reuptake inhibitors, selective serotonin reuptake inhibitors, and monoamine oxidase inhibitors; however, only one-third of patients respond to these therapeutics (Li, 2019), which are often associated with numerous side effects and a high risk of relapse after drug withdrawal, such as the 61.8% relapse rate in the case of fluoxetine (Andrews et al., 2012; Lee et al., 2019). Therefore, further study to develop novel effective antidepressants is urgently needed.

Several hypotheses to explain the pathology of depression, including glutamatergic excitotoxicity, monoamine system impairment, hypothalamic-pituitary-adrenal axis dysfunction, neuroinflammation, and neural plasticity and neurogenesis disruption, have emerged (Taniguti et al., 2019). Among these hypotheses, inflammatory processes have been suggested to be involved in the etiology of depression in many studies (Domingues et al., 2018). Increasing amounts of preclinical and clinical research have shown that proinflammatory cytokines might contribute to depression. The proinflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) play critical roles in the process of inflammation and can induce depressive disorders (Wright et al., 2005; Guo et al., 2019). The levels of IL-6 and TNF-awere reported to be increased in the blood and cerebrospinal fluid of MDD patients (Syed et al., 2018). Escherichia coli lipopolysaccharide (LPS), a commonly used proinflammatory endotoxin, can trigger microglial activation and induce immune activation and behavioral changes that are similar to the clinical symptoms of human depression (Adzic et al., 2015; Taniguti et al., 2019). Therefore, mice were administered LPS to serve as a model of central nervous system (CNS) inflammation and induce depression-like behaviors. Nuclear factor-kB (NF-kB), a major transcription factor, is involved in the activation of an exceptionally large number of genes in response to inflammation. Once stimulated by LPS, NF-kB translocates to the nucleus and regulates the expression of inflammatory cytokines such as TNF-a, interleukin-1b (IL-1b), and IL-6 (Lin et al., 2007; Domingues et al., 2018; Muhammad et al., 2019).

Fibroblast growth factor 21 (FGF21), a member of the fibroblast growth factor (FGF) family, is mainly expressed in the liver and functions as a hormone. After its secretion, FGF21 regulates a variety of pharmacological activities; whole-body energy metabolism, especially glucose and lipid metabolism; and insulin sensitivity (Xu et al., 2009; Gaich et al., 2013; Pan et al., 2018; Li, 2019). In addition, recent studies showed that the administration of FGF21, a regulator of metabolic function, had a therapeutic effect on mood stabilizers (Chang et al., 2018). In bipolar disorder (BD) patients, the FGF21 level was significantly enhanced after valproate treatment; however, there was no significant difference between the FGF21 levels of healthy control and BD patients, indicating that FGF21 may be a common regulator of the mood response (Chang et al., 2018). Moreover, a previous study reported a significant negative association between cerebrospinal fluid FGF21 levels and Beck Depression Inventory (BDI) scores in male Chinese subjects, but not in female (Liu et al., 2017). These results further demonstrate that FGF21 plays a role in mood regulation; however, the mechanism by which FGF21 mediates mood disorders is not clear.

In the current study, the effects of recombinant human FGF21 (rhFGF21) on the depression-like behaviors of LPS-induced mice models were evaluated. The NF-kB signaling pathway is involved in inflammatory events. Once stimulated by LPS, NFkB is phosphorylated and activated, and phosphorylated NF-kB translocates into the nucleus and binds a consensus sequence in targeted genes to regulate the expression of inflammatory cytokines such as TNF-a, IL-6, and IL-1b (Yang M. et al., 2018). Therefore, we speculated that the anti-depressive effect of rhFGF21 is mediated by NF-kB signaling pathway regulation. To confirm this hypothesis, we evaluated the expression levels of inflammatory cytokines in the hippocampus of an LPS-induced depression-like model and in primary microglia. Additionally, the NF-kB signaling pathway was analyzed to further explore the possible underlying mechanism of the antidepressant effects of rhFGF21.

### MATERIALS AND METHODS

### Reagents and Antibodies

rhFGF21 was supported from Key Laboratory of Biopharmaceutical, School of Pharmaceuticals Sciences, Wenzhou Medical University, that is produced and purified from Escherichia according to the reference (Wang et al., 2010). FGFR1 inhibitor PD173074 was purchased from Selleckchem (Houston, TX, USA). LPS was obtained from Sigma (Sigma-Aldrich, St Louis, MO). The following primary antibodies applied were purchased from Abcam (Cambridge, MA, USA): anti-FGFR1 (No. ab824), anti-p-FGFR1 (No. ab59194), and anti-b-actin (No. ab8227); anti-NF-kB (No. 8242) and anti-p-NF-kB (No. 3033s) were purchased from Cell Signalling Technology (Danvers, MA, USA); anti-BDNF (No. BS9896M) and anti-Iba1 (No. 019-19741) were purchased from Bioworld technology (Louis Park, MN, USA) and FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), respectively. The secondary antibodies used in this study were goat anti-rabbit IgG H&L (HRP) (No. ab6721) and Alexa Fluor ®488-conjugated donkey anti-rabbit (No. ab150073) purchased from Abcam (Cambridge, MA, USA).

### Animals

The experiments were conducted in male C57BL/6N mice (20– 25 g), which were purchased from the Animal Center of the Chinese Academy of Sciences (Beijing, China). The animal use and care protocol conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and was approved by the Animal Care and Use Committee of Wenzhou Medical University.

### Drug Administration and Animal Experimental Procedures

The experimental design and protocol used for the animal experiment in this study are illustrated in Figure 1A. The mice were divided into three groups randomly; (1) the control vehicle (normal saline, NP) administered group; (2) the LPS + NP treated group; (3) the LPS + rhFGF21 treated group. Mice were pretreated intraperitoneally (i.p.) with vehicle or rhFGF21 (0.75, 1.5, and 3 mg/kg) twice daily for three consecutive days. These doses were chosen because it has previously been shown that rhFGF21 with the dose of 1.5 mg/kg dramatically attenuated locomotor function deficits in mice (Jiang et al., 2018). One hour after the last rhFGF21 administration on day 3, the mice were treated i.p. with LPS (0.83 mg/kg) dissolved in sterile saline. The concentration of LPS was based on the results of previous studies (Tomaz et al., 2014; Taniguti et al., 2019; Wang et al., 2019a; Wang et al., 2019b). The animals were subjected to following behavioral tests 24 h after LPS. Then, the animals were deeply anesthetized with isoflurane and euthanized by decapitation. The hippocampal tissue was rapidly removed and stored at -80°C until assays. NF-kB, iNOS, and brain-derived neurotrophic factor (BDNF) protein levels were assessed by western blot analysis (n = 5). Levels of the proinflammation factors IL1-b, TNF-a, and IL-6 were determined by RT-PCR (n = 5). To detect Iba1, NF-kB, and FGFR1 by immunofluorescence localization (n = 4), mice were deeply anesthetized and subjected to cardiac perfusion with saline followed by perfusion with 4% paraformaldehyde (PFA). Their brains were rapidly removed

and post-fixed in 4% PFA overnight for further immunofluorescence analysis (Figure 1A).

### Behavioral Assay

Depression behavior was monitored by increased duration of immobility in the forced swim test (FST) and decreased sucrose preference; meanwhile, sickness behavior was measured by body weight loss, reduced food intake, and locomotor activity. It has been reported that LPS-induced depression-like behaviors can be dissociated from sickness from 24 h after LPS administration (Walker et al., 2013; Walker et al., 2019). The sickness responses and behavior (piloerection, ptosis and lethargy) was usually measured during peak period of sickness at 6 h after LPS administration (Silverman et al., 2013; Walker et al., 2019). In our behavioral experiment, we forced on examining the effects of rhFGF21 on depression-like behavior measured at 24 h after LPS, which include FST and locomotor activity monitored by tail suspension test (TST) and open field test (OFT) (n = 9) (Yu et al., 2019).

The TST, a behavioral test used to evaluate depressive-like behavior in animals, was conducted as previously described (Steru et al., 1985; Lee et al., 2019) with slight modifications. The TST was carried out in a soundproof box (40 × 20 × 60 cm) with cameras on the side and bottom of the box. Briefly, a string was fixed to each mouse with tape 1 cm from the tip of its tail. The mice were 20 cm above the ground, and the test was carried out for 6 min. The immobility time was recorded during 5 min of a 6-min observation period.

The FST is a common behavior test used to evaluate depression-like behavior. The immobility time after each mice ceased struggling and remained floating motionless in the water was recorded. A decrease in the duration of immobility is indicative of an antidepressant-like effect (Porsolt et al., 1977). In this test, mice were individually placed into a cylinder (25 cm in height, 10 cm in diameter) containing water at a depth of 19 cm of water at 25 ± 1°C. After a habituation period (2 min), the total amount of time each animal remained immobile during a 6 min session was scored as the immobility time, as described previously (Taniguti et al., 2019).

The OFT, a method used to evaluate the autonomous behavior, inquiry behavior, and tension of experimental animals in new environments, was carried out according to a previous study with few modifications (Ieraci and Herrera, 2006). In brief, mice were individually placed in the center of a black wooden box (50 × 50 × 50 cm) that served as the open field. Locomotion behavior indicated by the number of standing number was recorded for 5 min using a camera and analyzed using an open field experimental video analysis system (Smart 3.0, Panlab SMART video tracking system, Barcelona, Spain). The arena floor was cleaned with a 10% ethanol solution between trials.

### Primary Rat Microglia Culture

Primary rat microglia cultures were prepared from the cerebral cortices of 1–2-day-old neonatal Sprague-Dawley rat pups with mild trypsinization as previously described with minor modifications (Lin et al., 2017). Briefly, after removing the meninges of the brain, the cortical cortices were dissected and cut into small, 2-mm pieces, and tissues were digested with 0.25% trypsin for 30 min at 37°C. The tissues were suspended in DMEM/F12 containing 10% FBS and 1% penicillinstreptomycin and mechanically triturated with a plastic P1000 pipette tip. Then, the mixed cells were passed through a 70-mm nylon mesh cell strainer and plated on 6-well plates or cell culture dishes. After three days, the medium was completely replaced and changed every three days with fresh medium. After approximately 14 days, the mixed cells achieved 90% confluency, and microglia were isolated from mixed glial cultures via mild trypsinization according to our previous study (Lin et al., 2017). Mixed glial cultures were incubated with a trypsin solution containing 0.25% trypsin-EDTA for 30 min to detach a layer of cells. Primary microglial cells remained attached to the bottom of the plate and were used for further study.

### Western Blot Analysis

Total protein from brain hippocampal tissue and primary microglia was extracted using protein extraction reagents containing 1% protease and phosphatase inhibitors. Nuclear and cytoplasmic protein was purified using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai,China). The protein content of the sampleswas measured by a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). An equivalent amount of protein (30 mg) was separated on an SDS-PAGE gel and then transferred onto a Polyvinylidene Fluoride (PVDF) membrane. After being blocked with 5% nonfatmilkin Tris-buffered saline (TBS) containing 1% Tweenfor 2 h at room temperature, the membranes were further incubated with primary antibodies overnight at 4°C (a 1:400 dilution of anti-FGFR1, and 1:1,000 dilution of anti-p-FGFR1, anti NF-kB, antip-NF-kB, anti-BDNF, and b-Actin). After three washes with TBST, the membraneswere incubated with a 1:10,000 dilution of goat antirabbit IgG secondary antibody for 1 h at room temperature. Finally, the immunoreactive protein bands were developed and visualized with an enhanced chemiluminescence (ECL) kit (Biological Industries, Kibbutz Beit-Haemek, Israel), and the band densities were quantified using Image Lab 5.0 software (Bio-Rad, CA, USA).

### Immunofluorescence Analysis

Immunofluorescence analysis was performed to determine the localization of Iba1 in the mice brain hippocampal tissue. In brief, the whole brain was post-fixed by 4% PFA for 12 h, embedded in paraffin, and cut into section (5 µm thick), followed by mounted on slides. Sections were deparaffinized and rehydrated. Then tissue was incubated with 3% H2O2 for 15 min, followed blocked nonspecific binding in 5% bovine serum albumin (BSA) for 30 min at 37°C. Then sections were treated with primary antibodies anti-Iba1 (1:1,000), NF-kB (1:1,000), FGFR1 (1:1,000) overnight at 4°C, followed by incubation with AlexaFluor 488 donkey anti-rabbit secondary antibody (1:1,000) at 37°C for 1 h. Then, the nuclei were stained with DAPI for 7 min. The immunostained sections were observed and imaged using a Nikon ECLPSE 80i fluorescence microscope (Nikon, Tokyo, Japan). Three indexes of microglia activation (number, soma size, and process length) were measured according to the previous study (Tang et al., 2018). The density of Iba1 positive cells were automatically analyzed by Image-Pro plus 6.0 software (Bethesda MD, USA) at ×10 magnification in a defined area. The area of microglia soma and the microglia process length were measured at ×20 magnification by Image-Pro plus 6.0 software. For each group, at least six representstive images were taken from four mice.

### RNA Extraction and RT-PCR

Quantitative real-time PCR with SYBR Green dye was applied to measure whether rhFGF21 affected the mRNA expression level of pro-inflammation cytokines including IL-1b, TNF-a, and IL-6. Total RNA was extracted from mice brain hippocampal tissue and primary microglia by the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The residual genomic DNA was removed by RQ1 RNase-Free DNase (Promega, Fitchburg, WI, USA), and 1 mg of the total RNA from each sample was applied for complementary DNA (cDNA) synthesis using the PrimeScriptTM RT Reagent Kit (TaKaRa, Kusatsu, Japan). Real-time qRT-PCR was performed in 96-well plates using a quantitative PCR system (CFX ConnectTM Real-Time System, Bio-Rad, CA, USA). Each reaction mixture consisted of 10 ml of the SYBR Green PCR Master Mix Kit (Applied Biosystems, Carlsbad, CA, USA), 2 ml of the forward and reverse primers (5 pmol each), 25 ng of the cDNA, and diethylpyrocarbonate water for a final volume of <sup>20</sup> <sup>m</sup>l. The oligonucleotide PCR primer pairs are listed in Table 1, purchased from Sangon Biotech (Shanghai, China). The cycling program was an initial hold at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s. The mRNA expression levels of the target genes were normalized to the mRNA expression level of the housekeeping gene b-actin, and analyzed by the 2–DDCT method. The results are expressed as the means ± SEMs of duplicate samples from three independent experiments.

### Statistical Analysis

All data are presented as mean ± SEM from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 7 (GraphPad software Inc., San Diego, CA, USA). Statistical significance between groups was determined by one-way analysis of variance (ANOVA)followed byTurkey's test using. P < 0.05 was considered statistically significant.


### RESULTS

### Endogenous FGF21 Was Reduced in the Hippocampus of the LPS-Induced Mouse Brain

FGF21 plays an important role in mood regulation, and its levels were significantly increased in BD patients after treatment with the antidepressant drug valproate (Chang et al., 2018). Therefore, we detected the endogenous expression levels of both FGF21 protein and FGF21 mRNA. LPS treatment significantly decreased the endogenous FGF21 protein (Figures 1B, C) and mRNA (Figure 1D) expression levels compared to those in saline-treated mice (control group).

### Exogenous rhFGF21 Administration Alleviated Depressive-Like Behavior Induced by LPS

To evaluate the effects of rhFGF21 on depressive-like behavior induced by LPS, the OFT, FST, and TST were performed. Mice were pretreated with saline or rhFGF21 (0.75, 1.5, and 3 mg/kg, i.p.) for three days, followed by LPS administration (0.83 mg/kg, i.p.). In the OFT, a general measure of curiosity and detective behavior, the total distance traveled, line crossings, and number of standing events are used to assess locomotor activity. LPS significantly suppressed the total distance traveled and number of standing events compared with those in the control group (Figures 2A, B). Pretreatment with different concentrations of rhFGF21 significantly improved the decreased total distance traveled (Figure 2A) and number of standing events caused by LPS treatment (Figure 2B). In the FST, a putative indicator of behavioral despair, LPS administration markedly increased the mouse immobility time compared with that of the control group, and rhFGF21 treatment significantly decreased the increased immobility time induced by LPS (Figure 2C). In the TST, a classic method to assess mood, the time taken until the mouse remained immobile was measured. LPS significantly increased the immobility time during suspension, indicating that LPS induced depression-like behaviors. rhFGF21 administration significantly reduced immobility compared with that in the LPS-treated group (Figure 2D). As shown by behavioral analyses, pretreatment with rhFGF21 at doses of 0.75, 1.5, and 3 mg/kg significantly alleviated LPS-induced depression behavioral deficits; however, 1.5 and 3 mg/kg rhFGF21 had a greater effect than 0.75 mg/kg rhFGF21. Therefore, rhFGF21 at a dose of 1.5 mg/kg was applied in further studies. Moreover, rhFGF21 without LPS administration did not change the total distance traveled, or number of standing events in the OFT or the immobility time of the mice in the FST and TST (Figures 3A– D). These results demonstrate that rhFGF21 improved depression-like behaviors induced by LPS and was effective only in disease conditions.

### Exogenous rhFGF21 Administration Reduced LPS-Induced Microglial Activation

Microglia, the innate immune cells in the brain, responds to inflammation (Lenz and Nelson, 2018). In depressed patients who

committed suicide, microglial activation was observed, indicating that microglia play an important role in neuroinflammation in the brain during the pathogenesis of depression (Brites and Fernandes, 2015). The transfer of microgliafrom a ramified status to an activated status is marked by reduced process length and swollen soma (Kettenmann et al., 2011; Tang et al., 2018). To further investigate the antidepressant effect of rhFGF21 on the inflammatory response, microglial activation in the mouse hippocampus was assessed by immunofluorescence labeling to determine the number, soma area, and process length of microglia. Iba1 immunofluorescence assessment demonstrated that LPS exposure significantly enhanced the number of microglia (Figures 4A, B, D) and activated microglia, as indicated by an increased soma area (Figures 4A, B, E) and shorter ramified processes (Figures 4A, B, F) compared with the control group. Interestingly, after pretreatment with rhFGF21 for 3 days, LPS-induced changesinmicroglial numbers andmorphologiesin the hippocampus were significantly reversed (Figure 4).

### Exogenous rhFGF21 Administration Reduced the Production of Proinflammatory Cytokines Induced by LPS Through Inhibiting NF-kB Signaling Pathway

Microglial activation can induce the expression of inflammatory cytokines. Therefore, the expression levels of the critical proinflammatory cytokines IL-1b, TNF-a, and IL-6 were measured by RT-PCR, and iNOs expression was measured by western blotting. LPS exposure significantly triggered the mRNA expression of cytokines in the hippocampus, as indicated by the enhanced release of TNF-a, IL-1b, and IL-6. Conversely, rhFGF21 administration markedly reversed LPS-induced changes in the expression levels of these inflammatory cytokines (Figures 5A–C). As shown by western blot analysis, LPS administration significantly increased the production of iNOS, and pretreatment with rhFGF21 considerably reversed this increase (Figures 5D, E).

The expression of inflammatory factors in LPS-triggered activated glial cells is regulated by NF-kB signaling pathways (Chen G. et al., 2018; Muhammad et al., 2019). LPS activated the NF-kB signaling pathway, as reflected by the increased nuclear level of NF-kB protein. To investigate whether rhFGF21 can decrease proinflammatory cytokine expression through inhibiting the NFkB signaling pathway, cytoplasmic and nuclear proteins were extracted, and NF-kB was measured by western blotting with bactin and H3 used as cytoplasmic and nuclear housekeeping proteins, respectively. There were no significant differences in NF-kB expression levels in the cytoplasm of cells in the LPS and rhFGF21 pretreatment groups (Figures 5D, F). However, LPS treatment markedly increased nuclear NF-kB, and this increase was markedly reversed by pretreatment with rhFGF21 for 3 days (Figures 5D, G).

### Exogenous rhFGF21 Could Function Through FGFR1 Activation

FGFR1 is widely expressed throughout the nervous system and contributes to hippocampal nerve growth and long-term potentiation (Deng et al., 2019). To investigate whether the

effect of rhFGF21 on depression is mediated through the FGFR1 signaling pathway, we detected the expression levels of FGFR1 and p-FGFR1 by western blotting. LPS administration markedly reduced FGFR1 and p-FGFR1 levels in the hippocampus compared with those in the control group, and rhFGF21 treatment significantly rescued this decrease in hippocampal FGFR1 and p-FGFR1 (Figures 6A–C).

BDNF is a major representative of the brain neurotrophic factors, which are involved in cognition control. Cognitive dysfunction in depressive patients is due to decreased levels of BDNF (Domingues et al., 2018), and BDNF has been suggested to influence the response to antidepressant treatment (Hennings et al., 2019). Therefore, the effect of rhFGF21 on BDNF expression was measured. LPS treatment significantly reduced BDNF expression compared with that in the control group. Interestingly, rhFGF21 prevented the decrease in BDNF levels in the mouse hippocampus induced by LPS administration (Figures 6A, D).

### Exogenous rhFGF21 Administration Inhibited Inflammatory Cytokines Expression Through NF-kB Inhibition Mediated by FGFR1 Activation in Cultured Primary Microglia

To further evaluate the effects of rhFGF21 on the inflammatory response of microglia and the underlying possible mechanism of action of these effects, primary microglia were extracted and isolated from the cerebral cortices of 1–2-day-old neonatal Sprague-Dawley rat pups. Meanwhile, since FGF21 usually functions by activating its receptor, to further investigate the function of FGFR1, its receptor, the FGFR1-specific inhibitor PD173074 (10 nM) was applied. Levels of the inflammatory cytokines IL-1b, TNF-a, and IL-6 were assessed by RT-PCR. The mRNA levels of IL-1b, TNF-a, and IL-6 were higher in the primary microglia of the LPS-treated group compared with the control group, while rhFGF21 treatment significantly reduced IL-1b, TNF-a, and IL-6 upregulation induced by LPS. Additionally, PD173074 significantly reversed the rhFGF21 treatment-induced downregulation of TNF-a, IL-1b, and IL-6 mRNA (Figures 7A–C), indicating that rhFGF21 suppresses inflammatory cytokine expression through activating FGFR1 in microglia.

NF-kB signaling pathway activation is involved and critical to the development of depression as it triggers the production of proinflammatory mediators (Su et al., 2017). To further explore the anti-depressive effects mediated by the anti-inflammatory mechanism of rhFGF21 in microglia, the protein levels of p-NFkB and NF-kB in cultured primary microglia were analyzed by western blotting. The levels of p-NF-kB/NF-kB were significantly higher in the LPS-treated group than in the control group, and rhFGF21 could suppress NF-kB activation stimulated by LPS administration, while cotreatment with the FGFR1 inhibitor PD173074 and rhFGF21 significantly reversed the inhibitory effects of rhFGF21 on NF-kB (Figures 7D, E). Consistent with the western blot data, LPS significantly

stimulated NF-kB activation and translocated into nucleus. Exogenous rhFGF21 treatment obviously suppressed this translocation. Whereas PD173074 co-administration significantly reversed the effects of rhFGF21 on NF-kB (Figure 7G). Besides, the effect of rhFGF21 on BDNF expression was also analyzed. Consistent with the results in the animal study, LPS administration significantly reduced the level of BDNF compared with that in the control group; however, rhFGF21 markedly upregulated BDNF expression, and as expected, the FGFR1 inhibitor PD173074 significantly reversed this upregulation of BDNF (Figures 7D, F). To further confirm whether rhFGF21 functions through its receptor FGFR1, the immunofluorescence staining of FGFR1 was also performed. Consistent with the result in the animal model, LPS markedly suppressed FGFR1 activation, rhFGF21 rescued FGFR1

activation. Nevertheless, this was reversed by PD173074 coadministration (Figure 8). In summary, these results indicate that rhFGF21 inhibits proinflammatory cytokine expression through NF-kB inhibition mediated by FGFR1 activation.

### DISCUSSION

Studies have demonstrated the association of four FGFs, FGF2, FGF9, FGF21, and FGF22, with depression, and the correlation between these FGFs and depression has been well reviewed by Deng et al. (2019). FGF2 is a neurotrophic factor widely expressed in the CNS. Administration of a combination of the antidepressants olanzapine and fluoxetine upregulated FGF2

induced group.

FIGURE 5 | Exogenous rhFGF21 administration reduced levels of proinflammatory cytokines TNF-a, IL-1b and IL-6 induced by LPS through inhibiting NF-kB signaling pathway in the mouse hippocampus. (A) rhFGF21 decreased LPS-enhanced hippocampal pro-inflammatory cytokine TNF-a. (B) rhFGF21 decreased LPS-enhanced hippocampal pro-inflammatory cytokine IL-1b. (C) rhFGF21 decreased LPS-enhanced hippocampal pro-inflammatory cytokine IL-6. (D) Representative images of proinflammatory cytokine iNOS and NF-kB expression in cytoplasm and nucleus detected by western blot. (E) Densitometric analysis for the protein expression of iNOS in cytoplasm. (F) Densitometric analysis for the protein expression of NF-kB in cytoplasm. (G) Densitometric analysis for the protein expression of NF-kB in nucleus. Data are means ± SEM (n=5). # P < 0.05, ##P < 0.01, ###P < 0.001 compared to control group; \*P < 0.05, \*\*\*P < 0.001 compared to LPS-induced group.

levels (Maragnoli et al., 2004). Furthermore, FGF2 alleviated depression-like behaviors in rats (Turner et al., 2008). Chronic FGF-2 infusion blocked deficits in the sucrose preference test (SPT) and FST caused by chronic unpredictable stress (Elsayed et al., 2012). FGF9 is expressed mainly by neurons in the brain (Garcès et al., 2000). Exogenous FGF9 administration was found to increase depression-like behaviors, and exogenous FGF9 knockdown in the dentate gyrus improved anxiety-like behavior in rats (Aurbach et al., 2015). The effect of FGF9 was found to be opposite that of FGF2, which may be due to the inverse relationship between FGF9 expression and FGF2 expression, and FGF2 and FGF9 may act as physiological antagonists to mediate emotionality and vulnerability in mood disorders (Aurbach et al., 2015). FGF22 is expressed mainly in the skin and brain, and the deletion of endogenous FGF22 induced depression-like behavior, as shown by the FST, TST, and SPT (Williams et al., 2016). Recent studies have reported that FGF21 possesses various pharmacological activities including its antioxidant activity, suppression of apoptosis induced by endoplasmic reticulum stress (Liang et al., 2017), and anti-inflammatory activity (Wang Q. et al., 2018; Zhang et al., 2019). FGF21 weakly binds heparin and can pass through the blood-brain barrier by simple diffusion (Hsuchou et al., 2007). rhFGF21 has been demonstrated to have potent therapeutic activity in various neurological disorders, such as Parkinson's disease (Makela et al., 2014), Alzheimer's disease (Amiri et al., 2018; Chen et al., 2019), and ischemic stroke (Jiang et al., 2018; Yang X. et al., 2018). In the current study,

pretreatment with rhFGF21 at doses of 1.5 and 3.0 mg/kg significantly prevented the increase in immobility time induced by LPS administration in the FST and TST, indicating the antidepressant-like effect of this large molecule. Notably, the administration of rhFGF21 alone did not significantly alter behavior compared with the control group, demonstrating that rhFGF21 is effective under only disease conditions.

The central or peripheral administration of LPS, an endotoxin, results in depression in animal models. Therefore, LPS treatment is a validated approach to establish a depression model, and LPS-induced depression-like animal models are commonly used to assess the anti-depressive effects of molecules. Various studies have demonstrated LPS-induced behavioral impairments including increased immobile time in the FST and TST (Brites and Fernandes, 2015), which is consistent with the results of our study. Pretreatment with 1.5 mg/kg rhFGF21 for three days markedly prevented and ameliorated LPS-induced depression-like behaviors (the increased total distance traveled and number of line crossings revealed by the OFT and decreased immobility time revealed by FST and TST), indicating the potential antidepressant effects of rhFGF21.

An increasing number of studies have suggested that inflammation plays a crucial role in the process of MDD (Rush et al., 2016; Yang M. et al., 2018). The activation of microglia, a kind of macrophage in the brain, can trigger the expression of proinflammatory cytokines involved in the pathological development of neuroinflammation- and degeneration-related diseases. The hippocampus is a brain region with a high density of microglia. Some studies have shown that microglia is activated in neuroinflammation, contributing to the development of depression (Wang Q. et al., 2018). LPS administration induced depression-like behaviors, triggered microglial activation in the hippocampus, and upregulated proinflammatory cytokine expression (Brites and Fernandes, 2015). Activated microglia are the major source of proinflammatory cytokines, such as TNF-a, IL-1b, and IL-6, in the CNS (Yang M. et al., 2018). Preclinical and clinical studies have proposed that proinflammatory cytokines are associated with the pathogenesis of depression (Karson et al., 2013) and that an increase in pro-inflammatory cytokines contributes to depressive-like behaviors (Raison et al., 2006; Guo et al., 2019). In addition, various studies demonstrated increased serum levels of TNF-a, IL-1b, and IL-6 in patients with depression (Song et al., 2009; Brites and Fernandes, 2015). In the current study, rhFGF21 could inhibit the activation of microglia and reduce the expression levels of the proinflammatory cytokines TNF-a, IL-1b, and IL-6 in the hippocampus of a depressive-like mouse model induced by LPS administration and in primary microglia stimulated by LPS.

The NF-kB signaling pathway plays a critical role in the inflammatory response. Under normal conditions, NF-kB is

expression of BDNF. Data are means ± SEM (n = 5). ##P < 0.01, ###P < 0.001 compared to control group. \*\*P < 0.01, \*\*\*P < 0.001 compared to LPS treated group. & P < 0.05, &&P < 0.01, &&&P < 0.001 compared to LPS and rhFGF21 co-treated group.

bound to the inhibitor IkB, and exists in an inactive state in the cellular cytoplasm. However, once activated by an activator, such as LPS, NF-kB is phosphorylated to p-NF-kB, which is then released and translocated into the nucleus, where it promotes the expression of inflammatory cytokines (Luo et al., 2014; Yang M. et al., 2018). In the current study, we examined nuclear and cytoplasmic levels of NF-kB in the hippocampus of an LPSinduced depression mouse model and determined the expression levels of NF-kB and p-NF-kB in LPS-induced primary microglia to assess the effects of rhFGF21 on the NF-kB signaling pathway. rhFGF21 could reduce the increase in the ratio of p-NF-kB/NF- kB induced by LPS and decrease the enhanced level of nuclear NF-kB induced by LPS, indicating that rhFGF21 could inhibit NF-kB activation and translocation into the nucleus. These results indicated that rhFGF1 may exert an anti-inflammatory effect by mediating the NF-kB signaling pathway and that rhFGF1 is a novel antidepressant candidate.

FGF21 activity requires its binding to a receptor complex that consists of FGFR1 and the coreceptor b-Klotho (Min et al., 2018). Previous studies have demonstrated that FGF21/FGFR1 signaling plays a major role in increasing basal glucose uptake (Hsu et al., 2019), inducing macrophages through the Ox-LDL-

FIGURE 8 | Exogenous rhFGF21 rescued LPS-induced downregulated FGFR1 activity detected by immunofluorescence staining.

induced inflammatory response (Wang N. et al., 2018), attenuating neuronal apoptosis in the penumbra of rats after permanent middle cerebral artery occlusion (MCAO) (Zheng et al., 2019), and protecting the blood-brain barrier by increasing tight junction and adhesion junction proteins after traumatic brain injury (Chen J. et al., 2018). In the current study, to further investigate the functions of rhFGF21 in suppressing the NF-kB pathway to inhibit microglial activation and proinflammatory cytokine expression mediated through FGFR1 activation, the FGFR1 inhibitor PD173074 and rhFGF21 were used to treat primary microglia stimulated with LPS. rhFGF21 activated FGFR1, which suppressed proinflammatory cytokine expression and reduced the ratio of p-NF-kB/NF-kB expression stimulated by LPS in primary microglia. In contrast, PD173074 blocked these inhibitory effects of rhFGF21, indicating that rhFGF21 suppresses the NF-kB pathway to inhibit microglial activation and proinflammatory cytokine expression mediated through FGFR1 activation. Proinflammatory cytokines can reduce the expression of BDNF, and neurotrophic factors are critical in the pathogenesis of depression and brain neuroplasticity (Lee and Kim, 2010). Several reports demonstrated that LPS administration induced inflammatory cytokine expression and reduced BDNF and other neurotrophin expression, which could cause cognitive dysfunction. BDNF levels are much higher in the hippocampus than in other brain structures due to the great biological importance of the hippocampus in memory maintenance and its involvement with emotions. In addition, our study demonstrated that rhFGF21 could prevent LPS-induced BDNF downregulation by increasing BDNF expression in the mouse hippocampus and in primary microglia, which was reversed by its combination treatment with PD173074.

### CONCLUSION

In conclusion, the current study provides evidence that rhFGF21 treatment improves LPS-induced depression-like behaviors and decreases proinflammatory cytokine expression. In addition, the current study demonstrates that rhFGF21 can act as an antidepressant upon hippocampal microglial stimulation through inhibiting activation of the NF-kB signaling pathway by activating FGFR1. Accordingly, the present study indicates that rhFGF21 could be a potent candidate drug for antidepressant therapy. However, the underlying mechanism by which rhFGF21 affects activated microglia and inflammatory factors requires further study.

### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

### ETHICS STATEMENT

All animal use and care protocol conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and was approved by the Animal Care and Use Committee of Wenzhou Medical University.

### REFERENCES


### AUTHOR CONTRIBUTIONS

XW and LL designed the experiment and wrote the manuscript. LZ, JH, RG, and SY performed the animal experiments and analyzed the data. FL, DW, and KG performed the cells experiments and analyzed the data. YZ, AH, and XW supervised the study and contributed to the design of the study. All the authors read and approved the final version of the manuscript.

### FUNDING

This work was supported by the National Natural Science Foundation of China (No. 81771284, 81971180), Natural Science Foundation of Zhejiang Province (LQ19H090012) and Wenzhou Municipal Science and Technology Bureau Project (Y20160078).


through FGF21/FGFR1/PI3K/Caspase-3 pathway in rats. Exp. Neurol. 320, 113007. doi: 10.1016/j.expneurol.2019.113007

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor is currently organizing a Research Topic with one of the authors LL, and confirms the absence of any other collaboration.

Copyright © 2020 Wang, Zhu, Hu, Guo, Ye, Liu, Wang, Zhao, Hu, Wang, Guo and Lin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Temporospatial Expression of Fgfr1 and 2 During Lung Development, Homeostasis, and Regeneration

Tingting Yuan1\*† , Kylie Klinkhammer <sup>1</sup>† , Handeng Lyu1,2, Shan Gao1 , Jie Yuan<sup>1</sup> , Seantel Hopkins <sup>1</sup> , Jin-San Zhang<sup>2</sup> and Stijn P. De Langhe1\*

<sup>1</sup> Department of Medicine, Division of Pulmonary, Allergy & Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, United States, <sup>2</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China

#### Edited by:

Ramaswamy Krishnan, Harvard Medical School, United States

#### Reviewed by:

Reinoud Gosens, University of Groningen, Netherlands Erzsébet Bartolák-Suki, Boston University, United States

#### \*Correspondence:

Tingting Yuan tyuan@uabmc.edu Stijn P. De Langhe sdelanghe@uabmc.edu

† These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 13 November 2019 Accepted: 28 January 2020 Published: 02 March 2020

#### Citation:

Yuan T, Klinkhammer K, Lyu H, Gao S, Yuan J, Hopkins S, Zhang J-S and De Langhe SP (2020) Temporospatial Expression of Fgfr1 and 2 During Lung Development, Homeostasis, and Regeneration. Front. Pharmacol. 11:120. doi: 10.3389/fphar.2020.00120 Fgfr1 (Fibroblast growth factor receptor 1) and Fgfr2 are dynamically expressed during lung development, homeostasis, and regeneration. Our current analysis indicates that Fgfr2 is expressed in distal epithelial progenitors AT2, AT1, club, and basal cells but not in ciliated or neuroendocrine cells during lung development and homeostasis. However, after injury, Fgfr2 becomes upregulated in neuroendocrine cells and distal club cells. Epithelial Fgfr1 expression is minimal throughout lung development, homeostasis, and regeneration. We further find both Fgfr1 and Fgfr2 strongly expressed in cartilage progenitors and airway smooth muscle cells during lung development, whereas Fgfr1 but not Fgfr2 was expressed in lipofibroblasts and vascular smooth muscle cells. In the adult lung, Fgfr1 and Fgfr2 were mostly downregulated in smooth muscle cells but became upregulated after injury. Fgfr1 remained expressed in mesenchymal alveolar niche cells or lipofibroblasts with lower levels of expression in their descendant (alveolar) myofibroblasts during alveologenesis.

Keywords: lung, Fgf, homeostasis, development, regeneration

### INTRODUCTION

Fgfr1 and Fgfr2, two of the four fibroblast growth factor receptors, play important roles during lung development and regeneration, often mediating reciprocal signaling between the epithelium and mesenchyme via their ligand Fgfs (Jaskoll et al., 2005; MacKenzie et al., 2015; Balasooriya et al., 2017; Yuan et al., 2019). Our recent studies showed that Fgf10-Fgfr2b signaling is critical for generating basal cells and to drive alveolar epithelial regeneration after bleomycin injury in the lung (Yuan et al., 2019). Both Fgfr1 and Fgfr2 are also considered as potential targets for lung cancer therapy (Weiss et al., 2010; Theelen et al., 2016). However, most studies have focused on Fgfr1 or Fgfr2 signaling pathways. The temporospatial expression pattern of both Fgfr1 and Fgfr2 has not been carefully assessed.

In this study, using two mouse models featuring nuclear expression of cerulean under control of the Fgfr1 promoter and nuclear expression of mCherry under control the Fgfr2 promoter, we sought to explore the temporospatial expression pattern of these two receptors at the single cell level, during lung development, homeostasis and response to naphthalene or bleomycin injury. Since Fgf signaling regulates the expression of its receptor, this expression profile will not only provide a spatiotemporal map of which cells respond to Fgfr1 or Fgfr2 signaling at a given point in time, but

**137**

also to what extent. Note that both Fgfr1 and Fgfr2 have two isoforms, b and c, which are respectively thought to be expressed in the epithelium vs mesenchyme; however, our reporter line cannot distinguish between these two isoforms.

### Fgfr1 and Fgfr2 Expression During Embryonic Lung Development

We find that during early lung development, around E13.5, Fgfr1 is moderately expressed in both the epithelium (Sox9, Sox2) and mesenchyme (Acta2, Adrp) (Figures 1A, D and 2D, G) and is upregulated in Sox9<sup>+</sup> cartilage progenitors (Figure 2A). At E15.5, Fgfr1 is expressed at low levels in the epithelium and remains expressed in developing lipofibroblasts (Figures 1B and 2H), vascular smooth muscle cells, and cartilage progenitors (Figures 2B, E, H). By E18.5, Fgfr1 expression becomes relatively restricted to ADRP<sup>+</sup> lipofibroblasts aka mesenchymal AT2 niche cells (MANC) (Figure 2I), vascular smooth muscle cells (Figure 2F) and tracheal cartilage (Figure 2C), with low expression levels in the conducting airway (Figures 1C, F).

Interestingly, we found Fgfr2 expression to be homogenously expressed in the lung epithelium at E13.5 (Figures 1A, D) as well as in airway smooth muscle cells (Figure 2D), and Sox9<sup>+</sup> cartilage progenitors (Figure 2A). At E15.5, Fgfr2 is expressed in the epithelium with higher levels proximally vs distally (Figures 1B, E, G), airway smooth muscle cells (Figure 2E), and cartilage progenitors (Figure 2B). A downregulation of

are mean ± s.e.m. \*\*P < 0.01, as determined by a two-tailed t-test; n = 6 biological replicates for each experimental group. Scale bars, 100 µm.

FIGURE 2 | Mesenchymal Fgfr1 and Fgfr2 expression during embryonic lung development. (A–C) Immunostaining on E13.5, E15.5, and E18.5 Fgfr1-Cerulean; Fgfr2-mCherry proximal lungs for GFP (Fgfr1-Cerulean, green), RFP (Fgfr2-mCherry, red), Sox9 (white); (D–I) Immunostaining on E13.5, E15.5, and E18.5 Fgfr1- Cerulean;Fgfr2-mCherry lungs for GFP (Fgfr1-Cerulean, green), RFP (Fgfr2-mCherry, red), Acta2 (white), or ADRP (white). Lower panels for each individual image show GFP (green) and RFP (red) channels only without the far red channel. Scale bars, 100 µm, n ≥ 6.

Fgfr2 signaling distally around E15.5 is required for the lung to transition from a branching program into an alveolar differentiation program; as we have previously demonstrated that overexpression of Fgf10 starting from E15.5 onwards prevents this transition by inducing distal epithelial Fgfr2 signaling (Volckaert et al., 2013b; Volckaert et al., 2019). Upregulation of Fgfr2 in the proximal epithelium at this stage also coincides with the differentiation of the basal cell lineage (Volckaert et al., 2013b; Balasooriya et al., 2017; Volckaert et al., 2017; Volckaert et al., 2019).

At E18.5 we find Fgfr2 to be highly expressed in the developing AT2 cells (Figures 3B, G), basal cells (Figures 3C, G), distal airway clubs at the BADJ (Figures 1F and 3G) and in cartilage cells (Figure 2C), with lower levels of expression in AT1 (Figures 3A, G) and proximal club cells (Figures 3D, G) and surrounding airway smooth muscle cells (Figure 2F). Furthermore, we find that at

E18.5, there is no Fgfr2 expression in Foxj1+ ciliated cells or CGRP+ neuroendocrine cells (Figures 3E, F). These observations are consistent with our previous reports that overexpression of Fgf10 during late lung development blocks the differentiation of AT1 and ciliated cells in favor of AT2 and club or basal cells, respectively (Volckaert et al., 2013b; Volckaert et al., 2019).

### Fgfr1 and Fgfr2 Expression During Postnatal Lung Development

During postnatal lung development, we found strong Fgfr2 expression in AT2 cells and strong Fgfr1 signaling in adjacent mesenchymal alveolar niche cells or lipofibroblasts (Figures 4A–D, U–Y). Interestingly, Fgfr2 expressionwas still presentinAT1 cells but atlowerlevel thaninAT2 cells (Figures 4E–H,Y),whichis consistent with an important role for Fgfr2 signaling in AT2 stem cell maintenance (Yuan et al., 2019). We further found Fgfr2 expression in club cells (Figures 4M–P), but not in ciliated cells (Figures 4I–L). Expression of Fgfr1 and 2 was low in airway and vascular smooth muscle cells (Figures 4Q–T). However, alveolar myofibroblasts exhibited modest Fgfr1 expression at P7 and P14 (Figures 4Q, R, Y), which is consistent with a lineage relationship with lipofibroblasts (AlAlam et al.,2015;ElAgha et al.,2017;Li et al.,2018).

### Dynamic Fgfr1 and Fgfr2 Expression After Naphthalene or Bleomycin Injury

Next, to investigate the dynamic changes in both Fgfr1 and Fgfr2 expression after lung injury, we performed naphthalene and bleomycin injuries on adult mice at 8 weeks of age and traced the expression changes during lung regeneration. As expected, we found that Fgfr2 expression was pretty much gone in the airway at 3 and 7 days after naphthalene injury, consistent with a loss of club cells the main Fgfr2 expression cell type in the adult conducting airway (Figures 5A–C, E–G and S1) (Volckaert et al., 2011). However, upon the return of club cells, Fgfr2 expression was gradually restored by 14 days after naphthalene injury (Figures 5D, H). We furtherfound a modest increase in both Fgfr1 and Fgfr2 signaling in the airway smooth muscle cells upon naphthalene injury, consistent with an activation of this stem cell niche upon injury (Figures 5I–L) (Volckaert et al., 2011; Volckaert et al., 2013a; Lee et al., 2017). Remarkably, we found Fgfr2 upregulated in neuroendocrine bodies upon naphthalene injury, consistent with a role for Fgf10 signaling in regeneration of the airway epithelium by this distinct stem cell population (Figures 5M–Q) (Volckaert et al., 2011).

Lastly, we monitored the expression of Fgfr1 and Fgfr2 upon bleomycin injury. We found Fgfr2 to be higher in the distal airway club cells compared to proximal airway club cells at 3 and 6 weeks after bleomycin injury (Figures 6A–C, P). These findings are consistent with our previous report on the role of Fgf10-Fgfr2 signaling in alveolar epithelial regeneration by bronchial epithelial stem cells (Yuan et al., 2019). We found similarly high expression of Fgfr2 in regeneration AT2 cells upon bleomycin injury (Figures 6D–F, P and S2) and in proximal neo-basal cells but significantly lower expression of Fgfr2 in more distal neo-basal cells (Figures 6G–I, P), consistent with our previous finding that Fgfr2 is required for the development of neo-basal cells upon bleomycin injury and that increased Fgf10 signaling can drive these cells along the AT2 cell lineage whereas reduced Fgfr2 signaling leads to their differentiation into AT1 cells (Yuan et al., 2019). Interestingly, we found Fgfr1 expression

are mean ± s.e.m. \*\*P < 0.01, as determined by a two-tailed t-test; n ≥ 5 biological replicates for each experimental group. Scale bars, 100 µm.

in myofibroblasts upon bleomycin injury (Figures 6J–O) consistent with them being derived from lipofibroblasts (El Agha et al., 2017; Yuan et al., 2019).

### DISCUSSION

We found that during early lung development, Fgfr1 and Fgfr2 show different expression patterns. Fgfr1 is more dominantly expressed in mesenchymal cells; whereas, Fgfr2 is preferentially expressed in epithelial cell lineages (Figures 1–3). This trend continuous during the postnatal stage, with strong Fgfr2 expression in AT2 and basal cells, lower expression in club cells, and strong Fgfr1 expression in mesenchymal alveolar niche lipofibroblast cells and vascular smooth muscle cells (Figure 4). In the developing lung, Fgf10, expressed and released by the distal mesenchyme, binds and activates Fgfr2 on distal tip epithelial progenitors epithelium to keep them in a progenitor-like state by inducing Sox9 (Volckaert et al., 2013b; Volckaert et al., 2019). In this study, we detected that at E15.5, Fgfr2 expression becomes downregulated in the Sox9<sup>+</sup> distal epithelium (Figures 1B, G) and upregulated more proximally, indicating that downregulation of Fgfr2 signaling distally is required for the lung to transition from a branching program into an alveolar differentiation program.

After naphthalene injury, Fgf10 signaling is reactivated in the parabronchial smooth muscle cells (PSMCs), and Fgfr2 is upregulated in neuroendocrine bodies, suggesting that Fgf10 signaling may activate this reserve stem cell population upon injury (Figures 5M–P), consistent with previous findings (Volckaert et al., 2011; Volckaert et al., 2013a; Lee et al., 2017).

We recently discovered that Fgf10-Fgfr2b signaling is critical for the neo-basal cell generation and alveolar epithelial regeneration after bleomycin injury (Yuan et al., 2019). From this study, we find similarly high levels of Fgfr2 in distal airway club cells, AT2 cells, and proximal-neo basal cells supporting our previous findings. Intriguingly, lineage tracing data from our previous study indicate that the majority of neo-basal cells, which appear near BADJs after injury, are generated through the dedifferentiation of club cells and function as a transitional epithelial cell lineage to assist in alveolar epithelial regeneration (Yuan et al., 2019). The dynamic reduction in Fgfr2 expression in a subset of p63+ basal cells (Figures 6H, I, P) implies their direct differentiation into AT1 cells, whereas increasing Fgfr2 expression might help boost basal cell to AT2 cell reprogramming (Yuan et al., 2019). The differentiation of lipofibroblasts (LIFs) into myofibroblast (MYFs) is a classical feature during bleomycin injury (Yuan et al., 2018). Fgf10, as one of the most important fibroblast growth factors during lung development binds with high affinity to Fgfr2b but has a lower affinity to Fgfr1b. During homeostasis, in adult lungs, Fgf10 is expressed in the mesenchymal niches between the cartilage rings where normally basal cells reside in the trachea and in the lipofibroblast or mesenchymal alveolar niche cells adjacent to AT2 cells in the alveoli. Whole lung Fgf10 expression goes up after bleomycininjury due to the amplification ofmyofibroblasts yet on a per cell basis Fgf10 expression is reduced in myofibroblasts compared to the lipofibroblasts they are derived from (Yuan et al., 2019). It is possible that Fgf10 expressing mesenchymal alveolar niche lipofibroblasts express Fgfr1b and therefore, respond to Fgf10 signalingin an autocrinefashion. Future experimentswill need to be designed to assess this (Al Alam et al., 2015).

### MATERIALS AND METHODS

### Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead contact, SL (sdelanghe@uabmc.edu).

### Experimental Model Details

All mice were bred and maintained in a pathogen-free environment with free access to food and water. Both male and female mice were used for all experiments. Fgfr1Cerulean (JAX 030708), Fgfr2mCherry (JAX 030710) (Molotkov et al., 2017) mice were obtained from Jackson laboratories and crossbred to homozygosity. For bleomycin injury, adult 8 week old mice were intratracheally instilled with 50 µl bleomycin (1U/kg body weight for females, and 0.8U/kg body weight for males) as previously described (Yuan et al., 2019). For naphthalene injury, adult 8 week old mice were intraperitoneally injected with naphthalene dissolved in corn oil (325 mg/kg body weight for males and 300 mg/kg body weight for females) as previously described (Volckaert et al., 2011). All experiments were approved by the University of Alabama at Birmingham Institutional animal care and use committee.

### Immunohistochemistry and Fluorescence

All staining was done on paraffin sections of formalin-fixed lungs or tracheas. Immunofluorescent staining was performed with the following primary antibodies: goat anti-Scgb1a1 (1:200; clone T-18; sc-9772; Santa Cruz Biotechnology Inc.), rabbit anti-Scgb1a1 (1:500; WRAB-CCSP; Seven Hills Bioreagents), mouse anti-a-Actin (1:500; clone 1A4; sc-32251; Santa Cruz Biotechnology Inc.), chicken anti-GFP (1:250; GFP-1020; Aves Labs Inc.), rabbit anti-Keratin 5 (1:200; clone EP1601Y; MA5-14473; Thermo Fisher Scientific), chicken anti-Keratin 5 (1:500; clone Poly9059; 905904; BioLegend); rabbit anti-p63 (DN) (1:500; clone poly6190, 619002; BioLegend), 3862S; mouse anti-p63 (1:50; clone 4A4; CM163B; Biocare Medical), rabbit anti-RFP (1:200; 600-401-379; Rockland Immunochemicals Inc), mouse anti-RFP (1:200; sc-390909; Santa Cruz Biotechnology Inc.), rabbit anti-Sftpc (1:200; WRAB-9337; Seven hills bioreagents), mouse anti-Hop (1:100; Clone E-1; sc-398703; Santa Cruz Biotechnology Inc.), guinea pig anti-ADRP (Adipophilin) (1:200; 20R-AP002; Fitzgerald Industries), rabbit anti-Sox2 (1:1,000; WRAB-1236; Seven hills bioreagents), goat anti-Sox9 (1:500; AF3075; R&D systems), rabbit anti-CGRP (1:5,000; C8198; Sigma) and Mouse anti-Foxj1 (1:500, 14-9965- 82, Invitrogen).

After deparaffinization, slides were rehydrated through a series of decreasing ethanol concentrations and antigens unmasked by either microwaving in citrate-based antigen unmasking solution (Vector Labs, H-3000) or by incubating sections with proteinase K (7.5 mg/ml) (Invitrogen, 25530-049) for 7 min at 37°C. Tissue sections were then washed in TBS with 0.1% Tween-20 and blocked with 3% bovine serum albumin (BSA), 0.4% Triton X-100 in Tris buffered saline (TBS) for 30 min at room temperature followed by overnight incubation of primary antibodies diluted in 3% BSA, 0.1% Triton X-100 in TBS. The next day, slides were washed in TBS with 0.1% Tween-20 and incubated with secondary antibodies diluted in 3% BSA, 0.1% Triton X-100 in TBS for 3h at room temperature. All fluorescent staining was performed with appropriate secondary antibodies from Jackson Immunoresearch, except for mouse anti-Hop (1:500; A-21125; Thermo Fisher Scientific). Slides were mounted using Vectashield with (Vector Labs, H-1200) or without DAPI (Vector Labs, H-1000) depending on immunostaining.

### Microscopy and Imaging

Tissue was imaged using a micrometer slide calibrated Zeiss LSM800 Laser scanning confocal microscope using ZEN imaging software. Images were processed and analyzed using Zen blue and Adobe Photoshop software. Average immunostaining intensity was quantified after segmentation and thresholding for pixel intensity.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/ Supplementary Material.

### REFERENCES


### ETHICS STATEMENT

All experiments were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

### AUTHOR CONTRIBUTIONS

TY designed and performed experiments, analyzed data, and wrote and edited the manuscript. KK performed experiments, analyzed data, and edited the manuscript. HL, JY, SG, and SH performed experiments. JZ edited the manuscript. SDL conceived and led the project, performed experiments, analyzed data, and wrote and edited the manuscript.

### FUNDING

This study was supported by NIH R01 HL126732, HL132156, and HL146160 awards to SDL; and Cystic Fibrosis Foundation (CFF) YUAN19F0 awards to TY.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020. 00120/full#supplementary-material

FIGURE S1 | Fgfr1, Fgfr2 and Scgb1a1 expression after naphthalene injury. Immunostaining on ctrl and naphthalene injured adult Fgfr1-Cerulean;Fgfr2 mCherry lungs for GFP (Fgfr1-Cerulean, green), RFP (Fgfr2-mCherry, red), Scgb1a1 (white).

FIGURE S2 | Fgfr1, Fgfr2 and Sftpc expression after bleomycin injury. Immunostaining on ctrl and bleomycin injured adult Fgfr1-Cerulean;Fgfr2-mCherry lungs for GFP (Fgfr1-Cerulean, green), RFP (Fgfr2-mCherry, red), Sftpc (white).


epithelial regeneration by bronchial epithelial stem cells after lung injury. Stem Cell Rep. 12 (5), 1041–1055. doi: 10.1016/j.stemcr.2019.04.003

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Yuan, Klinkhammer, Lyu, Gao, Yuan, Hopkins, Zhang and De Langhe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Non-Mitogenic Fibroblast Growth Factor 1 Enhanced Angiogenesis Following Ischemic Stroke by Regulating the Sphingosine-1- Phosphate 1 Pathway

### Edited by:

Li Lin, Wenzhou Medical University, China

#### Reviewed by:

Yu-Jie Fu, Northeast Forestry University, China Changhong Xing, UT Southwestern Medical Center, United States Ning Liu, Tulane University, United States

#### \*Correspondence:

Ye Xiong xiongye2310@163.com Xue Wang xinyuw001@163.com

† These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 13 November 2019 Accepted: 22 January 2020 Published: 03 March 2020

#### Citation:

Zou Y, Hu J, Huang W, Ye S, Han F, Du J, Shao M, Guo R, Lin J, Zhao Y, Xiong Y and Wang X (2020) Non-Mitogenic Fibroblast Growth Factor 1 Enhanced Angiogenesis Following Ischemic Stroke by Regulating the Sphingosine-1-Phosphate 1 Pathway. Front. Pharmacol. 11:59. doi: 10.3389/fphar.2020.00059 Yuchi Zou1† , Jian Hu2† , Wenting Huang<sup>3</sup> , Shasha Ye<sup>2</sup> , Fanyi Han<sup>2</sup> , Jingting Du<sup>2</sup> , Mingjie Shao<sup>3</sup> , Ruili Guo<sup>2</sup> , Jingjing Lin<sup>2</sup> , Yeli Zhao2,4, Ye Xiong1\* and Xue Wang1,2\*

<sup>1</sup> The Frist Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, <sup>2</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>3</sup> School of the First Clinical Medical Science, Wenzhou Medical University, Wenzhou, China, <sup>4</sup> Engineering Laboratory of Zhejiang Province for Pharmaceutical Development of Growth Factors, Biomedical Collaborative Innovation Center of Wenzhou, Wenzhou, China

Ischemic strokes account for about 80% of all strokes and are associated with a high risk of mortality. Angiogenesis of brain microvascular endothelial cells may contribute to functional restoration following ischemia. Fibroblast growth factor 1 (FGF1), a member of FGF superfamily, involved in embryonic development, angiogenesis, wound healing, and neuron survival. However, the mitogenic activity of FGF1 is known to contribute to several human pathologies, thereby questioning the safety of its clinical applications. Here, we explored the effects and mechanism of action of non-mitogenic FGF1 (nmFGF1) on angiogenesis in mice after ischemia stroke and an oxygen-glucose deprivation (OGD) induced human brain microvascular endothelial cells (HBMECs) injury model. We found that intranasal administration nmFGF1 significantly promoted angiogenesis in mice after stroke, and significantly increased the formation of matrigel tube and promoted scratch migration in a dose-dependent manner in OGD-induced HBMECs in vitro. However, the co-administration of an FGF receptor 1 (FGFR1)-specific inhibitor PD173074 significantly reversed the effects of nmFGF1 in vitro, suggesting that nmFGF1 functions via FGFR1 activation. Moreover, nmFGF1 activated sphingosine-1-phosphate receptor 1 (S1PR1, S1P1) in mice after stroke in vivo. S1P1 protein antagonist VPC23019 and agonist FTY720 were used to confirm that nmFGF1 promotes angiogenesis in vitro partially through the S1P1 pathway. OGD induced downregulation of S1P1 expression. The S1P1 antagonist VPC23019 blocked the stimulatory effects of nmFGF1, whereas the S1P1 agonist FTY720 exerted effects comparable with those of nmFGF1. Furthermore, PD173074 reversed the effect of nmFGF1 on upregulating S1P1 signaling. In conclusion, nmFGF1 enhanced angiogenesis in mice following stroke and OGDinduced HBMECs through S1P1 pathway regulation mediated via FGFR1 activation. This new discovery suggests the potential therapeutic role of nmFGF1 for the treatment of ischemic strokes.

Keywords: ischemic stroke, nmFGF1, angiogenesis, migration, S1P1 pathway

### INTRODUCTION

Neurological disorders are the leading cause of disabilityadjusted life-years (DALYs) worldwide and the second leading cause of deaths following cardiovascular diseases. Stroke, a global health problem, was the largest contributor to global neurological DALYs in 2016, and ischemic stroke is known to account for about 80% of all stroke cases (Markus, 2011; GBD 2016 Neurology Collaborators, 2019). However, current therapeutic approaches for ischemia stroke include intravenous thrombolysis, surgical thrombectomy, and neuroprotection (Wang et al., 2019). Recombinant plasminogen activator (rtPA), as the only approved thrombolytic agent for the acute ischemic stroke treatment, can only be administrated up to 6 hours after stroke onset, and may enhance the risk of hemorrhagic transformation. Therefore, development of an effective therapeutic strategy for stroke is urgent.

Neurorestorative progression in stroke is characterized with neurogenesis, angiogenesis, and synaptic plasticity, and is beneficial for functional recovery after stroke (Gopurappilly et al., 2011; Chen et al., 2019a). Angiogenesis, the formation of new capillaries from the pre-existing blood vessels, is observed in the penumbra of brain infarct region in animal model following stroke and in the brain of patients with stroke (Ruan et al., 2015). In the early stages of ischemia, capillaries proliferate, and damaged cells release a various factors to enhance angiogenesis, thus resulting in the formation of new blood vessels and increase of cerebral blood perfusion (Arenillas et al., 2007; Wang et al., 2019), which considered as a defense system that restores nutrient and oxygen supplies to the ischemic brain tissue (Tomanek and Schatteman, 2000; Chen et al., 2019a; Kanazawa et al., 2019). However, endogenous ischemic tissue stimulated angiogenesis may not adequately compensate for the acute impaired circulation (Nariai et al., 1994; Wang et al., 2019). Therefore, angiogenesis promotion exogenously is a promising treatment strategy for ischemic stroke, thereby, facilitating survival of brain tissue following ischemia stroke.

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, and its receptors (S1PR1-5) highly expressed in various system including vascular, immune, nervous, and reproductive systems (Hla, 2004; Sartawi et al., 2017). S1P receptor 1 (S1PR1, S1P1) signaling is essential in endothelial cells (ECs) and regulates sprouting angiogenesis. S1PR1 is induced in angiogenic ECs and plays an important role in vascular development (Liu et al., 2000; Jung et al., 2012). On the contrary, blocking S1P signaling via S1PR1 inhibition may provide a new direction for antiangiogenic therapy to treat tumors (Liu et al., 2019; Rostami et al., 2019). It has been previously demonstrated that S1P1 activation could alleviate brain injuries in experimental ischemia stroke models such as transient middle cerebral artery occlusion (MCAO) (Hasegawa et al., 2010) and neonatal hypoxia-ischemia (Zhou et al., 2010). S1PR1 modulators involved in S1P1 signaling pathway improve microvascular circulation after thrombosis and exert beneficial roles in cerebral ischemia (Li et al., 2019). The above evidences indicated S1P1 activation could be considered as a good candidate target for ischemia stroke treatment.

Fibroblast growth factor 1 (FGF1) is a member of the FGF family and regulates various cellular processes such as angiogenesis, cell migration, cell differentiation, wound healing, and tube formation via binding to FGF receptors and heparin sulfate proteoglycans (Cheng et al., 2011; Wu et al., 2017; Kerr et al., 2019). However, the mitogenic activity of FGF1 is known to contribute to metastasis and tumorigenesis (Cronauer et al., 2003; Li et al., 2007). In the present study, the effects of nonmitogenic FGF1 (nmFGF1) derived from the deletion of the N terminal residues 1–27 of the full length wild-type FGF1 were investigated in an oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cell (HBMECs) injury model and an ischemia and reperfusion-injured MCAO mouse model. Previous studies have demonstrated that FGF1 could induce neurogenesis and angiogenesis in rats after ischemic stroke (Cheng et al., 2011) and rescue hippocampal neurons from apoptotic death induced by ischemia (Cuevas et al., 1998). However, the mechanism underlying the action of FGF1 on angiogenesis is yet unclear. Here, we investigated whether exogenous nmFGF1 administration could promote angiogenesis through the S1P1 signaling pathway.

### MATERIALS AND METHODS

### Reagents and Antibodies

NmFGF1 was produced in Escherichia coli and purified to be endotoxin free as previously described (Wu et al., 2005). The FGFR1-specific inhibitor PD173074 was purchased from Abcam (Cambridge, MA, USA). S1P1 antagonist VPC23019 was purchased from Cayman Chemical (Ann Arbor, MI). The primary antibodies applied in this study including anti-FGFR1 (No. ab824), anti-p-FGFR1 (No. ab59194), anti-S1P1 (No. ab11424), anti-CD31 (No. ab28364), and anti-b-Actin (No. ab8227) were purchased from Abcam (Cambridge, MA, USA), and anti-FGF1 (No. BM5544) was obtained from Bosterbio (Pleasanton, CA, USA). The secondary antibody used were goat anti-rabbit IgG H&L (HRP) (No. ab6721), and Alexa Fluor ®488-conjugated donkey anti-rabbit (No. ab150073) which were also purchased from Abcam (Cambridge, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and S1P1 agonist FTY720 were purchased from Sigma-Aldrich (St. Louis, MO, USA). EBM-2 medium was purchased from Lonza (Hopkinton, MA, USA). Matrigel matrix was obtained from Corning Inc (Tewksbury, MA, USA). All other chemicals were of analytical-reagent grade.

### Animals Grouping and Drug Administration

The experiments were conducted with male C57BL/6N mice (20–25 g), which were purchased from the Animal Center of the Chinese Academy of Sciences (Beijing, China). The animal use and care protocol conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and was approved by the Animal Care and Use Committee of Wenzhou Medical University.

After MCAO, the mice were randomly divided into two groups, mice subjected to MCAO treated with saline (MCAO + vehicle group) and nmFGF1 treatment MCAO-induced mice (MCAO + nmFGF1 group). For MCAO + nmFGF1 group, nmFGF1 was intranasally administrated at a dose of 0.75 mg/kg, followed by MCAO surgery. NmFGF was intranasally administrated once daily for 10 consecutive days, then the brain of animals were prepared for further immunofluorescence analysis (Figure 1A).

### Transient Focal Cerebral Ischemia and Reperfusion Model Preparation

A transient focal cerebral ischemia mouse model was generated by MCAO followed by reperfusion, as previously reported (Zhang et al., 2017) with minor modifications. Briefly, the mice were anesthetized by isoflurane, and a surgical incision was made in the midline neck. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully separated and ligated. An appropriate nylon monofilament for MCAO (Guangzhou Jialin Co., Ltd, Guangzhou, China) coated with 1% poly-L-lysine was inserted from the right ECA to ICA and gently advanced to the origin of the middle cerebral artery (MCA) until some resistance occurred after the movement of the nylon monofilament. The nylon monofilament was gently removed after 1 hour of occlusion to allow reperfusion. During surgery, the regional cerebral blood flow (CBF) was measured by Laser-Doppler flowmetry (Perimed, Jarfalla, Sweden) based on the previous study (Jiang et al., 2016; Wang et al., 2018). And the mice that did not show a significant CBF reduction of at least 75% of baseline levels after MCA occlusion, or that died after ischemia stimulation or reperfusion excluded from further experimental study.

### Immunofluorescence Analysis

To evaluate cell proliferation, 5-ethynyl-2-deoxyuridine (EdU) labeling was performed using BeyoClickTM EdU-488 kit (C0071S, Beyotime, Shanghai, China) according to the manufacturer's protocol. From day 6 to day 10, all groups of mice were treated with EdU [50 mg/kg intraperitoneal (i.p.) injection in saline] once daily for 4 consecutive days. After 4 hours, the mice were transcardially perfused with ice-cold phosphate-buffered saline (PBS; 0.1 M), followed by 4%

fibroblast growth factor 1 (nmFGF1) administration after middle cerebral artery occlusion (MCAO) in adult mice and analysis. (B) Schematic illustration of cell experimental procedure after oxygen-glucose deprivation (OGD)/R and analysis. (C) Representative bands of endogenous FGF1 expression detected by western blot. (D) Densitometric analysis for the protein expression of FGF1. Data are expressed as means ± SEM (n = 5). \*\*p < 0.01.

paraformaldehyde (dissolved in PBS). The brains were quickly excised for immunofluorescence staining. EdU staining was assessed with immunofluorescence.

Immunofluorescence staining was used to detect microvessel density. The whole brain tissue was post-fixed with 4% paraformaldehyde for 12 hours, embedded in paraffin, and cut into sections (5 µm thickness), followed by mounting on slides. The sections were treated with a primary antibody against CD31 (1:500 dilution) overnight at 4°C and then washed thrice with PBST at an interval of 5 minutes, followed by incubated with Alexa Fluor 488 or Alexa Fluor 647 donkey anti-rabbit/goat secondary antibodies (1:1000) for 1 hour at room temperature. Nuclear staining was performed with 4′,6-diamidino-2 phenylindole (DAPI) for 5 minutes. The stained sections were stored at 4°C. The immunostained sections were observed and imaged using a Nikon ECLPSE 80i fluorescence microscope (Nikon, Tokyo, Japan). Image-Pro plus 6.0 software (Bethesda MD, USA) was applied to analyze the images as previously described (Jiang et al., 2016). In brief, the density of the new blood vessel (EdU<sup>+</sup> cells), blood vessel (CD31<sup>+</sup> cells), or S1P1+ cells were automatically counted from three microscopic field randomly chosen from the peri-infarct regions in each three section from four animals of each group.

### HBMECs Culture and Treatment

HBMECs were purchased from Cell Systems Corporation (ACBRI376, Kirkland, WA, USA) and cultured in complete growth EBM-2 Medium with 10% FBS and penicillin/ streptomycin (100 U/ml) in an atmosphere containing 5% CO2 at 37°C. To assess the effects of nmFGF1 on OGD-induced HBMECs and the possible underlying mechanisms, the cells were divided into the following groups: control group, OGD treatment group, OGD+nmFGF1-treated group, and OGD+nmFGF1 +PD173074-treated group, OGD+nmFGF1+VPC23019-treated group, or OGD+nmFGF1+FTY720-treated group.

Control group is the HBMECs monolayers culture without OGD/R treatment. For the OGD/R treatment, the protocol was performed as described previously (Lin et al., 2018; Chen et al., 2019b). In brief, HBMECs cultured EBM-2 media were replaced with glucose free DMEM media, and then the cells cultured dish or plates were put in a specialized, humidified chamber (Heidolph, incubator 1000, Brinkmann Instruments, Westbury, NY) at 37°C, which contained an anaerobic gas mixture (90% N2, 5% H2, and 5% CO2), for 20 minutes to ensure anaerobic conditions, and for a further 4 hours incubation. Reperfusion was started by removing the culture dish or plates from the anaerobic chamber, and replacing the OGD medium with EBM-2 complete medium. Then the cells were allowed to reoxygenate in a regular incubator for 20 hours before further analysis. For nmFGF1-treated group, the cells were exposure to nmFGF1 containing medium with final concentration of 50, 100, and 200 nM during OGD/R. For OGD+ nmFGF1+PD173074-treated group, OGD+nmFGF1+VPC23019-treated group, or OGD +nmFGF1+FTY720-treated group, the cells were co-treated with nmFGF1 and PD173074 (50 nM), VPC23019 (40 nM), and FTY720 (50 nM) during OGD/R, respectively. Then these cells were applied for tube formation and wound healing assay. To explore the underlying mechanism of action, the FGFR1 and S1P1 expression level was assessed by western blot (Figure 1B).

### Cell Viability Assay

MTT assay was performed to determine cell viability as described previously (Huang et al., 2019). Briefly, HBMECs (1×10<sup>4</sup> cells/ well) were seeded in 96-well plates in complete growth EBM-2 medium and cultured overnight. After treatment, the medium was replaced with fresh medium containing 0.5 mg/ml MTT. After 3-hour incubation at 37°C, the medium was removed, followed by the formazan crystals dissolved in dimethyl sulfoxide. The absorbance was measured at 570 nm by a microplate reader with a reference setting of 630 nm.

### Tube Formation Assay

Tube formation is a classical in vitro model to assess the angiogenesis (Lu et al., 2019). To assess the effect of nmFGF1 on the angiogenic capacity of OGD-treated HBMECs, the tube formation assay was performed as described previously (Huang et al., 2019). In brief, Matrigel matrix was thawed at 4°C and added into each wells of a 48-well plate, and the plate was incubated 30 minutes to allow Matrigel solidification. The treated cells were seeded in the above Matrigel-coated plate (6×10<sup>4</sup> per well) triplicate, and incubated in a 37°C incubator for 16 hours, after which the number of tubes in two random fields from each well was counted under a bright field microscope.

### Wound Healing Assay

Wound healing assay used to evaluate cell migration across a scratch gap in vitro, is also a classical in vitro model of angiogenesis (Lu et al., 2019). After OGD/R treatment, the smooth scratches was made perpendicular to the well plate by a 10 ml pipette tip, and the width of each scratch wound should be as close to identical as possible. Afterward, the treated cells were cultured in medium with 1% FBS for further 16 hour incubation. Bright field images of migration were taken and compared with images of the cells before scratching. The relative migration distance was quantified by ImageJ software. The value of gap closure was calculated by the following equation: % of gap closure = (W0−Wn)/W0×%, where W0 is the gap width at 0 hour, and Wn is the gap width at 16 hours. Data were acquired from three independent experiments with each condition performed in triplicate.

### Western Blot Analysis

Ipsilateral hemisphere after MCAO with 1 hour reperfusion and HBMECs were lysed and proteins were extracted with RIPA lysis buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA)) with 1% protease and phosphatase inhibitor cocktail (P1261, Solarbio, Beijing, China) at 4°C. The supernatant was collected after cell lysate centrifuge, and the protein concentration was measured by a Bradford Protein Assay Kit. An equivalent amount of protein (80 µg) was separated on 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk at room temperature for 1 hour and were then blocked with primary antibodies, including those targeting FGFR1 (1:500), p-FGFR1 (1:500), S1P1 (1:1000), FGF1 (1:1000), and bactin (1:1000), and then incubated with goat anti-rabbit IgG secondary antibody (1:10000). Finally, the immunolabeling was detected by an enhanced chemiluminescence (ECL; GE Healthcare) detection system according to the manufacturer's instruction. The intensity of the bands was quantified using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA).

### Statistical Analysis

The results are expressed as mean ± SEM. Three or more replicates were performed for each experiment. Statistical differences among data from multiple groups were analyzed by one-way ANOVA followed by Tukey's posttest using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered statistically significant.

### RESULTS

### nmFGF1 Promoted Angiogenesis in Ischemic Boundary Following Ischemia and Reperfusion

To evaluate the effects of exogenous FGF1 on angiogenesis after ischemia stroke, we firstly assessed the changes of endogenous FGF1 expression after MCAO by western blot analysis. As the result shown, the expression level of endogenous FGF1 was significantly reduced after MCAO with 1 hour reperfusion. (Figures 1C, D).

EdU was intraperitoneally injected to mark proliferating cells, while CD31 was used as a marker of vessels. EdU- and CD31 immunoreactive cells are defined as markers of angiogenesis. To assess the effect of nmFGF1 on angiogenesis, the number of EdUpositive cells and vessel-associated EdU-positive cells was detected in peri-infarct corpus callosum at 10 days after reperfusion (Figure 2A). The number of EdU-positive cells (Figures 2B, C) and vessel-associated EdU-positive cells (Figures 2B, D) was significantly higher in nmFGF1 administration group than in MCAO + vehicle group. Vascular density was evaluated with CD31 immunofluorescence analysis. Quantitative analysis of CD31 labeling revealed that the number of vessels in the boundary region of ischemia at 10 days after reperfusion was significant higher in the mice treated with nmFGF1 than in those from MCAO + vehicle group (Figures 2E, F).

### nmFGF1 Promoted Angiogenesis via S1P1 Pathway Regulation

S1P signaling via the cell surface receptor S1P1 is involved in several physiological processes, including angiogenesis, cell survival, and cell proliferation (Reinhard et al., 2017; Xie et al., 2017). nmFGF1 may enhance angiogenesis following stroke via S1P1 activation; hence, we evaluated the effect of nmFGF1 on S1P1 expression in peri-infarct corpus callosum at 10 days after reperfusion (Figure 3A). In comparison with the vehicle-treated MCAO-induced mice, the intranasal administration of nmFGF1-treated MCAO-induced mice showed a significant

FIGURE 2 | Non-mitogenic fibroblast growth factor 1 (nmFGF1) promoted angiogenesis in ischemic boundary following ischemia stroke. (A) A representative image of hematoxylin-eosin (HE) staining in a coronal brain section at 10 days after middle cerebral artery occlusion (MCAO). Box illustrates the peri-infarct areas from the corpus callosum where images in B were taken. (B) Representative immunofluorescence images of DAPI and EdU staining along with the staining for the vascular endothelium marker CD31 at 10 days after stroke. (C) Quantitative analysis of EdU- labeled cells in ischemic boundary following stroke. (D) Quantitative analysis of EdU<sup>+</sup> /CD31<sup>+</sup> co-labeled cells in ischemic boundary following stroke. (E) Representative fluorescence image for vascular density analysis following staining with CD31 at 10 days after stroke. (F) Quantitative analysis of vascular density by CD31 labeling. Data are expressed as means ± SEM (n = 5). \*p < 0.05 and \*\*p < 0.01 as compared with the MCAO + vehicle group.

regulatory effect on the expression of S1P1 after 10 days (Figures 3B, C). The number of S1P1- and CD31-positive vascular ECs was markedly higher in these mice than in the vehicle-treated mice (Figures 3B, D). These results indicate nmFGF1 promoted angiogenesis through the activation of the S1P1 signaling pathway following stroke.

### nmFGF1 Enhanced Tube Formation and Wound Healing of HBMECs After OGD

To confirm our hypothesis that nmFGF1 promotes angiogenesis after stoke, OGD/R-induced HBMECs were used. We tested whether nmFGF1 imparts protection against OGD/R-induced cytotoxicity by assessing the viability of HBMECs with the MTT assay. To induce OGD/R, HBMECs were subjected to OGD for 4 hours, followed by 20 hours of reoxygenation treatment. In nmFGF1 treatment group, HBMECs were exposed to nmFGF1 at 50, 100, and 200 nM concentrations during OGD/R. The effect of nmFGF1 (100 nm) on cell proliferation and tube formation under normal oxygen conditions was also assessed. As a result, we found that 100 nM nmFGF1 had no effect on cell proliferation under normal oxygen conditions, and OGD/R exhibited no significant effects on cell viability. nmFGF1 at 50, 100, and 200 nM concentrations did not markedly change the viability of cells under OGD conditions (Figure 4A), indicating that the protective effect of nmFGF1 on cell angiogenesis after OGD/R was not mediated through an increase in cell viability. To elucidate the effect of nmFGF1 on angiogenesis after OGD, tube formation assay was performed. The results demonstrate that nmFGF1 at a dose of 100 nm significantly increased the number of tubes and tubular structures as compared with the control group comprising cells cultured under normal conditions (complete medium). However, OGD-induced HBMECs showed lower angiogenic capacity than the control cells. Interestingly, nmFGF1 at 100, and 200 nM could markedly rescue the angiogenesis of HBMECs after OGD, while low dose (50 nM) nmFGF1 treatment failed to impart obvious protection (Figure 4B). Quantitative analysis revealed that nmFGF1 (100 and 200 nM) significantly increased the tube formation ability of HBMECs with similar angiogenesis efficacy (Figure 4C).

EC migration is another classical method for vascular angiogenesis assessment. The effects of nmFGF1 at different concentrations (50, 100, and 200 nM) on cell migration were analyzed by the scratch wound healing assay. As a result, nmFGF1 at a dose of 100 nm could significantly increase EC migration as compared with the control treatment. While OGD condition induced migration defects, nmFGF1 (100 and 200 nM) treatment could obviously facilitate wound healing. Quantitative analysis revealed that 100 and 200 nM nmFGF1 exerted similar effects; however, 50 nM nmFGF1 had no significant effects on cell migration (Figures 4D, E). Based on the above results, 100 nM nmFGF1 was used in the subsequent experiment to study the underlying mechanism of action.

without affecting viability. (A) OGD and three concentrations of nmFGF1 (50, 100, and 200 nm) had no effect on cell viability. (B) Representative images to study the effect of OGD exposure and nmFGF1 treatment on tube formation ability of cells. (C) Quantification of the effects of nmFGF1 on the number of capillary-like tubes in OGD-treated cells. (D) Representative images to study the effect of nmFGF1 on wound healing in OGD-stimulated cells. (E) Quantification of the effects of nmFGF1 on wound closure in OGD-treated cells. Date are expressed as means ± SEM (n = 6). \*p < 0.05 as compared to the control group, # p < 0.05 as compared to the OGD-treated group.

### nmFGF1 Induced Angiogenesis in HBMECs After OGD via Activation of FGFR1 Signaling

FGFs and their receptors (FGFRs) are critical in many biological processes, including angiogenesis, wound healing, and tissue regeneration (Sun et al., 2017; Xue et al., 2018). To investigate whether FGFR1 signaling is involved in angiogenesis mediated by nmFGF1 in HBMECs after OGD, cells were treated with the combination of FGFR1 inhibitor PD173074 (10 nM) and nmFGF1, and the activation of FGFR1 was detected by measuring the phosphorylation of FGFR1 (p-FGFR1) using western blotting. As a result, we found that the ratio of p-FGFR1/FGFR1 was not changed after OGD treatment; however, nmFGF1 significantly upregulated the ratio of p-FGFR1/FGFR1. As expected, PD173074 prevented the increase in the ratio of p-FGFR1/FGFR1 (Figures 5A, B).

We also assessed angiogenesis and migration ability and found that PD173070 administration alone had no effects on the tube formation ability of HBMECs under normal culture conditions. On the other hand, 100 nM nmFGF1 treatment significantly improved the tube formation ability of OGD-induced cells, while cotreatment with nmFGF1 and PD173074 significantly suppressed this proangiogenic effect. PD173074 alone had no effect on angiogenesis of OGD-induced cells (Figures 5C, D). Consistent with the effect of nmFGF1 on HBMECs tube formation ability, treatment with 100 nM nmFGF1 significantly promoted HBMECs migration, which was significantly suppressed in the presence of PD173074; however, PD173074 alone had no effect on cell migration (Figures 5E, F). These results suggest that the protective effects of nmFGF1 on tube formation and wound healing of OCD-induced HBMECs could be dependent on FGFR1 signaling activation.

### nmFGF1 Induced Angiogenesis in HBMECs After OGD/R by Upregulating S1P1 Expression

To confirm whether S1P1 is necessary to mediate the angiogenesis effects of nmFGF1 on HBMECs, the cells were treated with the S1P1-specific inhibitor VPC23019 (40 nM) and agonist FTY720 (50 nM) in the presence of nmFGF1 during OGD/R (Lin et al., 2018), and tube formation and scratch wound healing assays were performed. The results show that VPC23019 and FTY720 administration alone had no effect on the tube number in control cells. While VPC23019 treatment alone failed to affect the tube formation ability of HBMECs subjected to OGD/R, the treatment with FTY720 markedly enhanced the number of tubes. VPC23019 significantly inhibited the tube formation ability of nmFGF1 in HBMECs during OGD/R, and FTY720 in combination with nmFGF1 increased the number of tubes (Figures 6A, B).

We examined the wound healing ability of cells after treatment with VPC23019 and FTY720. While nmFGF1 significantly promoted the migration of OGD/R-induced cells in the scratch wound healing assay, VPC23019 obviously

p < 0.05 as compared to the nmFGF1 treated group.

reversed this effect. S1P1 agonist FTY720 alone or in combination with nmFGF1 significantly increased the migration ability of cells. VPC23019 alone had no significant effects on the migration of HBMECs in both normal conditions and OGD/R environment (Figures 7A, B). These results suggest that nmFGF1 could rescue the OGD/R-induced S1P1 downregulation to enhance the tube formation and migration abilities of HBMECs under OGD/R conditions.

### nmFGF1 Rescued the OGD/R-Induced Downregulation of S1P1 Mediated by FGFR1 Activation

S1P signaling through its cell surface receptor S1P1 is involved in several physiological processes, including angiogenesis, cell survival, and cell proliferation (Reinhard et al., 2017; Xie et al., 2017). We investigated whether nmFGF1 affects S1P1 expression. HBMECs were subjected to OGD for 4 hours and reoxygenation for 4 hours, and were simultaneously treated with nmFGF1. The protein expression of S1P1 was measured with western blotting. As a result, we found that S1P1 protein level was significantly suppressed by OGD/R. However, nmFGF1 administration obviously rescued S1P1 expression (Figures 8A, B). Moreover, FGFR1-specific inhibitor PD173074 markedly reversed the nmFGF1-mediated increase in S1P1 expression level (Figures 8A, B). This result suggests that nmFGF1 upregulates S1P1 expression through FGFR1 activation.

## DISCUSSION

Angiogenesis is the formation of new microvessels from the preexisting vessels (Ruan et al., 2015; Hatakeyama et al., 2020). Stroke is the primary cause of disability, owing to the limited ability to restore the damaged brain tissue (Nih et al., 2018; Rust et al., 2019). After ischemia stroke injury, inflammatory response coupled with limited angiogenesis and neuronal growth leads to functional deficits. A model of cerebral ischemia-reperfusion was established toinvestigate the pathophysiology of stroke.MCAOmodeliswidely used for the induction of focal cerebral ischemia (Cotrina et al., 2017). OGD serves as an experimental approach to replicate ischemic stroke (Chen et al., 2019a). Angiogenesis is essential for brain tissue repair following ischemia stroke, as it may increase blood flow containing oxygen and metabolic nutrients to reach the affected brain regions (Hatakeyama et al., 2020). Several studies have focused on angiogenic therapy development for the recovery of injured brain following ischemia. In the current study, we explored the effects of nmFGF1 on angiogenesis in an MCAO ischemia mouse model and OGD-induced HBMECs in vitro. We found that nmFGF1 improved the angiogenesis in cerebral ischemia mice at day 10 after MCAO. We also demonstrate that nmFGF1 promoted angiogenesis and wound healing in OGDinduced HBMECs. In addition, we show for the first time that the angiogenesis effects of nmFGF1 were abolished by FGFR1- and S1P1-specific inhibitor, indicating that these effects were mediated through the activation of S1P1 signaling pathway and FGFR1.

FGF1 is unable to freely cross the brain upon systemic administration because of its filtration by the blood-brain barrier (BBB). However, intranasal administration may facilitate its direct entry into the central nervous system without exerting any adverse effects (Cheng et al., 2011). Previous studies have shown that the intranasal administration of FGF1 could enhance angiogenesis after stroke (Cheng et al., 2011). However, the mitogenic activity of FGF1 may contribute to several pathologies or metastasis and tumorigenesis (Cronauer et al., 2003; Li et al., 2007). For the safe application of FGF1, we applied nmFGF1 to mice after stroke and HBMECs after OGD/ R. As expected, nmFGF1 treatment could enhance angiogenesis after ischemia injury in mice and promote angiogenesis and wound healing in OGD/R-induced HBMECs.

Although several studies have reported the protective effects of FGF1 on angiogenesis, the underlying mechanism of action on angiogenesis is unclear. FGFR1 is expressed in the brain (Wang et al., 2017; Huang et al., 2019), and its activation may protect the BBB in OGD/R-induced HBMECs (Lin et al., 2018). Cotreatment with the FGFR1 inhibitor PD173074 reversed the positive effects of nmFGF1 on migration and angiogenesis, indicating that FGFR1 activation induced migration and angiogenesis of HBMECs stimulated with OGD/R. In addition, S1P and its five receptors (S1PR1-5) are widely expressed in several systems, including the vascular, nervous, and reproductive system (Sartawi et al., 2017). S1P signaling pathway has been implicated in angiogenesis and is required for vascular stabilization. Dysfunction in the S1P-S1PR signaling pathway induces various vascular defects (Fischl et al., 2019; Obinata and Hla, 2019). The temporal profile of changes of the S1P1 expression in the early phase after transient MCAO was documented by Hasegawa et al. (2013), that the expression of S1P1 was significantly decreased in the infarct cortex but preserved in the peri-infarct cortex at 24 hours after MCAO. It has been reported that S1P1 expression on ECs of leptomeningeal arteries and capillaries upregulated early after permanent MCAO, peaking at 6 hours, whereas a significant increase in the expression of S1P1 in neurons was observed from 24 hours later (Iwasawa et al., 2018). S1PR modulators exerted protective effects in preclinical and clinical studies following ischemic stroke. In addition, these agents may improve microvascular circulation in cerebral ischemia (Lin et al., 2018). It has been reported that S1P1 agonist LAS1238 significantly reduced infarct volume of mice model after ischemia/reperfusion, indicating S1P1 is potential target for ischemic stroke treatment (Brait et al., 2016). Besides, a

FIGURE 8 | Non-mitogenic fibroblast growth factor 1 (nmFGF1) rescued oxygen-glucose deprivation (OGD)/R-induced downregulation of S1P1 expression by FGFR1 activation. (A) Representative western blots for S1P1 expression. (B) Quantification of the relative expression level of S1P1. Date are expressed as the means ± SEM (n = 4). \*p < 0.05 as compared to the control group, # p < 0.05 as compared to the OGD-treated group, &&p < 0.01 as compared to the nmFGF1-treated group.

previous study reported that the activation of S1P signaling could protect the BBB after exposure of HBMECs to OGD/R (Lin et al., 2018). However, the effects of nmFGF1 on S1P signaling pathway are unknown. In our current study, we confirmed nmFGF1 significantly increased S1P1 expression in the ECs indicated by colocation of CD31 and S1P1 by immunofluorescence assay after 10 days following MCAO, that was consisted with previous study (Iwasawa et al., 2018). We treated OGD/R-induced HBMECs with S1P1 antagonist VPC23019 and S1PR1 agonist FTY720 in combination with nmFGF1. VPC23019 treatment alone had no effect on tube formation, while FTY720 markedly enhanced the number of tubes. In the presence of VPC23019, the tube formation ability of HBMECs induced by nmFGF1 was significantly inhibited during OGD/R, and FTY720 in combination with nmFGF1 increased the tube number. nmFGF1 exhibited similar angiogenesis ability with S1PR1 agonist FTY720, that was consisted with previous study (Shang et al., 2020). Although we did not perform the experiment to observe the effects of nmFGF1 on neuroprotection after stroke, combined with the previous study that FGF1 enhanced neurogenesis after stroke (Cheng et al., 2011), we can speculate one potential mechanism of nmFGF1 neuroprotection was that enhanced angiogenesis might be partially due to improvements in the microenvironment of vessel growth. As the data shown, PD173074 also inhibited the positive effects of nmFGF1 on S1P1 signaling. Taken together, nmFGF1 can promote angiogenesis through S1P signaling pathway mediated by FGFR1 activation.

### CONCLUSION

Our study provides the first evidence that intranasal nmFGF1 administration enhanced angiogenesis via S1P1 signaling pathway following stroke and highlights the need to better understand the mechanism underlying nmFGF1 function in ischemia protection. Taken together, these results suggest that nmFGF1 could be considered as potential angiogenic agent for ischemic stroke treatment.

### DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding authors.

### ETHICS STATEMENT

All animal use and care protocols conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes

### REFERENCES


of Health and were approved by the Animal Care and Use Committee of Wenzhou Medical University.

### AUTHOR CONTRIBUTIONS

XW and YX designed the experiment and wrote the manuscript. YuZ, JH, WH, and SY performed the animal experiments. FH, JD, RG, and MS performed the cells experiments. JL and YeZ analyzed the data. All the authors read and approved the final version of the manuscript.

### FUNDING

This work was supported by Wenzhou Municipal Science and Technology Bureau Project (Y20170224 and Y20170686), Natural Science Foundation of Zhejiang Province (LQ19H090012), and Medical and Health Science and Technology Program of Zhejiang Province (2018KY505).

Burden of Disease Study 2016. Lancet Neurol. 18 (5), 459–480. doi: 10.1016/ S1474-4422(18)30499-X


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a shared affiliation, though no other collaboration, with the authors at time of review.

Copyright © 2020 Zou, Hu, Huang, Ye, Han, Du, Shao, Guo, Lin, Zhao, Xiong and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Fibroblast Growth Factor 1 Ameliorates Diabetes-Induced Liver Injury by Reducing Cellular Stress and Restoring Autophagy

Zeping Xu1† , Yanqing Wu2† , Fan Wang3,4† , Xiaofeng Li <sup>1</sup> , Ping Wang<sup>1</sup> , Yuying Li <sup>1</sup> , Junnan Wu<sup>1</sup> , Yiyang Li <sup>1</sup> , Ting Jiang<sup>1</sup> , Xindian Pan<sup>5</sup> , Xie Zhang<sup>6</sup> , Longteng Xie<sup>7</sup> , Jian Xiao1\* and Yanlong Liu1,8\*

Edited by: Saverio Bellusci, University of Giessen, Germany

#### Reviewed by:

Hao Wang, RMIT University, Australia Huatao Chen, Northwest A&F University, China Cho-Ming Chao, German Center for Lung Research, Germany

#### \*Correspondence:

Jian Xiao xfxj2000@126.com Yanlong Liu benjaminlyl@wmu.edu.cn

† These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 20 August 2019 Accepted: 16 January 2020 Published: 03 March 2020

#### Citation:

Xu Z, Wu Y, Wang F, Li X, Wang P, Li Y, Wu J, Li Y, Jiang T, Pan X, Zhang X, Xie L, Xiao J and Liu Y (2020) Fibroblast Growth Factor 1 Ameliorates Diabetes-Induced Liver Injury by Reducing Cellular Stress and Restoring Autophagy. Front. Pharmacol. 11:52. doi: 10.3389/fphar.2020.00052 <sup>1</sup> School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Institute of Life Sciences, Wenzhou University, Wenzhou, China, <sup>3</sup> The Second Affiliated Hospital, Xinjiang Medical University, Urumqi, China, <sup>4</sup> Beijing Hui-Long-Guan Hospital, Peking University, Beijing, China, <sup>5</sup> School of Medicine, Hangzhou Normal University, Hangzhou, China, <sup>6</sup> Department of Pharmacy, Ningbo Medical Treatment Center, Li Huili Hospital, Ningbo, China, <sup>7</sup> Department of Infection Diseases, Ningbo Fourth Hospital, Xiangshan, China, <sup>8</sup> Center for Health Assessment, Wenzhou Medical University, Wenzhou, China

Background: Type 2 diabetes (T2D) is a metabolic dysfunction disease that causes several complications. Liver injury is one of these that severely affects patients with diabetes. Fibroblast growth factor 1 (FGF1) has glucose-lowering activity and plays a role in modulation of several liver injuries. Nevertheless, the effects and potential mechanisms of FGF1 against diabetes-induced liver injury are unknown.

Methods: To further investigate the effect of FGF1 on diabetic liver injury, we divided db/ db mice into two groups and intraperitoneally (i.p.) injected either with FGF1 at 0.5 mg/kg body weight or saline every other day for 4 weeks. Then body weights were measured. Serum and liver tissues were collected for biochemical and molecular analyses.

Results: FGF1 significantly reduced blood glucose and ameliorated diabetes-induced liver steatosis, fibrosis, and apoptosis. FGF1 also restored defective hepatic autophagy in db/db mice. Mechanistic investigations showed that diabetes markedly induced oxidative stress and endoplasmic reticulum stress and that FGF1 treatment significantly attenuated these effects.

Conclusions: FGF1-associated glucose level reduction and amelioration of cellular stress are potential protective effects of FGF1 against diabetes-induced liver injury.

Keywords: fibroblast growth factor 1, diabetes, liver injury, oxidative stress, endoplasmic reticulum stress

## INTRODUCTION

Type 2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance and pancreatic cell failure (Kasuga, 2006). Hyperglycemia resulting in glucotoxicity is the primary pathophysiological trigger of diabetes-induced complications (Wu et al., 2015a; Wu et al., 2016). Liver injury occurs commonly in patients with diabetes (Gezginci-Oktayoglu et al., 2009). Liver damage caused by diabetes is characterized by abnormal liver enzyme levels, steatosis, fibrosis, and cirrhosis. Diabetes-induced liver damage produces several pathological changes in the morphology and microstructure of hepatic tissues and functions, including vacuolization and lipid accumulation, resulting in significant damage (Wu et al., 2015a). There are many mechanisms mediating liver damage caused by diabetes, including hyperglycemia, oxidative stress, endoplasmic reticulum stress, and advanced glycation end-products (JaeMyoung et al., 2014). Therefore, it is important to detail these pathophysiological mechanisms and to explore effective therapeutic strategies to meliorate diabetes-induced liver injury.

Fibroblast growth factor 1 (FGF1) releases from cells through a nonclassical secretory pathway, acting on cells in various tissues, including liver and vasculature, where it exerts classic mitogenic activity (Jiang et al., 2016). FGF1 has an unique ability to lower blood glucose, and pharmacologically-relevant FGF1 (0.5 mg/kg) leads to impressive changes in several measurements, including blood glucose, nearly normalizing after 35 days in T2D micemodels with impaired insulin sensitivity (JaeMyoung et al., 2014). Administration of exogenous FGF1 stimulates glucose uptake in an insulin-dependent fashion and suppressed the hepatic production of glucose to achieve whole-body insulin sensitization in a mouse model of T2D (JaeMyoung et al., 2014). As an insulin sensitizer, FGF1 mediates homeostatic control of glycemia by acting on several pathways (Li, 2019). The glucose-lowering activity of FGF1 is dissociated from its mitogenic activity and is mediated predominantly via FGF receptor 1 signaling (JaeMyoung et al., 2014).

FGF1 also reverses hepatic lipid steatosis by improving lipid catabolism (Liu W. et al., 2016). Chronic treatment with rFGF1 led to reduced hepatic steatosis and augmented liver glycogen content (JaeMyoung et al., 2014). A recent study showed that FGF1 meliorated acetaminophen (APAP)-induced hepatotoxicity in mice through suppression of inflammation, apoptosis, and oxidative and endoplasmic reticulum stress (Wang et al., 2019). These findings suggest that FGF1 may have hepato-protective properties beyond its anti-steatotic and anti-inflammatory properties. Based on these studies, we aimed to investigate the effects and molecular mechanisms of FGF1 in diabetic-induced liver injury.

### MATERIALS AND METHODS

### Animal Experiments

Twelve-week-old male db/db mice and their nondiabetic db/m littermates were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Animals were maintained under 12:12 h light:dark cycle conditions. The db/db mice were divided into two groups and were intraperitoneally (i.p.) injected either with FGF1 (Guang et al., 2018) (0.5 mg/kg body weight, n = 8) or physiologic saline (n = 8) every other day for 4 weeks (Wu et al., 2018). After 4 weeks, body weights were measured. Then serum and liver tissues samples of these mice were collected for biochemical and molecular analyses.

### Biochemical Analysis

Blood glucose levels were monitored from tail blood samples using a blood glucose meter (Glucometer, SANNUO, China). Serum levels of alanine aminotransferase (ALT), triglyceride (TG), liver glutathione peroxidase (GSH-PX), and liver 4 hydroxynonenal (4-HNE) protein adducts were measured according to the manufacturer's instructions (Jian Cheng Biotechnology Co., Ltd. of Nanjing, China). Total antioxidant capacity (T-AOC), malondialdehyde (MDA), and superoxide dismutase (SOD) activity were also measured with various assay kits (Beyotime Biotechnology Corporation, Shanghai, China). Plasma glycosylated hemoglobin (GHbA1c) levels were measured using the Mouse Glycated Hemoglobin A1c (GHbA1c) ELISA Kit (Enzyme-linked Biotechnology Co., Ltd. Shanghai, China).

### Liver Triglyceride Assay

Hepatic triglyceride (TG) levels were determined as described previously (Liu Y. et al., 2016), using the TG reagent (Thermo Fisher Scientific, Middletown, VA).

### Pathology and Immunohistochemical Staining on Liver Tissue

Hematoxylin and eosin (H&E) staining and Masson's trichrome (MT) staining (Solarbio Science & Technology, Beijing, China G1340) were employed to evaluate the characteristics of liver tissues in histological changes and fibrosis. Immunohistochemical staining was used to further verify the deposition of collagen I (collagen I, 1:500; Abcam, Cambridge, UK) in fibrotic liver. The procedure was performed as described previously (Hao et al., 2009).

### Terminal Deoxynucleotidyl Transferase Deoxyuridine Triphosphate Nick End Labeling Assay

Liver sections of 5 mm were stained for terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) according to the manufacturer's instruction using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon, CA, USA).

### Western Blotting Analysis

Liver tissues were homogenized and determined as described previously (Wang et al., 2019). The blots were incubated with primary antibodies: cleaved caspase-3, Bax, Bcl-2, ATF6, GRP78, Nrf2, SOD2, collagen IA1, ATG5, p62 and LC3 (Abcam, USA), HO-1 (Santa Cruz Biotechnology, CA, USA), CHOP, P-IRE1a, P-eIF2a, P-PERK, PERK, FGFR1, P-AMPK and AMPK (Cell Signaling Technology, Danvers, MA, US), and a-SMA, TGF-b, and GAPDH (Proteintech, China) followed by incubation with their corresponding secondary antibodies: anti-rabbit and antimouse (Proteintech, China).

### Statistical Analysis

All data were expressed as mean ± standard error of the mean (SEM). Statistical differences were determined using one-way ANOVA (for comparison of two experimental conditions). Statistical significance was considered at P values < 0.05. Statistical calculations were done using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, USA).

### RESULTS

### FGF1 Treatment Reduced Blood Glucose and Ameliorated Hepatic Steatosis

A previous study showed that a single injection of FGF1 was sufficient to restore blood glucose levels to the normal range for more than 2 days in both db/db and DIO mouse models (JaeMyoung et al., 2014). In agreement with that study, our results showed that FGF1 treatment markedly reduced blood glucose levels in db/db mice (Figure 1B). HbA1c levels in db/db mice were markedly higher than those of db/m mice. No significant differences were found between db/db mice with FGF1 treatment and db/db mice with respect to HbA1c levels (Figure 1C). In addition, db/db mice had distinctly elevated plasma alanine aminotransaminase (ALT) levels, a liver injury marker, at 16 weeks of age compared with the db/m mice, an effect that was distinctly reduced by FGF1 treatment (Figure 1D). Body weights of db/db mice were significantly greater than those of db/m mice, an effect that was markedly decreased with FGF1 treatment (Figure 1E). Compared with db/ m mice, liver weights of db/db mice were markedly higher, an effect reversed by FGF1 treatment (Figure 1F). Although no significant difference was found between db/m mice and db/db mice with respect to the ratio of liver/body weight, the liver/body weight ratio in db/db mice was reduced by FGF1 treatment (Figure 1G). Pathological hepatic lipid accumulation is a characteristic of diabetes liver injury. As previously reported, accumulated fat interferes with insulin signaling pathways and consequently reduces the glucose content taken up from the bloodstream, an effect that is tightly connected to hepatic injury and steatosis in T2DM (Marchesini et al., 2001). In the present study, db/db mice had elevated plasma TG levels, a common feature of dyslipidemia accompanying with type 2 diabetes; this effect was diminished by FGF1 treatment (Figure 1H). Histological analyses revealed significant lipid accumulation in liver cells from db/db mice as shown by H&E staining. This effect was significantly attenuated by FGF1 treatment (Figure 1A).

The db/db mice had elevated liver TG levels, and FGF1 treatment ameliorated these increases (Figure 1I). Taken together, our results suggest that db/db mice had fatty livers, and FGF1 treatment reduced blood glucose and attenuated diabetes-induced hepatic steatosis.

### FGF1 Treatment Ameliorated Diabetes-Induced Liver Fibrosis by Reducing Deposition of Collagen

The expression of collagen is used as an index of liver injury. We graded the degree of hepatic fibrosis using Masson staining and collagen I immunohistochemical staining (Figures 2A, B). The areas of collagen fibers deposition were more significantly expressed in the central vein and surrounding sinus gaps in db/db mice than those in db/m control mice (Figures 2C). Consistent with the immunohistochemical results, western blotting results showed that protein levels of collagen IA1 in livers from db/db mice were significantly greater, but that were much lower in the FGF1 treatment group (Figures 2D, E). Furthermore, protein levels of a‐SMA and TGF-b were substantially greater in db/db mice livers (Figures 2D, F, G). All these changes in db/db mice were reversed by FGF1 treatment, suggesting that FGF1 inhibited diabetes-induced liver fibrosis by abolishing collagen accumulation in the liver.

### FGF1 Treatment Ameliorated Diabetes-Induced Hepatic Apoptosis

To determine the mechanisms by which FGF1 protects against diabetes-induced liver damage, we examined apoptosis pathways. Compared with liver cells in db/m mice, we found that liver cells of db/db mice were severely damaged, characterized by irregular shapes and chaotic arrangement of cells. Numbers of TUNEL-positive staining cells were substantially greater in liver cells from db/db mice than those in db/m liver cells, an effect that was inhibited by FGF1 treatment (Figures 3A, B). Apoptosis indicators were also measured (Figures 3C–F). Expression levels of cleaved caspase-3 and Bax were much higher in db/db mice than those in db/m mice (Figures 3C, E, F). By contrast, compared with the db/m mice, Bcl2, an anti-apoptotic protein (Yu et al., 2019), was expressed at lower level in db/db mice livers (Figures 3C, D). All these changes were reversed by FGF1 treatment. Taken together, these observations suggest that FGF1 ameliorated diabetesinduced hepatic apoptosis in db/db mice.

### FGF1 Treatment Restored Defective Hepatic Autophagy in Diabetic Mice

Previous studies showed that diabetes impaired hepatic autophagy and that obesity caused markedly decreased

FIGURE 2 | FGF1 treatment ameliorated diabetes-induced liver fibrosis by reducing deposition of collagen. (A) Masson staining of liver from db/m, db/db, and db/ db + FGF1 mice (10 x:scale bars = 100 mm). (B, C) Immunohistochemical staining of collagen I and collagen I positive area of liver from db/m, db/db and db/db + FGF1 mice (20 x:scale bars = 50 mm 40 x:scale bars = 25 mm). (D) Protein expression of collagen I, a-SMA, and TGF-b in livers from db/m, db/db, and db/db + FGF1 mice. (E–G) Intensities of collagen I, a-SMA, and TGF-b normalized to GAPDH. All data are presented as mean ± SEM, n = 8. \*P < 0.05 \*\*P < 0.01 \*\*\*P < 0.001 vs. the db/m group and db/db + FGF1 group.

FGF1 group.

autophagy in livers of both genetic and dietary mice models; this effect was demonstrated by decreased expression levels of LC3 and Atg5 (Yang et al., 2010). By contrast, p62 is involved in aggresome formation and is degraded through autophagy (Iwadate et al., 2014), with higher levels in ob/ob mice livers than in lean controls (Yang et al., 2010). To study the regulation of autophagy in the present diabetes model, we first examined expression patterns of molecular indicators of autophagy. In line with previous studies, we found that diabetes was characterized by lower expression of autophagy indicators in db/db mice livers, demonstrated by lower expression levels of LC3 and ATG5 proteins (Figures 4A, C, D). Levels of p62 protein level were substantially higher in db/db mice livers than that in db/m mice (Figures 4A, B). As expected, these changes were reversed by FGF1 treatment, suggesting that FGF1 treatment restored defective hepatic autophagy in db/db mice.

### FGF1 Treatment Blocked Diabetes-Induced Oxidative Stress in Liver

Oxidative stress is a molecular mechanism that characterizes diabetes complications (Yang et al., 2015). Fatty acids in the liver induce free radical formation, causing lipid peroxidation and inducing release of proinflammatory cytokines. The release of malondialdehyde and 4-hydroxynonenal correspondingly causes cell death, protein cross-linkage, and stellate cells activation, leading to collagen synthesis and fibrosis (Xiao et al., 2017). Oxidative stress is also a critical factor in the progression of nonalcoholic fatty liver disease associated with diabetes (Mohamed et al., 2016). In the present study, we determined whether FGF1 treatment reversed diabetesinduced oxidative stress by measuring glutathione levels and oxidative stress markers (Figure 5). Plasma malondialdehyde (MDA) levels were significantly greater in db/db mice livers than those in of db/m mice livers (Figure 5A), an effect that was reduced by FGF1 treatment. As expected, liver glutathione peroxidase (GSH-PX) levels were lower in db/db mice livers than those in db/m mice livers, an effect that was increased by FGF1 treatment (Figure 5B). There were no significant differences in plasma MDA levels and liver GSH-PX levels between db/db mice and db/db + FGF1 mice. 4 hydroxynonenal (4-HNE)-protein adducts and protein carbonyl content were measured to assess lipid peroxidation and protein oxidation levels, respectively (Valle et al., 2012). Consistently higher levels of carbonyl and 4-HNE adducts were observed in db/db mice, an effect that was decreased by FGF1 treatment (Figure 5C). Although no significant differences were found between db/m mice and db/db mice with respect to liver 4-HNE levels and plasma T-AOC levels, FGF1 treatment significantly increased liver 4-HNE levels and plasma T-AOC levels in db/db mice (Figures 5C, D). HO-1 is the rate limiting enzyme in heme degradation, being the first step, catalyzing degradation of pro-oxidant heme to carbon monoxide,

db/m group and db/db + FGF1 group.

biliverdin, and ferrous iron (Abraham and Kappas, 2008). HO-1 gene expression is regulated by the transcriptional activator Nrf2 (Jiying et al., 2004; Igarashi and Sun, 2006). In the present study, FGF1 treatment caused significant increases in expression levels of Nrf2 in db/db mice, which ameliorated diabetes-induced oxidative stress in liver tissue (Figures 5E, F). Furthermore, expression levels of HO-1 in db/db mice liver were also lower than those of db/m mice, an effect that was diminished by FGF1 treatment (Figures 5E, G). SOD2 protein expression levels in liver were also significantly lower in db/db mice than those in db/m mice, an effect that was markedly ameliorated in FGF1 treated db/db mice (Figures 5E, H). Taken together, these data suggest that FGF1 treatment markedly reversed diabetesinduced hepatic oxidative stress in db/db mice.

In addition, we also examined FGFR1-AMP-activated protein kinase (AMPK) signaling pathway. Western blotting results show that FGFR1 expression level in db/db + FGF1 liver was markedly higher than that in db/db liver, indicating that FGFR1 signaling was activated by FGF1 treatment (Supplement Figures 1A, B). AMPK, a serine kinase, has been reported to enhance cellular antioxidant capacity through inducing activation of Nrf2 and HO-1 (Lee et al., 2019). In the present study, as shown in Supplement Figures 1A, C, hepatic P-AMPK levels were significantly lower in db/db mice compared with the db/m mice. As expected, these changes were reversed by FGF1 treatment. Taken together, these data suggest that FGF1 treatment markedly reversed diabetes-induced hepatic oxidative stress by regulating FGFR1-AMPK pathway.

### FGF1 Treatment Suppressed Diabetes-Induced Endoplasmic Reticulum Stress in Liver

Diabetes-induced liver injury causes changes in microstructure and morphology of liver tissues, leading to changes of hydrophilic and hydrophobic domains in endoplasmic reticulum, an effect that was closely associated with changes of cellular endoplasmic reticulum (ER) polarity. These data suggest that differences in ER polarity can suggest degree of diabetesinduced liver injury (Xiao et al., 2017). Proteins enter the ER as unfolded polypeptide chains, also called the unfolded protein response (UPR) (Ron and Walter, 2007). Elevation in the phosphorylation of eIF2a and PERK (P-eIF2a and P-PERK) is indicative marker of ER stress in diabetes mice. Furthermore, P-IRE1a, an early and crucial indicator of ER stress, plays an important role in mediating the UPR response under ER stress (Wang et al., 2013). To measure FGF1 treatment mediated inhibition of diabetes-induced ER stress in liver, we measured expression levels of ER stress protein markers. Hepatic protein levels of glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), and C/EBP‐homologous protein (CHOP) were significantly greater in db/db mice and these changes were reversed by FGF1 treatment (Figures 6A–D).

Notably, we identified dramatically higher P-IRE1a, P-eIF2a, and P-PERK protein expression levels in livers of db/db mice than in those of db/m mice, an effect that was inhibited by FGF1 treatment (Figures 6A, E–G). Taken together, these data suggest that FGF1 treatment significantly suppressed diabetes-induced ER stress.

### DISCUSSION

In the present study, we demonstrated the glucose-lowing effect of FGF1 in db/db mice and its effects on the progression of liver injury in T2DM. These observations were associated with reduced plasma ALT, liver, and plasma TG levels, and decreased ER stress and oxidative stress as well as hepatic apoptosis. Furthermore, hepatic autophagy was corrected by FGF1 treatment.

The db/db mouse model is characterized by leptin receptor deficiency, and displays hyperinsulinemia, hyperglycemia, and glucosuria phenotypes (Razzoli et al., 2015). Previous studies have shown that FGF1 has therapeutic potential for insulin resistance and type 2 diabetes. A single injection of 0.5 mg/kg rFGF1 corrected hyperglycemia in ob/ob mice. This effect of FGF1 is dose-dependent, and even at the maximal dose (2.0 mg/ kg) it does not produce hypoglycemia. Moreover, FGF1 has no effect on blood glucose or insulin levels in normoglycemic chowfed mice. Furthermore, chronic rFGF1 treatment in mice with diet-induced obesity (DIO) also caused pronounced and sustained lowering of blood glucose levels and increased insulin sensitization (JaeMyoung et al., 2014). The glucoselowering effects of FGF1 are mediated predominantly via FGF receptor 1 signaling. Injection of agonistic anti-FGF receptor 1 antibodies into obese diabetic mice induced acute and sustained amelioration of hyperglycemia, accompanied by significant improvement in hyperlipidemia, hyperinsulinemia, and hepatosteatosis (Wu et al., 2015b). In the present study, treatment of FGF1 for 4 weeks significantly reduced glucose levels in the circulation. Our results further confirmed the therapeutic potential of FGF1 for insulin resistance and type 2 diabetes.

Previous studies have shown that recombinant FGF1 (rFGF1) effectively improved hepatic damage and inflammation in ob/ob mice and choline-deficient mice, two etiologically-different models of NAFLD. Studies have reported that rFGF1 reduced steatosis in the periportal zone first, and subsequently improved hepatic lipid catabolism (Liu Y. et al., 2016). Consistent with these findings, we confirmed that FGF1 administration ameliorated diabetes-induced liver damage. We showed that FGF1 treatment reduced plasma ALT levels, ameliorated liver lipid accumulation as well as hepatic apoptosis and liver fibrosis and corrected hepatic autophagy. Furthermore, we demonstrated that FGF1 treatment blocked diabetes-associated collagen accumulation. All these findings are consistent with previous observations demonstrating the hepatic-protective effects of FGF1 in various animal models. Mechanistic studies demonstrated that induction of cellular stress in diabetic liver was inhibited by FGF1 treatment, suggesting that reduction of cellular stress was a potential molecular mechanism in course of FGF1 treatment for diabetes-induced liver injury.

Previous studies reported that FGF1 inhibited oxidative stress and consequently blocked diabetes-induced cardiomyopathy (Wu et al., 2016). Elevated oxidative stress is a major causal factor in diabetes-associated complications (Wu et al., 2011). Liver tissues from diabetic rats also showed significantly increased levels of reactive oxygen species (ROS), and dramatically lower levels of GSH, catalase, and superoxide dismutase (SOD) (Borderud et al., 2014). In the resting state, Nrf2 is in a non-free and non-active state of continuous degradation. When stimulated by electrophiles or ROS, Keap1 is uncoupled from Nrf2, allowing Nrf2 to transfer to the nucleus, and the Maf protein in the gene. Binding to the heterodimer recognizes and binds ARE, initiates transcription of the downstream protective protein genes, and enhances cell's ability to resist oxidative stress (McMahon et al., 2006). Nrf2 signaling may compensatorily increase the response to acute stress (Stewart et al., 2003), modulating expression of several oxidative damage genes, including HO-1 and SOD2 that are protective in diverse models of liver diseases (Ge et al., 2015; Peng et al., 2018). In our study, diabetes markedly resulted in down-regulation of Nrf2, HO-1 and SOD2, an effect that was reversed by FGF1 treatment. Furthermore, FGF1 treatment regulated FGFR1-AMPK signaling pathway. The abovementioned data suggest that FGF1 treatment ameliorated hyperglycemia and subsequently oxidative stress during diabetes-induced liver injury via regulation of the FGFR1- AMPK-Nrf2/HO-1 signaling pathway.

A previous report showed that ER stress induced the production of reactive oxygen species (ROS) (Peng et al., 2018), thereby leading to oxidative stress (Hyung-Ryong et al., 2009). ER stress occurs when misfolded proteins accumulate in the ER lumen, and this is referred to as the unfolded protein response (UPR) (Hyung-Ryong et al., 2009). The UPR is made up of three main branches, controlled by ER membrane proteins: activating transcription factor (ATF), inositol-requiring enzyme (IRE), and protein kinase-like ER kinase (PERK), all of which are suppressed by binding to GRP78 (Hummasti and Hotamisligil, 2010). Elevated ER chaperone proteins are biological indicators of ER stress. In mammalian cells, 2 kinases, the inositol-requiring enzyme-1a (IRE1a) and PERK are responsible for activation of UPR. In diabetes, ER stress is caused by increased secretory demands to compensate for insulin resistance and is further aggravated by a fatty acid-induced ER dysfunction in b-cells (Saltiel and Kahn, 2001). Under these conditions, IRE1a activation induces messenger RNA (mRNA) splicing of transcription factors, whereas PERK activation inhibits protein translation and increases the expression of proapoptotic C/EBPhomologous protein (CHOP) via phosphorylation of eukaryotic initiation factor 2a (eIF2a) (Zinszner et al., 1998). In accordance with these data, we found that expression levels of ER stress markers, including CHOP, GRP78, and ATF6, were elevated in db/db mice. Protein expression levels of P-IRE1a, P-eIF2a, and P-PERK were much higher in livers of db/db mice than in those

of db/m mice, an effect reversed by FGF1 treatment. Taken together, these data suggest that FGF1 exerted effects on diabetes-induced liver injury by suppressing diabetes-induced ER stress.

According to previous studies, overload of misfolded and/or aggregated proteins in ER lumen leads to ER dysfunction, resulting in ER stress and apoptosis (Varadarajan et al., 2012). Apoptosis is a primary characteristic of the pathogenesis of liver disease. In accordance with results of previous studies, we found that diabetes boosted apoptosis-related proteins expression levels including Bax and cleaved caspase3, and decreased Bcl2 protein expression level, all of which were reversed by FGF1 treatment. Hepatic apoptosis is regulated by autophagic activity. In addition, cross-talk between autophagy and apoptosis have been manifested by regulatory genes sharing common pathways. These regulatory genes include ATG5 and Bcl2 (Kewei, 2015). Previous studies showed that reduction in expression of autophagy-related protein ATG5, and ATG protein is an essential mediator of the autophagic process, resulting in defective insulin responsiveness. In the current study, we demonstrated that FGF1 increased defective hepatic autophagy in db/db mice via regulation of autophagy related proteins, including LC3II, ATG5 and p62 (Figure 4).

In summary, our findings expand the notion that FGF1 controlled hyperglycemia and normalized blood glucose levels in db/db mice. Treatment with FGF1 for 4 weeks significantly reduced blood glucose levels. These observations were associated with reduced diabetic liver damage. The protective effect of FGF1 on diabetes-induced injury was demonstrated by significantly lower plasma ALT activity levels, TG levels, and hepatic lipid accumulation as well as suppressed cellular stress, reduced diabetes-induced hepatic apoptosis, and restored defective hepatic autophagy. We confirmed that inhibition of cellular stress and restoring autophagy are the potential molecular mechanisms of FGF1 inhibition of diabetes-induced liver injury (Figure 7). One limitation is that we did not record food intake; this will be measured in a future study to fully understand the role of FGF1 in reducing glucose effect and ameliorating diabetes-induced liver injury.

### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

### ETHICS STATEMENT

The animal study was reviewed and approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University & the Laboratory Animal Centre of Wenzhou Medical University.

### AUTHOR CONTRIBUTIONS

YAL and JX conceived and designed the research. ZX performed the experiments and wrote the paper. YW gave important and thoughtful advices and performed the experiments. FW performed the statistical analysis and supplied a lot of technology and served as a fund assistant. XL, PW, YUL, JW, YIL, TJ, XP, XZ, and LX provided assistance with the experiments. All authors discussed the drafting of the manuscript.

### FUNDING

This work was partly supported by the National Natural Science Foundation of China (81300311), the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences, the Technology Support Project of Xinjiang (2017E0267), the Xinjiang Tianshan Youth Project for Outstanding Young Scientists (2017Q007), Natural Science Foundation of Xinjiang Province (2018D01C228), Zhejiang public welfare technology research project (LGF19H030008),

### REFERENCES


Ningbo Huimin project (2016C51004), and Ningbo Natural Science Funding (2018A610376), Project of Medical Technology of Zhejiang Province (2020KY908), and Natural Science Foundation of Xinjiang Uyghur Autonomous Region (2018D01C228).

### ACKNOWLEDGMENTS

We thank Prof. Xiaokun Li for his advice on experimental design.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020. 00052/full#supplementary-material

SUPPLEMENT FIGURE 1 | FGFR1-AMPK signaling pathway was activated. (A) Protein expression of FGFR1, P-AMPK and AMPK in liver from db/m, db/db and db/db + FGF1 mice. (B) Intensities of FGFR1 normalized to GAPDH. (C) Intensity of P-AMPK normalized to AMPK. All data are presented as mean ± SEM, n = 8. \*P < 0.05 \*\*P < 0.01 \*\*\*P < 0.001 vs. the db/m group and db/db + FGF1 group.

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HO-1 and TGF-beta1/Smad3 pathway. Molecules 23 (1), 8–9. doi: 10.3390/ molecules23010215


apoptosis. Am. J. Obstet. Gynecol. 212 (6), 802 e801–808. doi: 10.1016/ j.ajog.2015.01.017


Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Xu, Wu, Wang, Li, Wang, Li, Wu, Li, Jiang, Pan, Zhang, Xie, Xiao and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Role of Fibroblast Growth Factor 10 Signaling in Duodenal Atresia

Matthew L. M. Jones1,2,3, Gulcan Sarila<sup>1</sup> , Pierre Chapuis<sup>2</sup> , John M. Hutson1,4,5 , Sebastian K. King1,2,3,4 and Warwick J. Teague1,2,3,4 \*

<sup>1</sup> F. Douglas Stephens Surgical Research Laboratory, Murdoch Children's Research Institute, Melbourne, VIC, Australia, <sup>2</sup> Discipline of Surgery, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia, <sup>3</sup> Department of Paediatric Surgery, The Royal Children's Hospital, Melbourne, VIC, Australia, <sup>4</sup> Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia, <sup>5</sup> Department of Urology, The Royal Children's Hospital Melbourne, Melbourne, VIC, Australia

Introduction: Duodenal atresia (DA) is a congenital bowel obstruction requiring major surgery in the first week of life. Three morphological phenotypes are described, reflecting increasing degrees of obstruction and discontinuity of the duodenum. The cause of DA is not known. Tandler's original "solid cord" hypothesis conflicts with recent biological evidence, and is unable to account for differing DA types. In humans, a genetic etiology is supported by the association between Trisomy 21 and DA, and reports of familial inheritance patterns. Interruption of FGF10/FGFR2b signaling is the best demonstrated genetic link to DA in mice, with 35–75% of homozygous knockout embryos developing DA.

#### Edited by:

Saverio Bellusci, University of Giessen, Germany

#### Reviewed by:

Stefano Rivetti, University of Giessen, Germany Denise Al Alam, University of Southern California, United States

> \*Correspondence: Warwick J. Teague warwick.teague@rch.org.au

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 22 November 2019 Accepted: 24 February 2020 Published: 10 March 2020

#### Citation:

Jones MLM, Sarila G, Chapuis P, Hutson JM, King SK and Teague WJ (2020) The Role of Fibroblast Growth Factor 10 Signaling in Duodenal Atresia. Front. Pharmacol. 11:250. doi: 10.3389/fphar.2020.00250 Purpose: This review examines the current evidence surrounding the etiology of DA. We focus on research regarding FGF10/FGFR2b signaling and its role in duodenal and other intestinal atresia. Further, we outline planned future research in this area, that we consider necessary to validate and better understand this murine model in order to successfully translate this research into clinical practice.

Conclusion: Determining the etiology of DA in humans is a clinical and scientific imperative. Fgf10/Fgfr2b murine models represent current science's best key to unlocking this mystery. However, further research is required to understand the complex role of FGF10/FGFR2b signaling in DA development. Such complexity is expected, given the lethality of their associated defects makes ubiquitous interruption of either Fgf10 or Fgfr2b genes an unlikely cause of DA in humans. Rather, local or tissue-specific mutation in Fgf10, Fgfr2b, or their downstream targets, is the hypothesized basis of DA etiology.

Keywords: duodenal obstruction, congenital intestinal atresia, fibroblast growth factor 10, fibroblast growth factor receptor 2b, morphogenesis

### INTRODUCTION

Duodenal atresia (DA) is a congenital foregut malformation affecting 1 in 5,000–10,000 live births (Escobar et al., 2004). Three morphological types of DA are described, reflecting increasing degrees of obstruction and discontinuity; type 1: bowel continuity but luminal obstruction or stenosis, type 2: bowel discontinuity with a connecting "bridge" of tissue, and, type 3: bowel discontinuity

**170**

examples of type 1 DA (F); type 2 DA (G); and type 3 DA (H) including demonstration of an intact esophagus, in the presence of tracheal atresia (H). Annotations denote scale bars, and location of the pylorus, P. Arrows indicate location of an atresia. Figures (E–H) reused from Teague et al. (2018), held under the CC-BY 4.0 license.

with complete separation (Skandalakis and Gray, 1994; **Figures 1A–D**). Duodenal atresia was first described in humans by Calder (1733), but it was not until the early twentieth century that Vidal and Ernst performed the first reported successful surgical repair (Vidal, 1905; Ernst, 1916). Nowadays, DA is routinely surgically repaired in the first week of life, with reestablishment of duodenal continuity and minimal postoperative morbidity or mortality (Zani et al., 2017).

The cause of DA is not known. In 1900, Julius Tandler published a hypothesis on the origins of DA, based on his studies of normal duodenal development (Tandler, 1900). Tandler meticulously dissected 11 human embryos and theorized, on the basis of his macroscopic observations, that normal duodenal development includes a "solid cord" phase due to exuberant endodermal growth. As such, he proposed that failure of this "solid cord" to re-canalize may result in DA in humans. Even then, Tandler cautioned readers, stating: "It is clear to me that the opinion represented here does not exceed the status of a new hypothesis, and it is not meant to exceed this." (Nichol et al., 2011). Notwithstanding, Tandler's "solid cord" hypothesis is generally accepted worldwide by the majority of pediatric surgeons. This is despite conflicting developmental biology findings (Cheng and Tam, 1998; Botham et al., 2012), and failure of Tandler's theory to account for the morphological variations of DA (Merrot et al., 2006).

As will be discussed in detail in this review, murine studies have shown that interruption of the Fgf10-Fgfr2b signaling axis may result in DA in 35–75% of knockout mice (Fairbanks et al., 2004b; Kanard et al., 2005; Botham et al., 2012; Reeder et al., 2012b; Teague et al., 2018). This also supports a genetic etiology for DA in humans, however, no specific genetic cause has been demonstrated to date. In humans, the association between DA and Trisomy 21 is well recognized, with DA occurring in 20% of cases (Khan et al., 2017). Yet, animal models of Trisomy 21 fail to demonstrate DA or other associated gastrointestinal malformations (Delabar et al., 2006). Again in humans, an autosomal recessive inheritance pattern for familial cases has been proposed, albeit based on a limited number of isolated and historical case reports (Fonkalsrud et al., 1969; Berant and Kahana, 1970; Best et al., 1989; Gross et al., 1996; Lambrecht and Kluth, 1998). Finally, whilst associations between DA and specific chromosomal anomalies have been reported (Mora et al., 2014; Zamfir et al., 2016), none relate to Chromosome 21, or to Chromosome 5 which houses the FGF10 gene in humans.

### FGF SIGNALING IN GASTROINTESTINAL DEVELOPMENT

The mammalian fibroblast growth factors (FGF) family contains 22 genes, 18 of which have been shown to bind and activate tyrosine kinase FGF receptors (FGFR), using either heparinlike molecules or klotho cofactors receptor binding (Ornitz and Itoh, 2015). These 18 FGFs have been grouped into six subfamilies based on biochemical function, sequence homology and evolutionary traits (Ornitz and Itoh, 2001). The FGF7 sub-family is comprised of FGF3, FGF7, FGF10, and FGF22, and preferentially binds and activates FGFR2b (the IIIb splice variant of the FGFR2 receptor). This receptor is expressed in the epithelial layer and can be activated by FGF7 and FGF10 ligands (Ibrahimi et al., 2001). These ligands show no binding activity with the mesenchyme-expressed FGFR2c variant (Igarashi et al., 1998; Zhang et al., 2006). Conversely, FGF8 ligand preferentially binds and activates FGFR2c, with no binding activity toward FGFR2b (Macarthur et al., 1995; Ornitz et al., 1996).

The activation of FGFR2b by FGF10 is associated with mesenchymal/epithelial interactions which instruct branching and budding morphogenesis. This is a key signaling pathway for normal development of the gastrointestinal tract [e.g., stomach (Spencer-Dene et al., 2006), duodenum (Kanard et al., 2005), and large intestine (Burns et al., 2004)] and its derivatives [e.g., lung (Park et al., 1998)]. Indeed, gastrointestinal development involves multiple signaling pathways and their associated transcription factors, including FGF10-FGFR2b, which are summarized in **Figure 2A** and reviewed elsewhere (Kim and Shivdasani, 2016; Danopoulos et al., 2017).

FGF10-FGFR2b binding is implicated in the activation of multiple intracellular signaling pathways, including the ERK1/2, MAP kinase (MAPK) pathway, to induce downstream activation of proliferation and cell survival genes (Luo et al., 2008; Yang et al., 2011; **Figure 2B**). TBX4/TBX5 may induce Fgf10 expression to promote region-specific stimulation of the mesenchymal proliferation at the initial sites of foregut growth. This growth depends on the regulation of the HOX and TBX genes, which are mediated by retinoic acid (RA) (Wang et al., 2006). Potentially, RA acts on TBX genes, such as TBX4 and TBX5, as a co-factor to activate FGF10 transcription and, as a result, mediates the FGF10-FGF8 positive feedback loop which is required for outgrowth (Nishimoto et al., 2015). Embryos that are RA-deficient fail to outgrow due to defective FGF10 signaling, resulting in a lack of FGF-target genes in the epithelium. Interestingly, the development of the foregut may be partly restored in the defective embryos by introducing RA supplementation early on in development (between E7.5 and E8.5), suggesting that RA regulation is upstream of FGF10 (**Figure 2B**; Wang et al., 2006).

Within the cecum and proximal colon FGF9 binds to the mesenchyme-expressed FGFR2c receptor to direct cecal mesenchyme expansion via the expression of PITX2. In conjunction with this, FGF10 acts to trigger the budding of the epithelium into the cecal mesenchyme, again through mediation of PITX2 expression. Thus, the interaction between FGF10 and PITX2 is critical for the maintenance and the proliferation of the epithelial progenitor cells of the colon (**Figure 2B**). During embryonic development, FGF10 is also expressed in the mesenchyme of the distal colon, where it binds and activates FGFR1b and FGFR2b to promote proliferation, differentiation and survival of the epithelium. This activation maintains progenitor cells within the embryonic small intestine by targeting E26 transformation-specific (ETS)-Family genes such as ETV4, ETV5, and CCND1 through the MAPK pathway (Nyeng et al., 2011; **Figure 2B**).

The homozygous deletion of the Fgf10 or Fgfr2b gene results in mice with multiple organ defects, including lung, limb and mammary glands (with failure to form buds 1, 2, 3, and 5). The resultant mice are non-viable after birth due to lung agenesis (De Moerlooze et al., 2000; Mailleux et al., 2002).

### Fgf10/Fgfr2b SIGNALING IN DUODENAL ATRESIA

Fairbanks et al. (2004b) were the first to identify a link between the Fgf10/Fgfr2b signaling pathway and intestinal atresia in mice. Using Fgfr2b knockout mice, they observed DA in 35% of null embryos when examined at gestational stage E18.5. These authors reported examples of both type 1 DA (57%) and type 3 DA (43%), but no type 2 DA (Fairbanks et al., 2004b). The same group then turned their attention to interruption of the ligand instead of receptor, knocking out Fgf10 (Kanard et al., 2005). Interestingly, Fgf10 knockout embryos were found to have a similar penetrance of DA (38%) compared to Fgfr2b knockouts, but with notably different relative frequency of DA types; type 1 DA (8%), type 3 DA (92%), and still no type 2 DA (Kanard et al., 2005). Further emphasizing the importance of the Fgf10/Fgfr2b pathway in intestinal development, deletion of either Fgf10 or Fgfr2b also resulted in a more distally located intestinal atresia, i.e., colonic atresia (Fairbanks et al., 2004a). In contrast with the incomplete penetrance of DA, colonic atresia was present in 100% of Fgf10 and Fgfr2b knockout mice embryos (Fairbanks et al., 2004a).

Importantly, the morphology of the duodenum is normal in Fgf10 hypomorphic mice, with no examples of DA in this setting of Fgf10 under-expression (Teague et al., 2018). Thus, even reduced Fgf10 signaling is sufficient for morphologically normal duodenal development. Similarly, Sala et al. (2006) found normal colonic development in Fgf10 hypomorphic mice, whereas complete loss of Fgf10 gene function resulted in colonic atresia. In the colon, Fgf10 expression was shown to be expressed first distally before extending proximally, and was found to be critical for the maintenance and proliferation of the epithelial progenitor cells in the colon. Furthermore, crypt formation seen within the proximal colon in the absence of Fgf10 suggested other signaling pathways were active in the development of this region, reinforcing the early anatomical and functional regionalization of the GI tract early during development. Another important finding of Sala et al. (2006) was that atresia occured without and associated loss of mesenchyme. This finding makes vascular compromise an unlikely mechanism for colonic atresia in Fgf10

FIGURE 2 | Schematic illustrations of the role of FGF10 in the developing and developed murine intestine. (A) The expression-dependent changes of key transcription factors in the foregut, midgut and hindgut mediated by differing levels of FGF10 and Wnt-signaling expression; hematopoietically expressed homeobox protein (HHEX), SRY (sex determining region Y)-box 2 (SOX2) in the foregut; pancreatic and duodenal homeobox 1 (PDX1), Sonic Hedgehog (SHH), homeobox genes (HOX), Bone morphogenetic proteins (BMP) in the midgut; Caudal-Type Homeobox Protein (CDX), Indian hedgehog (IHH) in the hindgut. (B) Key FGF10-pathways involved the development of the duodenum, cecum/proximal colon and distal colon; Fibroblast growth factor (FGF) 8, 9, and 10; Tyrosine kinase fibroblast growth factor receptors (FGFR) 2b, 2c; extracellular signal-regulated kinases (ERK1/2); mitogen-activated protein kinase (MAPK); Thromboxanes B4 (TXB4); Thromboxanes B5 (TBX5); ETS translocation variant 4 (ETV4); ETS translocation variant 5 (ETV5); Cyclin D1 (CCND1); Sonic Hedgehog (SHH).

knockouts, as a vascular event would be expected to cause loss of both mesenchyme and epithelium.

The impact of Fgf10 over-expression has also been investigated in both ex vivo and in vivo models. Torashima et al. (2016) showed that the addition of Fgf10 to tissueengineered small intestine (TESI) resulted in increased size and weight of the sample, and increased villi height and crypt depth. In this setting, epithelial differentiation was not inhibited by over-expression of Fgf10, a finding attributed to the lack of normal feedback inhibitory mechanisms in the ex vivo TESI samples. Nyeng et al. (2011) studied in vivo over-expression of Fgf10 within the developing duodenum. In these embryos, Fgf10 over-expression caused attenuation of cell differentiation and expansion of the progenitor niche along with upregulation of

FGFR signaling targets known to exert feedback inhibition on the FGF pathway (e.g., Sprouty2, Etv4, and Etv5) (Minowada et al., 1999; Nyeng et al., 2011). Conversely, complete loss of Fgf10 led to the premature differentiation of all enteric cell lineages in the duodenum (Nyeng et al., 2011). These findings support the inference that Fgf10 is necessary to prevent premature differentiation of enteric cell lineages, and so maintain the epithelial progenitor cell population during development. In extension of this conclusion, these authors hypothesized that premature differentiation and resultant depletion of the progenitor cell population is responsible for the epithelial deficiencies of DA in Fgf10 knockout mice (Nyeng et al., 2011).

An alternative to the premature differentiation hypothesis, is that proposed by Botham et al. (2012). These investigators concluded that exaggerated endodermal apoptosis, rather than terminal differentiation of progenitor cells was the root cause for DA in Fgfr2b knockout mice. Apoptosis was seen in the endoderm at E10.5, disappearance of the endoderm in the atretic region by E11.5, followed by involution of the mesoderm in the absence of further apoptosis at E13.5. This timeline corresponded with first recognition of DA in these Fgfr2b knockout mice, also at E13.5. Comparisons were also made between the duodenum of Fgfr2b knockout mice and otherwise normal, human embryo samples. In mice, loss of endoderm is complete at an earlier stage of development (E11.5, Carnegie Stage 16). In humans, however, "epithelial crowding" with corresponding apparent loss of duodenal lumen is not seen until Carnegie Stage 17 with "recanalization" only at Carnegie Stage 18. Thus, there is a temporal dislocation between luminal occlusion in atretic murine and normal human duodenal development (Matsumoto et al., 2002; Botham et al., 2012). The timeline for murine DA is supported by the findings of Nichol et al. (2012), who showed murine duodenal narrowing evident from E11.5 and DA from E13.5.

Yet another hypothesis is that of Merei (2004), who proposed that intestinal atresia may be the result of aberrant notochord signaling. Reeder et al. (2012a) used Fgfr2b knockout mice, reporting 42% penetrance of DA and 100% penetrance of colonic atresia. These investigators refuted the role of aberrant notochord signaling as the basis for murine DA, showing neither disruption of sonic hedgehog (Shh) expression nor notochordal discontinuity in Fgfr2b knockouts. Furthermore, organ culture of explanted wildtype murine gut in the presence of supraphysiological concentrations of Shh failed to induce atresia in either the duodenum or colon.

One striking feature of the DA reported in the aforementioned, seminal studies is the morphological types of DA reported in Fgf10 and Fgfr2b knockout mice (Fairbanks et al., 2004b; Kanard et al., 2005; Botham et al., 2012; Reeder et al., 2012b). In humans, the distribution of DA types shows similar proportions of type 1 and type 3 DA, and few cases of type 2 DA; e.g., 54% type 1, 4% type 2, and 42% type 3 DA (Khan et al., 2017). In contrast to this, when collated together, 93% of all murine DA cases reported in these studies were examples of type 3 DA, i.e., the most severe type of DA with complete discontinuity of the duodenum (**Figure 1D**; Fairbanks et al., 2004b; Kanard et al., 2005; Botham et al., 2012; Reeder et al., 2012b). Also, none of these studies reported any examples of type 2 DA in mice.

Of relevance to DA type and the severity of malformation is the work by Reeder et al. (2012b). Reeder et al. (2012b) investigated a role for retinaldehyde dehydrogenase 2 (Raldh2), a gene which encodes the enzyme responsible for the final step of vitamin A conversion to RA. Raldh2 is critical for normal posterior foregut development (Wang et al., 2006), and is underexpressed in the duodenum of Fgfr2b knockout mice (Botham et al., 2012). Therefore, Reeder et al. combined heterozygous Raldh2 deletion with homozygous deletion of Fgfr2b with the hypothesis of generating a more severe DA phenotype than with homozygous deletion of Fgfr2b alone. Contrary to this hypothesis, addition of Raldh2 haploinsufficiency resulted in a reduced rather than increased penetrance of atresia (21 vs 45%), and a shift toward less severe atresia types: Fgfr2IIIb−/−; Raldh2± (80% type 1 DA, 20% type 2 DA, and no type 3) versus Fgfr2IIIb-/-; Raldh2+/+ (7% type 1 DA, 0% type 2 DA, and 93% type 3 DA). These authors suggested that signaling downstream of Fgf10/Fgfr2b, i.e., Raldh2, was responsible for the phenotypic variations in DA type morphology.

We have recently reported a more human-like distribution of DA types, including type 2 DA, in two strains of CRISPR-derived Fgf10 knockout mouse (Teague et al., 2018). Of note, these newly reported strains; tm1 (B6- Fgf10<c.[464\_470dup;506\_645del]APNMu>); and tm2 (B6-Fgf10<c.495\_507delAPNMu>) (Eppig et al., 2006) each demonstrate all three DA types (**Figures 1F–H**), and a significantly higher penetrance of DA than previous animal models [74% (Teague et al., 2018) versus 35–45% (Fairbanks et al., 2004b; Kanard et al., 2005; Botham et al., 2012; Reeder et al., 2012b). The basis for such differences in type-distribution and penetrance when compared with previously reported Fgf10 knockout mouse strains remains unclear. The various strains are of consistent background, namely C57/Bl6 (Fairbanks et al., 2004b; Kanard et al., 2005), and whilst the genetics differ, each represents a nonsense mutation (Eppig et al., 2006). We hypothesize that signaling molecules and pathways downstream of Fgf10-Fgfr2b are moderating the morphology and penetrance of the DA phenotype in these various strains.

Our hypothesis is supported by the findings of Al Alam et al. (2012). While an FGF9/Pitx2/FGF10 signaling axis has been proposed within the lung (De Langhe et al., 2008), Al Alam et al. demonstrated that the same signaling axis is active in developing gut (Al Alam et al., 2012). Using tissue specific Fgf9 and Pitx2 conditional knockout they demonstrated that, within the cecum, FGF9 controls mesenchymal Pitx2 expression and, in turn, Fgf10 expression. They also found that Pitx2 induced the expression of Fgf10, even in the absence of Fgf9, suggesting that Pitx2 is downstream of Fgf9. In further attempts to define pathways upstream of Fgf10 involved in atresia, Reeder et al. investigated the role that Shh plays with Fgfr2b (Reeder et al., 2014). Using a Matrigel culture system and focusing on the colon of Fgfr2b knockout mice, they found a downregulation of Shh at E11.5 throughout the entire colon, and then loss of Foxf1 expression 12 h later in the mid-to-distal colon prior to its

involution. They also found that the exogenous addition of Shh to the Fgfr2b deficient colon failed to prevent the formation of atresia, suggesting that the loss of Shh is not a critical event in atresia formation.

### FORTH-COMING RESEARCH INTO DUODENAL ATRESIA ETIOLOGY

Based on the currently available literature regarding Fgf10- Fgfr2b signaling in duodenal atresia, we hypothesize that the etiology of DA in humans is genetically driven, and that the causative genetic changes are downstream of Fgf10- Fgfr2b. This downstream locus may account for both the incomplete penetrance of DA in the murine model, and the different morphological types (i.e., severity) of DA. It may also explain why human DA patients lack the nonsurvivable associations of Fgf10 deletion such as pulmonary agenesis. Therefore, we plan to test this hypothesis by further investigating the up- and down-regulation of genes and pathways in atretic and morphologically normal duodenum. Coordinated RNA sequencing, validation, and protein interaction analyses are planned, focusing on the duodenum of CRISPR-derived Fgf10 knockout mice with and without duodenal atresia, as well as additional wildtype controls. We consider that all three of these mice populations are necessary to discern candidate genes and pathways responsible for DA within the murine model. Also, given the complete penetrance of colonic atresia in the setting of Fgf10-Fgfr2b signaling interruption, we plan additional experiments to delineate the up- and down-regulation of genes and pathways in atretic and morphologically normal colon. We consider the results of colonic analyses may aid discernment of the genetic changes which hold true relevance for atresia formation, amongst the high volume of genetic changes we expect to observe in this model. Furthermore, we plan to test the aforementioned dose-dependence hypothesis, by scrutinizing gene expression differences between the three morphological types of DA in our CRISPR-derived Fgf10 knockout mice.

In order to determine the translational relevance of the genetic analysis within our murine DA model, we plan to study the genetic profile of a thought-provoking subset of human DA patients with known associated anomalies akin to Fgf10-Fgfr2b signaling-related defects, e.g., craniofacial, limb, and lung anomalies. We consider this human DA subset to be at particular "risk" of demonstrating an underlying genetic basis for their DA related to the FGF10-FGFR2b signaling pathway. For this analysis, we will screen our institutional DA patient database for case with potential Fgf10-Fgfr2b signaling-related defects, and perform detailed clinical genetic phenotyping and exon sequencing of phenotypically homogenous patient groups. During the subsequent bioinformatic and genetic analyses, we will pay particular attention those genes and pathways evidenced to be associated with DA in our murine model.

Ultimate translation of these further studies will be to determine the cause of DA in humans, which will have immediate and therapeutic application in the clinical context of antenatal counseling. Also, these may provide the bases for future hypothesis-driven clinical targets and trials to ameliorate or prevent the development of DA in humans.

### SUMMARY

Interruption of FGF10-FGFR2b signaling is the best demonstrated genetic link to DA in mice. This corresponds in importance, and hypothesis-formulating relevance, to the association between DA and Trisomy 21 in humans. FGF10 haploinsufficiency and FGF10 mutations have been linked to human disease, principally to craniofacial syndromes (Entesarian et al., 2005; Milunsky et al., 2006; Rohmann et al., 2006), chronic respiratory disease (Klar et al., 2011), but no human gastrointestinal tract atresia cases to date (Tatekawa et al., 2007). In contrast to this narrative of haploinsufficiency, homozygous Fgf10 invalidation appears necessary for DA in our animal models (Teague et al., 2018), albeit accompanied by non-survivable anomalies such as lung agenesis. Therefore, we hypothesize that DA is caused by conditional tissue-specific deletion in Fgf10, Fgfr2b or their downstream targets (e.g., spatially limited to the duodenum or foregut). Constitutional deletion of this nature is less likely to explain DA in humans for reasons of non-survivable anomalies as detailed previously.

We consider determining the etiology of DA in humans to be a clinical and scientific imperative. Therefore, further study is required to understand the exact role of FGF10-FGFR2b signaling in this. Murine models of DA based on interruption of this signaling will be key to unlocking the mystery of DA in humans. Parallel genetic studies of human DA patients will enable translation of model-derived lessons from bench to bedside.

### AUTHOR CONTRIBUTIONS

MJ conceived the project, conducted the review of literature, analyzed the evidence, and wrote and finalized the manuscript. GS provided specific scientific input into the analysis of evidence, and assisted in writing and finalizing the manuscript. PC mentored MJ and assisted in finalization of the manuscript. JH mentored MJ, and assisted in the analysis of evidence and finalization of the manuscript. SK mentored MJ, and assisted in the review of literature, analysis of evidence, and finalization of the manuscript. WT mentored MJ, conceived the project, and assisted in the review of literature, analysis of evidence, and writing and finalization of the manuscript.

### FUNDING

Our laboratory's past and future work included in this review is supported by a Royal Australasian College of Surgeons (RACS) Foundation in Surgery Small Project Grant, a Colorectal Surgical Society of Australia and New Zealand (CSSANZ) Foundation Research Grant, and an Australian New Zealand Association

of Paediatric Surgeons (ANZAPS) Douglas Stephens Research Grant. MJ is supported in his higher degree research through the John Brooke Moore Scholarship at the University of Sydney. In addition, WT and SK are both generously supported by The Royal Children's Hospital Foundation, Melbourne.

### REFERENCES


### ACKNOWLEDGMENTS

The authors would like to acknowledge Mr. Bill Reid, The Royal Children's Hospital Creative Studio, for his assistance in the art production of the figures for this review.


mutation. J. Pediatr. Surg. Case Rep. 2, 492–494. doi: 10.1016/j.epsc.2014. 10.003


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Jones, Sarila, Chapuis, Hutson, King and Teague. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# SIRT1 Mediates Effects of FGF21 to Ameliorate Cisplatin-Induced Acute Kidney Injury

Qiongzhen Chen<sup>1</sup>† , Junfeng Ma<sup>2</sup>† , Xiaoning Yang<sup>2</sup> , Qinyao Li<sup>2</sup> , Zhuofeng Lin2,3 \* and Fanghua Gong2,3 \*

<sup>1</sup> College of Life and Environmental Science, Wenzhou University, Wenzhou, China, <sup>2</sup> School of Pharmacy, Wenzhou Medical University, Wenzhou, China, <sup>3</sup> Engineering Laboratory of Zhejiang Province for Pharmaceutical Development of Growth Factors, Biomedical Collaborative Innovation Center of Wenzhou, Wenzhou, China

Acute kidney injury (AKI) is a common complication in cancer patients. Kidney function is closely related to patients' quality of life and tumor prognosis. Cisplatin is a highly effective anti-tumor drug. However, the use of cisplatin is limited by its nephrotoxicity. It has been reported that FGF21 has a renal-protective function, but the mechanisms by which it does so remain unclear. In this study, we show that the expression of FGF21 is significantly upregulated in both in vitro and in vivo cisplatin-induced AKI models. Administration of recombinant FGF21 to cisplatin-induced AKI mice resulted in significantly decreased blood urea nitrogen (BUN) and serum creatinine levels, as well as significantly reduced protein levels of kidney injury molecule-1 (TIM-1), C-caspase 3, and Bax. H&E-stained kidney sections from cisplatin-induced AKI mice treated with recombinant FGF21 showed a relatively normal renal tissue structure, a reduced number of necrotic sites and vacuolar changes, and decreased casts, suggesting alleviated renal tubular injury. Experiments with an AKI cell model (cisplatin-treated HK-2 cells) yielded similar results as the mouse model; recombinant FGF21 significantly downregulated protein expression levels of TIM-1, C-caspase 3, and Bax. Furthermore, administration of recombinant FGF21 to cisplatin-treated AKI models significantly increased SIRT1 expression, and the beneficial effects of FGF21 on kidney injury were reversed by SIRT1 knockdown. Collectively, our results suggest that SIRT1 mediates the protective effect of FGF21 on cisplatin-induced kidney injury.

Keywords: acute kidney injury, renal tubular injury, fibroblast growth factor 21, sirtuin1, cisplatin

### INTRODUCTION

Acute kidney injury (AKI) is a clinical syndrome characterized by an abrupt decline in kidney function, a decrease in glomerular filtration rate, and the accumulation of nitrogenous waste in the kidney (Vanmassenhove et al., 2017). Many factors contribute to the induction of AKI, including drugs, hypovolemia, sepsis, and surgical injury (Motwani et al., 2018). Hence, AKI is widely distributed among clinical departments (Basile et al., 2016). In hospitalized patients, AKI incidence has reached 5–7% and is still rising, with a mortality rate of 40%. AKI is also important risk factor for the development of chronic kidney disease (Coca et al., 2012; Oh et al., 2017).

#### Edited by:

Zhouguang Wang, Albert Einstein College of Medicine, United States

### Reviewed by:

Chen Li, Charité Medical University of Berlin, Germany Yingnyu Gao, Marshall University, United States

#### \*Correspondence:

Zhuofeng Lin zhuofenglin@wzmc.edu.cn Fanghua Gong gongwenheng@163.com

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 18 October 2019 Accepted: 21 February 2020 Published: 10 March 2020

#### Citation:

Chen Q, Ma J, Yang X, Li Q, Lin Z and Gong F (2020) SIRT1 Mediates Effects of FGF21 to Ameliorate Cisplatin-Induced Acute Kidney Injury. Front. Pharmacol. 11:241. doi: 10.3389/fphar.2020.00241

**178**

**Abbreviations:** AKI, acute kidney injury; CIS, cisplatin; kidney injury molecule-1, TIM-1; SIRT1, sirtuin1; FGF21, fibroblast growth factor 21; CKD, chronic kidney disease; NC, negative control; BUN, blood urea nitrogen; C-caspase 3, cleaved caspase-3.

Cisplatin (CDDP, cis-Diaminodichloroplatinum) is used as a chemotherapeutic drug for the treatment of many different types of cancer. However, about one-third of the tumor patients develop AKI several days after cisplatin treatment. Therefore, the clinical application of cisplatin is frequently limited by its adverse effects (Fernández-Martínez et al., 2016; Kim et al., 2017; Hori et al., 2017; Oh et al., 2017; Li Z. et al., 2018).

Fibroblast growth factor 21 (FGF21) is mainly expressed in the liver, adipose tissue, and pancreatic islet, and is also expressed in muscle and kidney tissues (Nishimura et al., 2000; So and Leung, 2016; Li, 2019). Due to a lack of the typical heparin-binding domain found in the other family members, FGF21 enters the blood circulation through the endocrine system (Fisher and Maratos-Flier, 2016). FGF21 activity depends on its binding to β-Klotho protein and FGF receptors (FGFRs) and the formation of a stable FGF21/β-Klotho/FGFR complex, which regulates the biological effects of downstream signaling molecules (Kuro, 2019). Previous studies showed that serum levels of FGF21 are significantly increased in both AKI and CKD, and FGF21 concentration is correlated with the severity of kidney injury (Li F. et al., 2018; Suassuna et al., 2019). However, numerous studies have demonstrated that FGF21 functions in alleviating inflammation and in the regulation of oxidative stress and autophagy-induced apoptosis (Lu et al., 2019; Suassuna et al., 2019). Recent works also showed that FGF21 significantly reduces urinary albumin levels in type II diabetic mice and alleviates kidney injury (So and Leung, 2016; Zhao et al., 2017).

Sirtuins are NAD-dependent deacetylases that function in multiple biological processes, such as proliferation, DNA repair, mitochondrial energy homeostasis, and anti-oxidation. Mammals contain seven sirtuins (SIRT1–7), located in different subcellular compartments. SIRT1 is one of the most widely studied sirtuins in kidney. It inhibits cell apoptosis, inflammation, and fibrosis by targeting the deacetylation of p53, FoxO1, and NF-κB, and hence functions in protecting cells (Kim and Lee, 2015; Phillipson-Weiner et al., 2016; Wang et al., 2017, 2019; Zhang et al., 2017; Morigi et al., 2018; Sun et al., 2018; Abdolvahabi et al., 2019).

In this study, we found that recombinant human FGF21 (rhFGF21) exerted significant beneficial effects on tubular epithelial cells and alleviated the decline in renal function in cisplatin-induced AKI. In addition, we found that SIRT1 expression was significantly upregulated in renal tubular epithelial cells and renal tissues during the process of cisplatininduced kidney injury. rhFGF21 treatment further increased the expression of SIRT1, resulting in decreased apoptosis in cells and improved kidney function.

### MATERIALS AND METHODS

### Cell Culture

Human renal proximal tubule cells (HK-2) were kindly provided by the Stem Cell Bank at Chinese Academy of Sciences. Cells were cultured in DMEM/F-12 media supplemented with 10% fetal bovine serum (FBS, Gibco, United States), 100 U/ml penicilin, and 100 µg/ml streptomycin. HK-2 cells were transfected with lentivirus siRNA against SIRT1 (LV-SIRT1-RNAi) (Genechem, Shanghai, China) and Control-RNAi (Genechem, Shanghai, China) for 72 h according to the manufacturer's instructions. HK-2 cells were pretreated with/without rhFGF21 (5 nM; provided by Key Laboratory of Biotechnology Pharmaceutical Engineering, Wenzhou Medical University) for 1 h, and cultured in the presence of cisplatin (20 µM, Sigma-Aldrich, United States). Cells were collected at different time points (12, 18, and 24 h).

### Mice and AKI Model

Healthy C57BL/6 male mice (∼25 g, 8 wk of age, purchased from Shanghai Slac Laboratory Animal Co., Ltd.) were kept at the barrier facility of the Laboratory Animal Centre of Wenzhou Medical University. All animals were kept for 1 week before the experiments started. All animal procedures were carried out in accordance with the principles of the Basel Declaration and Recommendations. Two experiments were conducted. The first experiment consisted of three different groups representing three different time points (24, 48, and 72 h). The experimental mice were randomly divided into three subgroups at each time point, i.e., the control group (NC), cisplatin model group (CIS), and treatment group (FGF21 + CIS) (n = 5 in each subgroup). Based on the procedure described by Li F. et al. (2018), we used a high dose (30 mg/kg) of cisplatin in a single intraperitoneal injection to establish the AKI mouse model. The control group was injected with the same volume of normal saline. As for the FGF21 + CIS group, rhFGF21 was dissolved in PBS and stored at 4◦C, and mice were pretreated with rhFGF21 at 0.5 mg/kg intraperitoneally 1 h prior to cisplatin injection. rhFGF21 was administered every 24 h based on the in vivo elimination rate of rhFGF21 after cisplatin injection, and the same volume of physiological saline was injected into other groups. Mice were euthanized at different time points (24, 48, and 72 h) after cisplatin injection. In the second experiment, we randomly divided the experimental mice into four groups (n = 5 per group), i.e., the control group (Ctrl-RNAi), the model group (Ctrl-RNAi + CIS), the treatment group (Ctrl-RNAi + rhFGF21 + CIS), and the SIRT1 knockdown group (LV-SIRT1-RNAi + rhFGF21 + CIS). LV-SIRT1-RNAi or Ctrl-RNAi (1.6 × 10<sup>9</sup> TU/kg) (Zheng et al., 2017) was administered by tail vein injection 72 h before cisplatin treatment, followed by the same procedure as in the first the experiment. Mice were euthanized 72 h after cisplatin injection. Blood and kidney tissues were collected and used for biochemical and histopathological examinations. Serum creatinine levels were measured using a Creatinine Assay Kit (Sarcosine Oxidase method); blood urea nitrogen (BUN) levels were measured by a Urea Nitrogen Kit (Urease method). The kits were obtained from the Nanjing Institute of Bioengineering (Nanjing, China).

### Immunoblotting

Analysis was performed to measure the protein levels in kidney tissue and HK-2 cells. Protein in total cell lysates was extracted and quantified by BCA Protein Assay. Protein was separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules). After blocking the membrane in a TBST solution containing 10% (w/v) non-fat milk for 1.5 h at room temperature, different proteins were probed with corresponding antibodies

overnight at 4◦C. The following antibodies were used: anti-FGF21 (1:1000, Abcam), anti-SIRT1 (1:1000, Abcam), anti-TIM-1 (1:20,000, Abcam), anti-Bax (1:10,000, Abcam), anti-cleaved caspase-3 (1:1000, Abcam), anti-β-Klotho (1:1000, Abcam), antiβ-actin (1:1000, Cell Signaling Technology), and anti-GAPDH (1:1000, Cell Signaling Technology). Subsequently, membranes were washed three times with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Protein signals were detected using a Tanon 5200 Chemiluminescence Imaging system (Tanon-5200) and bands were quantified using ImageJ software.

### Histology and Immunohistochemistry

Prior to histological analysis, kidney tissues were fixed in 4% paraformaldehyde for over 24 h, embedded in paraffin, cut into 5 µm sections, stained with hematoxylin and eosin (H&E), sealed with neutral resin, and observed under an optical microscope. Tissue damage and necrosis were assessed by microscopy (×400) in 10–20 randomly selected fields of view. Tissue damage was scored by the percentages of renal tubular injury, cell debris, brush border loss, and cast formation (0, no damage; 1, less than 25% damage; 2, 25–50% damage; 3, 50–75% damage; 4, more than 75% damage).

For immunohistochemistry analysis, 5 µm kidney tissue sections were incubated with anti-FGF21 (Abcam; Cambridge, United Kingdom) and anti-SIRT1 (Abcam; Cambridge, United Kingdom), followed by the secondary antibody (ZSGB-Bio, Beijing, China) and DAB. For each mouse, 10–20 randomly selected fields of view were scanned under an upright microscope. The percentages of positive staining regions of FGF21 or SIRT1 were quantified using Image-Pro 6.0.

### Immunofluorescent Staining

For immunofluorescent staining analysis, cells were fixed with 4% paraformaldehyde and incubated with Triton X-100 (1%) for 15 min. The cells were incubated with anti-FGF21 and anti-SIRT1 overnight at 4◦C, incubated with fluorescent-probe-conjugated goat anti-rabbit at room temperature for 1 h, stained with DAPI (Abcam, Cambridge, United Kingdom), and mounted. Images were captured using a laser confocal microscope (Nikon, Ti-E and A1 plus) and quantitatively analyzed using Image-Pro 6.0.

### Apoptosis Assay

Five-micrometer-thick tissue sections were used for TUNEL staining with the apoptosis detection kit (Boster Biological Technology Co., Ltd., Wuhan, China). Briefly, the slides were deparaffinized, rehydrated, and treated with proteinase K (20 mg/ml) for 15 min at room temperature. The slide was preliminarily incubated with labeling buffer (digoxigenindUTP) at room temperature for 2 h. Then blocking solution incubated 30 min at room temperature, and added the reaction mixture containing anti-digoxigenin antibody and Fluorescein-Streptavidin (FITC, green), incubated at 37◦C for 30 min. DAPI was used for nuclear counterstaining.

The HK-2 cells (1 × 10<sup>6</sup> cells/well) were seeded on sixwell chamber slides. After different treatments, the slides were detected with the apoptosis detection kit (CY3, red). DAPI was used for nuclear counterstaining. TUNEL-positive cells were imaged under a laser confocal microscope (Nikon, Ti-E and A1 plus).

### Statistical Analysis

Data analysis was performed using GraphPad Prism 6.0 software. All data are expressed as mean ± SEM of three independent experiments; the Student'st-test or one-way ANOVA was used for statistical comparisons of different groups. The Pearson test was performed to determine the linear correlation of two variables. P < 0.05 indicated a significant statistical difference.

### RESULTS

### Cisplatin Induces HK-2 Cell Apoptosis in vitro and AKI in vivo

HK-2 cells were treated with cisplatin for 12, 18, and 24 h. The expression of the apoptosis-related proteins C-caspase 3 and Bax increased in a time-dependent manner (**Figures 1A–C**). TUNEL analysis showed nucleus aggregation and significantly increased numbers of apoptotic cells in HK-2 cells after 24 h as compared with control cells (P < 0.001, **Figures 1D,E** and **Supplementary Figure S1**).

As for in vivo experiments in mice, we found that both BUN levels and serum creatinine levels began to rise 24 h after cisplatin treatment, reaching a maximum at 72 h. Both serum creatinine and BUN concentrations at 72 h were about 4.5 fold greater than in the control group, indicating that kidney function was significantly impaired (P < 0.001, **Figures 1F,G**). Analysis of kidney tissue showed that the protein levels of the AKI markers TIM-1, C-caspase 3, and Bax increased significantly in a time-dependent manner (**Figures 1H–K**). Renal pathological changes were evaluated 72 h after cisplatin administration using H&E staining of mouse kidney paraffin sections. The control group displayed an intact tubular structure, the renal tubules were shaped regularly and arranged neatly, glomerular space was normal without obvious damage, the tubular epithelial cells were arranged in high density, nuclei were round and large, nucleoli were clear and distributed evenly, and no obvious apoptosis and necrosis were found. On the contrary, cisplatin administration resulted in renal tubular damage, necrosis, a large amount of cell debris, vacuolar changes, multiple sites of casts, increased glomerular space, and tissue damage; the average renal tubular injury score was ∼3.5 (**Figures 1L,M**).

Collectively, these findings indicate that cisplatin causes HK-2 cell apoptosis in vitro, and induces AKI in vivo, with an impaired tubular structure and a reduced glomerular filtration rate.

### Cisplatin Induces a Significant Upregulation of FGF21 in Renal Tubular Cells in vitro and in vivo

Because FGF21 plays an important role in energy metabolism and cellular stress response, we examined the expression of FGF21 in HK-2 cells after cisplatin treatment. FGF21 expression was significantly upregulated after 12 h; the highest FGF21 levels were

FIGURE 1 | Cisplatin induces tubular cell damage in in vitro and in vivo AKI models. (A–C) Representative immunoblotting analysis of C-caspase 3 and Bax in HK-2 cells. Protein levels of C-caspase 3 and Bax were quantified by densitometry, with β-actin as protein loading control. (D,E) TUNEL staining in HK-2 cells (scale bar: 100 µm; DAPI: blue, TUNEL: red), and quantitative analysis of TUNEL staining. (F) Changes in mouse BUN levels. (G) Changes in mouse serum creatinine levels. (H–K) Representative immunoblotting analysis of TIM-1, C-caspase 3, and Bax in mouse renal tissue. Expression levels of TIM-1, C-caspase 3, and Bax were quantified by densitometry, with GAPDH as protein loading control. (L) H&E staining of mouse renal tissue (scale bar: 100 µm). (M) Histopathological scores of H&E staining of mouse kidney tissue. \*\*\*P < 0.001, \*\*P < 0.01, \*P < 0.05.

observed at 24 h (P < 0.001, **Figures 2A,B**). Expression levels of the FGF21 receptor β-Klotho also increased (**Figures 2A,C**). Immunofluorescence staining revealed a significant increase in intracellular protein levels of FGF21 after 24 h of cisplatin treatment (P < 0.001, **Figures 2D,E**).

Levels of FGF21 and FGF21 receptor β-Klotho in the AKI mouse model were measured at different time points after cisplatin administration (24, 48, and 72 h). Consistent with the cell culture experiments, in vivo FGF21 expression increased in a time-dependent manner,

and β-Klotho was significantly upregulated 72 h after cisplatin administration (P < 0.001, **Figures 2F–H**). Immunohistochemistry results showed that 72 h after cisplatin administration, the levels of FGF21 were significantly increased in the area of renal tubular injury, especially in the areas with severe tissue damage or necrosis (P < 0.001, **Figures 2I,J**).

Collectively, our results indicate that cisplatin induces the upregulation of FGF21 expression in renal tubular cells in vitro and in vivo.

### rhFGF21 Significantly Attenuates Cisplatin-Induced Acute Injury of Renal Tubular Cells in vitro and in vivo

HK-2 cells were pretreated in vitro with rhFGF21 for 1 h before cisplatin treatment. We found that rhFGF21 downregulated the expression of C-caspase 3 and Bax differently at different time points (12, 18, and 24 h) compared with cisplatin-treated cells (**Figures 3A–C**). Moreover, TUNEL staining results indicate that the apoptosis rate of cisplatin-treated HK-2 cells was >50%. In comparison, rhFGF21 pretreatment reduced the percentage of apoptotic cells to less than 20% (**Figures 3D,E**).

Similarly, we studied the protective effects of rhFGF21 on renal tissues in the mouse AKI model. Our results indicate that at 24 h after rhFGF21 administration, there was no significant difference in BUN and serum creatinine levels among the three groups. At 48 h and later, the administration of rhFGF21 significantly decreased the BUN and serum creatinine levels (**Figures 4A,B**). In addition, immunoblot analysis demonstrated that mice treated with both cisplatin and rhFGF21 had significantly lower levels of TIM-1, C-caspase 3, and Bax than those treated with cisplatin alone (**Figures 4C–F**). Renal pathological changes were evaluated 72 h after cisplatin administration by H&E staining of mouse kidney paraffin sections. Microscopic evaluation of the control group showed that the tubular structure was unchanged, tubular epithelial cells were arranged in high density, and no obvious tissue damage was observed. In contrast, intraperitoneal injection of cisplatin resulted in impaired tubular structure, accompanied by increases in glomerular space and casts, a large amount of cell debris, and vacuolar changes, and the average renal tubular injury score was ∼3. rhFGF21 treatment reversed cisplatin-induced renal injury.

**183**

Although tissue damage was more severe than in the control group, rhFGF21 treatment resulted in a relatively normal tissue structure, fewer necrotic sites and vacuolar changes, and fewer casts as compared with cisplatin-treated mice. The average tissue damage score was reduced to ∼1 by rhFGF21 (**Figures 4G,H**).

These experimental results demonstrate that rhFGF21 plays a cytoprotective or prosurvival role during cisplatin treatment in HK-2 cells and mouse AKI.

### rhFGF21 Upregulates SIRT1 Expression in Renal Tubular Cells in vivo and in vitro

SIRT1 is a key player in cisplatin-induced AKI and nephrotoxicity (Kim et al., 2019; Zhang et al., 2019). In cisplatin-treated HK-2 cells, SIRT1 protein levels were upregulated, similarly to FGF21 (**Figures 5A,B**). In cisplatin-treated HK-2 cells, SIRT1 expression was increased, and rhFGF21 further promoted SIRT1 expression (**Figures 5C,D** and **Supplementary Figures S2A–D**). Quantification of immunofluorescence signals confirmed that rhFGF21 promotes the expression of SIRT1 (**Figures 5E,F**).

In the AKI mouse model, SIRT1 expression was not significantly increased at 24 h, but it was increased at 48 h and later (**Figures 5G,H**). Consistent with our in vitro study, we found that rhFGF21 treatment upregulated SIRT1 expression in AKI mice at different time points (P < 0.05 at 24 h, **Figures 5I,J**). Our immunohistochemistry results indicate that cisplatin induced expression of SIRT1 72 h after cisplatin injection, and rhFGF21 further promoted renal SIRT1 expression (**Figures 5K,L**).

Collectively, these results indicate that rhFGF21 can upregulate SIRT1 expression in injured renal tubular cells in a timely manner. Hence, the underlying mechanisms by which rhFGF21 attenuates cisplatin-induced AKI may involve SIRT1.

### SIRT1 Mediates FGF21-Induced Attenuation of Cisplatin-Induced HK-2 Cell Apoptosis and Mouse AKI

SIRT1 plays an important role in the repair of organ and cell damage. Our previous experiments showed that cisplatin induces the expression of SIRT1 in HK-2 cells and mouse kidney tissue. Moreover, rhFGF21 further upregulates the expression of SIRT1 and improves cisplatin-induced AKI. To determine whether rhFGF21 prevents cisplatin-induced AKI by activating SIRT1, we examined the function of rhFGF21 in HK-2 cells after SIRT1 knockdown. Results indicate that SIRT1 expression was effectively downregulated after LV-SIRT1-RNAi transfection in HK-2 cells. SIRT1 protein levels in HK-2 cells were reduced by approximately 80% after LV-SIRT1-RNAi transfection compared with Ctrl-RNAi (**Figures 6A,B**).

Next, we knocked down SIRT1 in HK-2 cells; we found that SIRT1 knockdown significantly reversed the beneficial effect of rhFGF21 on cisplatin-induced apoptosis (**Figures 6C,D**). Compared with the rhFGF21 + cisplatin group, SIRT1 knockdown cells treated with rhFGF21 + cisplatin showed significantly higher expression levels of C-caspase 3 and Bax (**Figures 6C,E,F**). In cells treated with rhFGF21 + cisplatin, TUNEL analysis showed a significant increase in the percentage of apoptotic cells after SIRT1 knockdown (**Figures 6G,H**). These results indicate that the beneficial effects of rhFGF21 on cell damage are significantly decreased in SIRT1-knockdown HK-2 cells.

Similar results were obtained in our in vivo experiments. SIRT1 expression in C57BL/6 mice was effectively knocked down after LV-SIRT1-RNAi transfection (**Figures 7A,B**). In mice treated with rhFGF21 + cisplatin, BUN and serum creatinine levels were significantly increased (**Figures 7C,D**). When SIRT1 expression was inhibited (**Figures 7E,F,J,K**), BUN and serum creatinine levels were higher than in rhFGF21 + cisplatin mice (**Figures 7C,D**). Immunoblot analysis showed that SIRT1 knockdown increased TIM-1, C-caspase 3, and Bax levels in mouse kidney compared with rhFGF21 + cisplatin mice (**Figures 7E,G–I**). H&E staining showed that rhFGF21 could not effectively ameliorate kidney damage after SIRT1 knockdown, and kidney tissue exhibited tubular structure disorder, a large amount of cell debris, vacuolar changes, increased casts and glomerular space, and aggravated tissue damage (**Figures 7L,M**). Furthermore, TUNEL analysis was performed using cisplatin-treated mice after SIRT1 knockdown. We found that the percentage of apoptotic cells in renal tissue was higher than 50%, even after rhFGF21 treatment (**Figures 7N,O**).

In conclusion, these results indicate that SIRT1 knockdown reduces the protective effects of rhFGF21 on cisplatin-induced AKI both in vitro and in vivo.

### DISCUSSION

In this study, we discovered that cisplatin induces acute tubular cell injury in vitro and in vivo. Cisplatin also increases FGF21 and SIRT1 protein levels in kidney. Our results indicate that rhFGF21 administration attenuates cisplatininduced nephrotoxicity. We further explored the involvement of FGF21/SIRT1 in renal protection.

Two phenomena cause cisplatin-induced AKI, i.e., cisplatin accumulation in tubular cells and renal tubular cell injury (Fernández-Martínez et al., 2016; Oh et al., 2017). Tumor patients are prone to AKI during cisplatin treatment. Their clinical manifestations include a decrease in glomerular filtration rate, an increase in BUN and serum creatinine levels, and electrolyte disorders (Arany and Safirstein, 2003). Our results indicate that both BUN levels and serum creatinine levels began to rise 24 h after cisplatin treatment, reaching a maximum at 72 h in mice. Currently, levels of serum creatinine, BUN, cystatin-C, TIM-1, and NGAL are routinely measured to investigate cisplatininduced AKI models (Holditch et al., 2019). Our studies also indicate the protein levels of the AKI markers TIM-1, C-caspase 3, and Bax increased significantly in a time-dependent manner.

Previous studies have shown that FGF21 regulates glucose and lipid metabolism to maintain energy balance and metabolic homeostasis. It is mainly expressed in the liver, adipose tissue, and pancreatic islet under physiological conditions (So and Leung, 2016). It has been shown that the serum FGF21 concentration is significantly increased in patients with diabetic nephropathy, chronic kidney disease, and long-term dialysis (Shahbazi et al., 2015; Li Y.Z. et al., 2018). This is possibly related to the decline in kidney function. Studies also showed that FGF21 has beneficial effects on tissue damage caused by metabolic abnormalities and responses to various cellular and metabolic stresses. It was significantly upregulated as an effective catabolic factor in order to antagonize metabolism and energy imbalance (Gómez-Sámano et al., 2017; Luo et al., 2017). In addition, it has been indicated that upregulation of serum FGF21 levels may delay the progression of diabetic nephropathy and chronic kidney disease (Li F. et al., 2018; Suassuna et al., 2019). Many studies have previously shown that loss of FGF21 causes pathologic damage in multiple animal models, including decreased productivity of pancreatic beta cells and insulin sensitivity (Lee et al., 2018; Wanders et al., 2017), promotion of the development of fatty liver in mice followed by aggravation of liver fibrosis (Singhal et al., 2018), and even aggravation of cisplatin-induced AKI (Holditch et al., 2019). Similarly, overexpression of FGF21 or administration of exogenous FGF21 regulates metabolism, controls the stress response, and attenuates organ damage (So and Leung, 2016; Kang et al., 2018; Lu et al., 2019; Suassuna et al., 2019). Our results are consistent with previous reports. Cisplatin induces a significant upregulation of FGF21 in renal tubular cells in vitro and in vivo. Administration of rhFGF21 in vitro and in vivo reduced cisplatin-induced tubular cell death, significantly decreased BUN and serum creatinine levels, and downregulated the expression of TIM-1, C-caspase 3, and Bax in kidney tissue. We further have confirmed that increased FGF21 expression during AKI can protect the kidneys.

A previous study demonstrated that FGF21 may protect kidney tubular cells by suppressing P53 (Li F. et al., 2018). Here, we investigated the role of SIRT1 from a new perspective. SIRT1 is a key molecule involved in kidney injury (Kim et al., 2019; Zhang et al., 2019). Our study provides both in vitro and in vivo evidence to show that SIRT1 expression is upregulated in damaged tubular cells during cisplatin-induced AKI. Previous studies have demonstrated the beneficial effects of SIRT1 in many organs, including the liver (Zhu et al., 2014; Abd Elwahab et al., 2017), heart (Li et al., 2019), bone (Hou et al., 2019), and

\*\*P < 0.01, \*P < 0.05.

kidney (Kim et al., 2019). In the ischemia-reperfusion AKI model, SIRT1 targets the deoxyacetylation of p53, FoxO1, and NF-κB to regulate the stress response, inflammation, cell senescence, and apoptosis (Grabowska et al., 2017; Zhang et al., 2017; Abdolvahabi et al., 2019; Wang et al., 2019). In addition, SIRT1 mediated inhibition of tubular cell apoptosis has been shown to be the primary mechanism of renal protection (Liao et al., 2019; Ryu et al., 2019; Zhang et al., 2019). Our data also suggest that such a mechanism is important during cisplatin-induced AKI.

Recently, pharmacologic studies have suggested that FGF21 improves cardiac function and alleviates Ang II-induced cardiac hypertrophy in a SIRT1-dependent manner (Li et al., 2019). Similarly, studies have reported that the anti-senescence effect of FGF21 on human umbilical vascular endothelial cells (HUVECs) is SIRT1-dependent (Yan et al., 2017). These findings imply that SIRT1 may contribute to the beneficial effects of FGF21. However, further experiments are needed to verify this hypothesis in cisplatin-induced AKI. In the present study, we demonstrate that rhFGF21 induces the expression of SIRT1, further attenuates renal tubular cell injury, and inhibits apoptosis. Moreover, the renal protective effects of rhFGF21 are decreased upon SIRT1 knockdown, suggesting that SIRT1 mediates the beneficial effects of FGF21 on cisplatin-induced AKI.

In summary, our results demonstrate that FGF21 ameliorates cisplatin-induced nephrotoxicity through SIRT1, preventing kidney failure and tubular cell damage.

### DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

### ETHICS STATEMENT

All animal experimentation was carried out in accordance with the principles of the Basel Declaration and recommendations

### REFERENCES


### AUTHOR CONTRIBUTIONS

QC, JM, XY, and QL performed experiments. FG and ZL coordinated the study and oversaw all experiments. FG wrote the manuscript. All authors discussed the results and commented on the manuscript.

### FUNDING

This research was supported by the Zhejiang Provincial Natural Science Foundation of China under grant LY17H160048 and National Key R&D Program of China (2017YFA0506000).

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2020.00241/full#supplementary-material

FIGURE S1 | TUNEL staining in HK-2 cells at different time points. HK-2 cells were treated with cisplatin for 12 h, 18 h, and 24 h. TUNEL staining in HK-2 cells (scale bar: 100 µm; DAPI: blue, TUNEL: red).

FIGURE S2 | rhFGF21 upregulated SIRT1 expression in HK-2 cells. (A–D) Representative immunoblotting analysis of SIRT1 in HK-2 cells. Protein levels of SIRT1 were quantified by densitometry, with β-actin as protein loading control. \*\*\*P < 0.001, \*\*P < 0.01, \*P < 0.05.



**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Chen, Ma, Yang, Li, Lin and Gong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Fibroblast Growth Factors in the Management of Acute Kidney Injury Following Ischemia-Reperfusion

Lian-Cheng Deng1† , Tahereh Alinejad1† , Saverio Bellusci 1,2\* and Jin-San Zhang1,2\*

<sup>1</sup> Center for Precision Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, <sup>2</sup> Institute of Life Sciences, Wenzhou University, Wenzhou, China

Ischemia-reperfusion injury (IRI), which is triggered by a transient reduction or cessation of blood flow followed by reperfusion, is a significant cause of acute kidney injury (AKI). IRI can lead to acute cell death, tissue injury, and even permanent organ dysfunction. In the clinic, IRI contributes to a higher morbidity and mortality and is associated with an unfavorable prognosis in AKI patients. Unfortunately, effective clinical drugs to protect patients against the imminent risk of renal IRI or treat already existing AKI are still lacking. Fibroblast growth factors (FGFs) are important regulators of key biological and pathological processes, such as embryonic development, metabolic homeostasis and tumorigenesis through the regulation of cell differentiation, migration, proliferation and survival. Accumulating evidence suggests that altered expression of endogenous FGFs is associated with IRI and could be instrumental in mediating the repair process. Therefore, FGFs have been proposed as potential biomarkers in the clinic. More importantly, exogenous FGF ligands have been reported to protect against renal IRI and display promising features for therapy. In this review, we summarize the evidence and mechanisms of AKI following IRI with a focus on the therapeutic capacity of several members of the FGF family to treat AKI after IRI.

#### Edited by:

Zhouguang Wang, Albert Einstein College of Medicine, United States

### Reviewed by:

Sandra Donnini, University of Siena, Italy Michele Samaja, University of Milan, Italy

### \*Correspondence:

Saverio Bellusci saverio.bellusci@innere.med. uni-giessen.de Jin-San Zhang Zhang\_JinSan@wmu.edu.cn These authors have contributed equally to this work

†

#### Specialty section:

This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology

Received: 05 November 2019 Accepted: 19 March 2020 Published: 08 April 2020

#### Citation:

Deng L-C, Alinejad T, Bellusci S and Zhang J-S (2020) Fibroblast Growth Factors in the Management of Acute Kidney Injury Following Ischemia-Reperfusion. Front. Pharmacol. 11:426. doi: 10.3389/fphar.2020.00426 Keywords: fibroblast growth factors, ischemia-reperfusion injury, acute kidney injury, protection, therapy

### INTRODUCTION

Acute kidney injury (AKI) is a syndrome and significant cause of death among hospitalized patients. AKI is characterized by a rapid decline in renal function and predisposes the transition of AKI patients to chronic kidney disease (CKD) and end-stage renal disease (ESRD) (Chawla et al., 2014). Ischemia-reperfusion injury (IRI) is the primary cause of AKI worldwide related to different clinical

Abbreviations: AKI, acute kidney injury; AKT, protein kinase B; CKD, chronic kidney disease; CSA-AKI, cardiac surgeryassociated AKI; DAMP, damage-associated molecular pattern; ERK, extracellular signal-regulated kinase; ER, endoplasmic reticulum; ESRD, end-stage renal disease; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FRS-2, fibroblast growth factor receptor substrate 2; HMGB1, high mobility group protein 1; HSPG, heparin sulphate proteoglycan, ICK, intracellular kinase domain; IL-1b, Interleukin-1 beta; IL-6, Interleukin-6; IR, ischemia reperfusion; IRI, ischemia reperfusion injury; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor kappa-B; PI3K, phosphatidylinositol-4,5 bisphosphate 3-kinase; PLCg: phospholipase C gamma; ROS, reactive oxygen species; TGF-b: transforming growth factor beta; TLR2; toll-like receptor 2; TLR4, toll-like receptor 4; TM, trans-membrane domain; TNF-a, tumor necrosis factor alpha; UPR, unfolded protein response.

circumstances such as shock, low cardiac output, and organ transplantation (Park et al., 2017; Ronco et al., 2019). Under these conditions, the reestablishment of blood flow after transient obstruction of circulation leads to renal injury. A multinational, multicenter study on critically affected patients confirmed the prevalence of AKI (5.7% of the patients included in the study) and association with a high mortality rate (Uchino et al., 2005). Another independent study shows an even higher percentage of patients admitted to hospital with such a complication, 10–15% of all hospitalisations (Al-Jaghbeer et al., 2018), the number is as high as 50% in patients in the intensive care unit (ICU) 50% (Hoste et al., 2015).

The disease mechanisms underlying the etiology and pathogenesis of AKI are complex and include mitochondrial dysfunction, reactive oxygen species (ROS), endoplasmic reticulum stress (ERS), autophagy, inflammation, apoptosis, and necrosis (Verma and Molitoris, 2015; Duann et al., 2016; Figure 1A). Even though renal IRI is known to be the predominant cause of morbidity and mortality, no effective treatment is currently available. Therefore, much attention has been dedicated to seeking novel therapeutic strategies for AKI. Intense research efforts using animal models have shed light on the pathophysiology of AKI. It has been reported that the expression of Fibroblast growth factors (FGFs) and their receptors (FGFRs) are increased in the context of AKI (Wai et al., 2013; Tan et al., 2017). Furthermore, AKI is more severe upon FGFR deficiency or blockade of its signalling (Villanueva et al., 2008; Xu and Dai, 2017). Some studies demonstrated that renal function recovered after administering various medications, including growth factors and cell transplantation (Ichimura et al., 1996; Patel et al., 2012). These results show that FGFs and their receptors are important for AKI.

In the context of IRI, several growth factors have been reported to exhibit protective effects as well as therapeutic potential as they not only prevent the damages from occurring, but also improve functional recovery after the damages are done (Figure 1A, Table 1). New functions of FGFs have recently emerged. Evidence for FGF1, FGF2, FGF7, and FGF10 to trigger mitogenic and antiapoptosis activities correlates with their ability to enhance the survival and outgrowth of various kidney cell types, such as collective cells, tubule cells, and glomerular cells. Progress has also been made in understanding the roles and associated mechanisms of FGFs in AKI (Figure 1B).

Here, we will summarize the available data on the roles and known mechanisms of FGFs in the pathogenesis, prevention, and repair of AKI with a focus on IRI. The discussion will first provide a succinct overview of FGF/FGFR signalling specificity and function, followed by a detailed summary of the published work on the roles of FGFs/FGFRs in the AKI, impact of endogeneous and recombinant FGFs as prevention and therapeutic measures for the IRI, pathophysiological processes and conclude with a highlight for future research to better understand underlying mechanism and FGFs in AKI disease and provide viable strategies to prevent IRI or avert progression to CKD.

### A SUCCINCT OVERVIEW OF FGF LIGANDS AND THEIR RECEPTORS

In mammalians, the FGF system consists of 18 ligands signalling through their specific FGFRs (Goetz and Mohammadi, 2013; Ornitz and Itoh, 2015, Figure 2A). The molecular weight of vertebrate FGFs ranges from 17 to 34 kDa and the proteins consist of a central core of 140 amino acids and 12 antiparallel b chains. The sequence similarity among different members is between 30% and 60%. FGFs are structurally related and functionally relevant, they elicit a redundant but also distinct repertoire of biological activities. FGFs are further divided into several subfamilies based on their sequence identity, receptor binding specificity and biological activities. These are the FGF1 subfamily, including FGF1 and FGF2; FGF4 subfamily, which includes FGF4, FGF5, and FGF6, the FGF7 subfamily made of FGF3, FGF7, FGF10, and FGF22, the FGF8 subfamily, which consists of FGF8, FGF17, and FGF18, as well as the FGF9 subfamily, which is made of FGF9, FGF16, and FGF20 (Ornitz and Itoh, 2015, Table 2). Finally, the FGF19 subfamily includes FGF19, FGF21, and FGF23 (Li, 2019). Heparin sulphate proteoglycan (HSPG) binding domains and N-terminal signalling peptides for secretion are common features shared by FGFs.

In most cases, FGFs are secreted into the extracellular space via the classical secretory pathway. FGFs have been described to act both in an autocrine and paracrine fashion. They signal through their specific trans-membrane receptors consisting of five members, FGFR1-5. FGFRs bind their ligands with high affinity and various degrees of specificity (Figure 2A). All FGFRs has a single pass trans-membrane domain (TM) and two intracellular kinase domains (ICKs) except for the atypical FGFR5, which has no enzymatic activity (Trueb, 2011). The extracellular segments of FGFRs are composed of three Ig-like domains (IgGI-IgGIII), and between the first and second Ig-like domains is the acid-box domain that determines ligandspecificity. The ICKs harbor the catalytic activity of receptors as well as autophosphorylation sites, which interact with intracellular substrates (Table 2).

### THE ROLE OF FGFS IN AKI

Many FGF ligands and their associated receptors are found to be highly expressed during kidney development. Genetic ablation and transgenic overexpression in mouse models, as well as exposure to exogenous FGF ligands, have established the critical regulatory roles of multiple FGF ligands in kidney development, particularly those that signal through FGFR1 and FGFR2 (Walker et al., 2016; Figure 1B). Some of the developmentally important FGFs/FGFRs expressed early during kidney development get re-expressed or activated in the regeneration phase after IRI (Villanueva et al., 2006; Tan et al., 2017). Currently, most of the FGFs reported to participate in AKI pathogenesis or reveal protective/therapeutic potentials are derived from FGF1, FGF7, and FGF19 subfamilies (Figures 1A, B), which we will be discussing in more detail.

and pathogenesis of AKI. The interactions of these complex disease mechanisms can lead to CKD and ESRD. FGFRs can be activated by endogenous FGFs and co-ligands following IRI. Exogenous recombinant FGFs such as FGF2 and FGF10 can protect again IRI and inhibit the transition of AKI to CKD and ESRD via regulating this complex pathogenesis and repair process. (B) The expression of several FGFs including FGF1/2/7/10 is induced upon IRI and is capable of promoting tubular epithelial cell proliferation through a paracrine effect. Furthermore, FGF1/2 mediated activation of FGFRs can inhibit the apoptosis of tubular epithelial cells and promote the transformation of tubular epithelium to mesenchymal cells. Exogenous stem cells can ease IRI by producing FGF1/2. Increased FGF1/2 can further support the survival of stem cells. FGF23 is produced by osteoblasts in bone in response to local and systemic factors and targets the kidney to create multiple endocrine networks. FGF23 also impacts macrophage infiltration through adjusting the immune system after IRI.

### MULTIFACETED ROLES OF FGF1/2 IN AKI

FGF1 (or Acidic FGF) and FGF2 (or basic FGF) are the prototypic members of the FGF family that have a similar broad range of biological activities. Both are in vitro mitogens for most of the ectodermal- and mesodermal-derived cell lines. Numerous studies have shown that intravenous administration of exogenous FGF1 or FGF2 can improve the physiological functions of different organs after IRI.


TABLE 1 | Fibroblast growth factors (FGFs) directly involved in regulating acute kidney injury (AKI).

FGF1 is an autocrine/paracrine regulator known to act on cells from various organs and tissues, including liver, vasculature, and skin. FGF1 exerts significant beneficial effects in different organs after IRI (Fu et al., 1995; Cuevas et al., 2000; Chen et al., 2005; Wang et al., 2010). The skeletal muscle damage protection provided by FGF1 may arise from its ability to regulate extracellular and intra-cellular calcium ions concentrations (Fu et al., 1995). FGF1 can promote small intestine epithelial cell proliferation in IRI in rats and its activity is associated with the activation of ERK1/2. Intravenously administered FGF1 could also alleviate IRI-induced apoptosis in rat intestinal tissues (Weng et al., 2004). In other studies, FGF1 was reported to activate PI3K/AKT-mediated suppression of oxidative stress and inflammation, especially for diabetic nephropathy (Pena et al., 2017). FGF1 can protect hepatic and renal functions after intestinal IRI (Weng et al., 2004). Fu et al. showed that the protective effects of FGFs might originate from the nonmitogenic effects of FGFs at the early stage and the mitogenic effects at the late stage of tissue repair (Fu et al., 2004). Cuevas et al. demonstrated a role for FGF1 after acute kidney damage following IRI by inhibiting neutrophil infiltration (Cuevas et al., 1999).

FGF2 is another vital protein for ureteric bud formation, and also necessary for the induction of mesenchymal cells aggregation. The mesenchymal aggregates cannot give rise to epithelial cells in the absence of FGF2. In the adult mice, FGF2 exerted a protective function against IRI in several organs such as the retina, brain, spinal cord, heart, and intestine. FGF2 also has a beneficial role in renal IRI. The primary mechanism is via the activation of the PI3K/AKT and ERK1/2 pathways. The expression of FGF2 is not observed in the adult kidney during homeostasis, but a strong induction is observed in the regeneration phase of AKI. FGF2 reduces the expression of renal damage markers such as ED-1 and a-smooth muscle actin and participates in the regeneration process after ischemic acute renal failure (Villanueva et al., 2006). Xu et al. revealed the same results upon the ablation of the receptor FGFR2 in fibroblasts (Xu and Dai, 2017). Such genetic manipulation ameliorates kidney fibrosis after IRI in mice. On the other hand, inhibition of FGFR2 sensitize kidney damage and suppresses nephrogenic protein expression (Villanueva et al., 2008). Another study demonstrated that FGF2 protects against renal IRI by attenuating mitochondrial damage and proinflammatory signals (Tan et al., 2017). Functional studies

FIGURE 2 | Mechanism of FGFs signalling during AKI after IRI. (A) FGFs interact with FGFRs with HS (and klotho for FGF23) as cofactor after IRI. The interactions induce activation of the RAS-MAPK, PI3K-AKT, and PLCg pathways. These pathways mediate antiapoptosis, differentiation, proliferation, and cell motility. (B) HMGB1, a nuclear transcription factor protein is released upon IR injury. Circulating HMGB1 can interact with TLRs to promote inflammatory cytokine secretion. Increased IL-1b, IL-6 and TNF-a in turn, activate NF-kB and further enhance inflammation. FGF2 may inhibit inflammation through robust protection of renal tubular cells from IR-induced apoptosis and subsequent release of HMGB1. FGF2 and FGF10 may inhibit autophagy via activation of PI3K/AKT and MAPK signalling. On the other hand, the endocrine FGF23 binds FGFR4 to activate the calcium channel and contribute to renal fibrosis. A large amount of calcium ion influx results in ROS activation, which also leads to increase in TGF-b expression and its down stream signalling to promote fibrosis.


TABLE 2 | Overview of Mammalian FGF subfamilies, receptor specificity and physiological functions.

have shown that FGF2 promotes proliferation on a variety of renal cell types, especially interstitial fibroblasts. FGF2 facilitates the epithelial to mesenchymal transition of tubular epithelial cells and contributes in the initial stage to an increase in the stroma population. The expression of FGF2 is increased in the process of stem cell treatment of AKI (Patel et al., 2012). We propose that both endogenous and exogenous FGF2 react to the transplanted stem cells after injury. The mechanism of stem cell therapy is not only the replacement of dead cells but more importantly, the role it plays through the secretion of growth factor as well as antiinflammatory molecules.

### EXOGENOUS FGF10 PROTECTS AGAINST IRI

FGF7 and FGF10 were initially isolated as a keratinocyte growth factor 1 and 2, respectively. Both are expressed in the kidney, but unlike other FGFs, which are paracrine factors that are expressed exclusively in the mesenchyme. They interact primarily with the "b" isoforms of FGFR2 (FGFR2b), which is an alternatively spliced RNA isoform containing a unique domain (called IIIb) in the third IgG-like loop (Zhang et al., 2006). FGF7 and FGF10 have been documented to play an essential role in the development, growth, differentiation, and homeostasis of the muscosal lining of the urinary tract (Qiao et al., 1999; Walker et al., 2016).

Both FGF7 and FGF10 bind FGFR2b, but exert largely distinct physiological functions. FGF7 is implicated in both the induction of basal urothelial cell proliferation and the expansion of transitional epithelium. It is an efficient growth and differentiation factor during development and wound healing. FGF7 also has a significant effect on the kidney. Compared to wild type littermates, Fgf7 knockout mice display smaller kidneys with fewer ureteric buds and nephrons. Earlier reports showed that the expression of both FGF-7 and FGFR2b is induced and segregated between interstitial and epithelial cells in response to chemically-induced proximal tubular damage. The activation of this mesenchymal to epithelial paracrine signaling is implicated in the regulation of tubular repair process (Ichimura et al., 1996). Additionally, renal IRI is found to promote FGFR2 phosphorylation together with the selective upregulation of FGF7 and FGF2. Recombinant FGF7 administration can induce FGFR2 expression and promote bladder progenitor proliferation (Vinsonneau et al., 2010; Figure 1B), but no study on FGF7 and renal IRI has been published.

FGF10 is a multifunctional growth factor playing crucial roles in the development of multiple organs and tissues, including the kidney (Itoh, 2016). In contrast to the Fgf7-null mice, which do not display significant developmental abnormalities, the inactivation of Fgf10 in mice causes broad developmental defects including limb bud induction, lung as well as kidney agenesis (Ohuchi et al., 2000). Similar to FGF7, FGF10 also signals via interaction with its high-affinity receptor FGFR2b. Deletion of Fgf10 in mice led to kidney dysgenesis characterized by fewer collecting ducts and nephrons. Overexpression of a soluble dominant-negative FGFR2b isoform in transgenic mice revealed more striking defects, including renal aplasia or severe dysplasia (Celli et al., 1998). Intra-tracheal administration of FGF10 in rats with IR (Ischemia-Reperfusion) induced lung injury significantly diminished lung edema, the release of inflammatory cytokines, immune infiltration, and protein exudation. Activation of PI3K pathway has been reported to underlie FGF10-mediated protection against I/R-induced endothelial cell apoptosis and barrier dysfunction (Fang et al., 2014). More recent studies provided further evidence that exogenous recombinant FGF10 mediates protection against renal IRI through suppression of excessive autophagy and ER stress (Tan et al., 2018; Tan et al., 2020), which will be further discussed later.

### FGF23 AS BIOMARKERS OF AKI AND CKD

FGF23 belongs to the FGF19 subfamily of endocrine FGFs which play important roles in interorgan crosstalk that governs a broad spectrum of metabolic homeostasis (Degirolamo et al., 2016; Li, 2019). Although there are a number of reports on the protective effect of FGF21 on the myocardial IRI, there has yet to be any published research directly addressing the role of either FGF19 or FGF21 in renal IRI. FGF23 is mainly produced by osteocytes and possibly osteoblasts. Significantly, it is dramatically increased in CKD and ESRD, and has been proposed as a biomarker for adverse outcomes in patients with CKD and ESRD (Isakova, 2012; Christov et al., 2019 Figure 1B). Similar to FGF19 and FGF21, FGF23 binding to FGFRs and the subsequent activation of FGF signal transduction requires a co-receptor, Klotho (Kurosu et al., 2006). The structure of FGF23 ternary complex together with a-klotho extracellular domain and the FGFR1c ligand-binding domain has recently been solved (Smith et al., 2014; Chen et al., 2018). FGF23 controls renal phosphate reabsorption, modulates the production of parathyroid hormone (PTH) and 1,25-(OH)2-vitamin D. It also participates in mineral homeostasis. FGF23 acts on the kidney to increase renal phosphate excretion and to decrease 1,25-dihydroxy vitamin D (1,25D) production (Li et al., 2019; Musgrove and Wolf, 2020).

FGF23 levels rose acutely in patients who underwent cardiac surgery and developed AKI, even before a significant increase in serum creatinine (Christov et al., 2013). In the current era of heightened awareness of the dire need for early diagnosis of AKI, FGF23 has been touted as a potential marker of the complex AKI syndrome (Leaf et al., 2017; Leaf et al., 2018). Shaker et al. reported that the change of plasma FGF23 concentrations at 24 hours after cardiac bypass surgery was associated with the severity of renal injury, whose sensitivity was 100%, and specificity was 97.1% suggesting that FGF23 could have a role as an early biomarker of AKI and predicts adverse outcomes among patients with established AKI (Shaker et al., 2018; Volovelsky et al., 2018). A recent prospective investigation in a large cohort of patients with CKD stages have identified elevated serum levels of interleukin-6, C-reactive protein, and FGF23 as independent risk factors for mortality in CKD (Munoz Mendoza et al., 2017). Circulating levels of FGF23 are increased in human AKI and CKD of various settings, and correlate to poor survival in patients across infants, children, and adults (David et al., 2017; Czaya and Faul, 2019). Therefore, serum FGF23 level is proposed to be an even more significant parameter than creatinine to assess the severity of the AKI (Christov et al., 2019).

Other studies demonstrate that tubule-derived FGF23 might amplify myofibroblast activation in AKI (Smith et al., 2017b). FGF23 is found to augment profibrotic signalling cascades in injury-primed renal fibroblasts via activation of FGFR4 and upregulation of the calcium transporter, a transient receptor potential cation channel. This function was independent of a-Klotho. Restoration of a-Klotho, as upstream regulators, can regulate the off-target effects of FGF23 (Smith et al., 2017a). Both FGF23 and a-Klotho have been proposed as prognostic biomarkers of AKI and also targets of therapeutic intervention for CKD or CVD after AKI. The effects of different FGF family members in the context of AKI is summarized (Table 1).

### FGFRS INVOLVED IN AKI

The FGF ligands signal through four receptors (FGFR1-4) and an atypical, kinase inactive FGFR5 or FGFRlL (Ornitz and Itoh, 2015). Some of FGFs/FGFRs are important during normal kidney development and also during postnatal repair. FGFs stimulate kidney cell fate determination, migration, and differentiation during organogenesis (Bates, 2011; Trueb et al., 2013) and regulate the proliferation, mobilization, and regeneration during repair after injury (Strutz, 2009; Gallegos et al., 2019).

FGFRs elicit different functions in mammalian development and diseases. Conditional knockout of Fgfr1 in ureteric bud and metanephric mesenchyme did not lead to any kidney development defects (Revest et al., 2001; Poladia et al., 2006). Ablation of Fgfr2 led to the formation of a smaller kidney with fewer nephrons (Sims-Lucas et al., 2011). The combined inactivation of Fgfr2 and Fgfr1 knockout in mice led to severe kidney aplasia (Sims-Lucas et al., 2011). General knockout of Fgfr3 and Fgfr4 did not affect early kidney development (Colvin et al., 1996; Weinstein et al., 1998). It has been previously reported that a given FGF ligand can bind multiple receptors and that conversely, the same receptor can bind different ligands, therefore allowing a very complex set of biological activities downstream of FGF/FGFR activation (Li, 2019). FGFR1, for example, can bind to most of FGFs except FGF7 and FGF18. FGFR2b, on the other hand, binds only FGF1 and the members of the FGF7 subfamily. Due to potential redundancy in FGFRs, their role in acute kidney injury is still unclear. However, exogenous FGF ligands, through the activation of the FGFR signalling pathway, are protective against renal damage. During IR-induced acute renal injury, increased expression of FGF activates FGFR phosphorylation and recruitment of FRS-2 and PLC-g. The classical downstream PI3K/AKT and MAPK signalling pathways are subsequently activated to control cell proliferation, differentiation and apoptosis as well as cell migration and other processes. This leads to decreased renal injury caused by IRI (Figure 2A).

Although IRI mainly affects renal tubular cells, IR-induced pathogenic process and its repair involve interactions between interstitial cells, infiltrated inflammatory cells and epithelial cells. Interestingly, a recent study using a mouse model with fibroblastspecific ablation of Fgfr2 gene indicated that FGFR2 expression in fibroblast may contribute to kidney fibrosis after IRI through promoting renal fibroblast activation and proliferation (Xu and Dai, 2017).

### IMPACT OF FGFS ON PATHOPHYSIOLOGY OF IRI

IRI represents a frequent underlying cause for both ischemic heart disease and AKI. Compared to the numerous publications on a broad spectrum and relatively extensive research on FGFs in ischemic heart injury, only a limited number of reports are available on FGFs (mainly FGF2, FGF10, and FGF23) in renal IRI and AKI. Additionally, most of FGF23-related studies focus on its role as a key metabolic regulator and an injury or prognosis biomarker. Consequently, FGF23 is a proinjury factor rather than a protective or therapeutic agent for AKI. Therefore, our following discussions will only focus on FGF2 and FGF10, both of which exhibit potent protection against IRI and therapeutic potential via impacting several key pathophysiological mechanisms.

### FGF2 PROTECTS AGAINST IR-INDUCED TUBULAR CELLS DEATH

Acute cell death of proximal renal tubules and inflammatory response of both innate and adoptive nature are hallmark features of IRI. Apoptosis plays an important role in the IR-induced pathogenesis and is a validated parameter to evaluate the cellular damage induced by ischemia. The protective role of FGF2 against IRI is well documented for ischemic myocardial infarction. Studies in genetically engineered mouse models established the antiapoptotic effect of endogenous FGF2 toward ischemic cardiac injury. FGF2 also exerts a positive impact on the repair process that may involve its activation of both MAPK and PKC pathways (House et al., 2003; House et al., 2005; House et al., 2007). Furthermore, under cardiac IRI conditions mimicking clinical acute myocardial infarction, endogenous FGF2 is considered an essential acute cardioprotective factor and a longer term proangiogenic factor (House et al., 2015). The protective effect of FGF2 against IRI and in promoting repair was also appreciated in renal IRI (Villanueva et al., 2008; Tan et al., 2017). Interestingly, FGF2 is found to be expressed early during kidney development, and gets re-expressed upon IRI and participates in the recovery process by promoting the expression of morphogen proteins to accelerate the repair process in the kidneys (Villanueva et al., 2006). Additionally, treatment with antisense oligoes targeting FGFR2 led to a significant increase in tubular TUNEL positive cells and expression of damage markers in an AKI model, whereas the expression of morphogenic proteins and cellular mitosis was inhibited (Villanueva et al., 2008). Our study demonstrated that exogenous recombinant FGF2 also exhibited robust protection against IRI and significantly improved animal survival in a rat IRI model (Tan et al., 2017).

Under hypoxic conditions, FGF2 may alleviate oxidative stress and IR-induced mitochondrial DNA damage and proapoptotic alteration of Bcl2/Bax expression and caspase-3 activation. The remarkable protective effect of FGF2 owns, at least in part, to its ability to preserve the integrity of the mitochondrial ATP-sensitive potassium channel (Tan et al., 2017). It is worth noting that, besides the major FGF2 protein isoform (18 kD, low molecular weight), there exist at least 4 other isoforms of higher molecular weight, which are reported to exert different or even opposite effects on apoptosis (Kardami et al., 2007; Liao et al., 2010; Manning et al., 2013) via different mechanisms. Besides apoptosis, other types of cell death such as necrosis, necroptosis, pyroptosis, ferroptosis, have been also been implicated to underlie the tubular cell damage, the potential role of FGFs on these additional pathways and the their interplay remain to be characterized (Xu and Han, 2016; Han and Lee, 2019; Hu et al., 2019). On the other hand, renal recovery from AKI requires the replacement of injured cells by new ones that can restore tubular epithelial integrity. In this regard, FGF2 may also facilitate the repair process of IRI, as post-IRI administration of FGF2 also exhibited effective protection of IRI and improved animal survival (Tan et al., 2017). The results collectively indicate that FGF2 has promising clinical potential for the prevention and treatment of IR-related AKI.

### FGF10 INHIBITS EXCESSIVE AUTOPHAGY AND ER STRESS

Autophagy is an evolutionarily conserved pathway that leads to lysosomal degradation of cytoplasmic substrates, such as damaged organelles and cytoplasmic proteins. The autophagic response is triggered under various stress conditions including nutrient starvation, hypoxia, and growth-factor deprivation, as well as ER stress and oxidant injury, most of which are involved in the pathogenesis of AKI. Knockout of Atg5 or Atg7 in proximal tubule led to accumulation of deformed mitochondria, ubiquitinpositive inclusion bodies increased apoptosis and worsened renal dysfunction upon IRI suggesting a role of autophagy to the normal homeostasis of the kidney and renoprotective effect in IR injury (Jiang et al., 2010). However, some studies also report that autophagic response exacerbates renal IRI (Kaushal and Shah 2016; Tan et al., 2018). Therefore, it is likely that autophagy may exhibit both protective and detrimental properties in renal IRI, depending on the duration, the phase, and even the extent of the IRI.

FGF can inhibit autophagy through the mTOR pathway and block differentiation during organogenesis (Zhang et al., 2012; Cinque et al., 2015). Different FGFs may have distinct activities to regulate autophagy. FGF21 protects cardiomyocytes by promoting autophagic flux with hypoxia/reoxygenation injury (Ren et al., 2019). On the other hand, FGF2 and FGF10 can alleviate IRI by suppressing excessive autophagy via PI3K/AKT and MAPK signaling in the kidney and other organs (Wang et al., 2015; Sun et al., 2018; Tan et al., 2018, Figure 2B). Based on the analysis of renal tissues for their LC3, Beclin-1 and SQSTM1 expression and localization, FGF10 treatment was found to significantly suppress autophagic phenotype, which was highly activated during IRI, whereas co-treatment of FGF10 with Rapamycin partially reversed such renoprotective effect, suggesting the involvement of mTOR pathway in the process. This study established that exogenously administered recombinant FGF10 is protective against IR-induced functional and tissue damage to the kidney, at least partially through mitigating excessive autophagy.

Recent studies further showed that IRI is accompanied with excessive activation of ER stress, which is involved in hypoxia injury-induced apoptosis of renal tubular epithelial cells. ER, a specialized organelle for protein synthesis, folding and trafficking, is highly sensitive to the intracellular microenvironment changes. Hypoxia and oxidative stress are intrinsic to IRI, which disturb ER functions and lead to impaired protein folding (Walter and Ron, 2011; Cao and Kaufman, 2014). Excessive accumulation of unfolded proteins activates Unfolded Protein Response (UPR), a cellular stress response mechanism to improve the protein folding efficiency while reducing mRNA translation along with protein expression (Schuck et al., 2009). Although ER stress plays an important role in cell growth and differentiation, excessive activation of ER stress and UPR can activate apoptotic signaling (Tabas and Ron, 2011; Hetz, 2012), which is mainly mediated by C/ EBP homologous proteins (CHOP), a master regulator of maladaptive ER stress-induced apoptosis. FGF10 effectively alleviated IRI evoked expression of ER stress-related proteins in the kidney including CHOP, GRP78, XBP-1, and ATF-4 and ATF-6, which may contribute to inhibit IR-induced activation of proapoptotic caspase-3 and Bax expression. Results from IRI model in vivo and in vitro cell culture experiments together indicate that FGF10 attenuates renal tubular epithelial cell death via inhibiting excessive ER stress, which is, at least in part, mediated by MEK-ERK1/2 signaling pathway (Tan et al., 2020). Therefore, FGF10 contributes to restore the balance between the adaptive pathway and the apoptotic pathway of UPR by inhibiting excessive ER stress.

### ROLE OF FGFS ON INFLAMMATION

The inflammatory response is an integral component in the initiation and exacerbation of AKI. Although a crucial element of the repair process, excessive activation of inflammatory signals and cytokine secretion may impose further damage to renal parenchyma cells. FGFs may exhibit different impacts on the inflammatory process of IRI. Contrary to the protective effects of FGF2 and FGF10 against IRI/AKI, FGF23 appears to be a deleterious factor.

Under normal physiology, FGF23 is mainly produced in bone by osteoblasts/osteocytes in response to local and systemic factors and targets the kidney to create multiple endocrine networks (Bergwitz and Juppner, 2010). Although some studies suggest that FGF23 interacts with the immune system, it is not clear whether FGF23 directly regulates immune cell functions or indirectly impacts immune responses through FGF23 regulation of 1,25-dihydroxyvitamin-D. FGF23 is increased in CKD. Macrophages do not regularly express FGF23 or a-Klotho, but in acute inflammation, FGF23 stimulates proinflammatory responses in M1 macrophages and blocks the transition to M2 macrophages (Figure 1B). In addition, FGF23 is proposed to activate FGFR2 in polymorphonuclear leukocyte to directly decrease their recruitment. Given the link of increased serum FGF23 to various tissue injuries, as well as evidence that the sources of FGF23 and control of its production in AKI and CKD differ from those in the physiologic conditions, mediators of inflammation contributing to elevated FGF23 have recently been proposed as potential drug targets, in addition to repurposing existing strategies to target FGF23 (Musgrove and Wolf, 2020).

High mobility group box 1 protein (HMGB1) is a highly conserved nuclear protein that functions as an architectural chromatin-binding factor and regulator of gene transcription. HMGB1 assumes diverse roles as an immuno-modulator in the form of a cytokine molecule or as nuclear chromatin and transcription regulator. HMGB1 can be activated and gets released from damaged parenchymal cells as a sterile inflammatory molecule and a major damage-associated molecular pattern (DAMP). Translocation of HMGB1 from the nucleus to the cytoplasm and subsequent release to the extracellular milieu is reported to promote inflammatory response via the activation of TLRs (Yang et al., 2010). HMGB1-TLRs pathway has long been recognized as an essential and early mediator in renal IRI and an attractive target of AKI and other disease therapies (Li et al., 2011; Zhang et al., 2016; Vijayakumar et al., 2019).

Recent efforts to explore the potential protective effect of exogenous FGFs on renal IRI illustrated that, besides promoting proliferation and inhibiting apoptosis, both FGF2 and FGF10 effectively inhibited IRI-induced release of HMGB1 from the nucleus to the extracellular domain, which is associated with a marked decrease in the expression of inflammatory cytokines such as TNF-a, IL-1b, and IL-6 following IRI (Tan et al., 2017; Tan et al., 2018, Figure 2B). This novel function of FGFs on the inflammatory cascade may be related to the inhibition of HMGB1-mediated TLR2 and/or TLR4 signaling (Leemans et al., 2009; Wu et al., 2010; Chen et al., 2017). It has been reported that FGF10 ameliorates cerebral ischemia injury via inhibiting NF-kB-dependent neuroinflammation and activating PI3K/AKT survival signalling pathway (Li et al., 2016). Whether the inhibition of HMGB1 and associated inflammatory cytokine release by FGFs is due to their protective effect against renal damage and therefore less DAMP release, or through some other mechanism(s) remains to be further elucidated.

### CONCLUSION AND PROSPECT

Extensive research in the past years have established FGFs as vital regulators in tissue repair and regeneration, as well as in metabolic homeostasis. Along with advances in our understanding of FGF biology and their regulation of various pathophysiologic processes comes the inspiration of harnessing their power for potential disease therapies.

In this review, we summarized the available data on the FGFs that have shown promising features related to IRI either as a preventative/therapeutic agent or a biomarker. We highlighted some functional and biological aspects that constitute the promising features of FGFs, particularly FGF2 and FGF10, in averting IR-induced tubular cell death and inhibition of overt inflammatory response, which may all contribute to their net beneficial effect on reducing the IRI and promoting recovery.

Admittedly, the exploration of FGFs as preventative or therapeutic agents in the management of clinical IRI is still in its rudimentary stages. Most of the published work has been conducted in pre-clinical settings. These widely used animal models, including the bilateral IR that is considered to best resemble clinical AKI, still have certain limitations in fully mimicking human AKI, which can be caused by numerous and complicated clinical conditions. Additionally, the underlying pathophysiology of IRI and how FGFs impact key biological and pathophysiological processes,

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### AUTHOR CONTRIBUTIONS

J-SZ and L-CD conceived the study. L-CD, TA, SB, and J-SZ drafted and revised the manuscript.


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Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor is currently organizing a Research Topic with several of the authors SB, J-SZ, and confirms the absence of any other collaboration.

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