Huangkui Capsule Ameliorates Renal Fibrosis in a Unilateral Ureteral Obstruction Mouse Model Through TRPC6 Dependent Signaling Pathways

Renal fibrosis is the final common pathological manifestation of almost all progressive chronic kidney diseases (CKD). Transient receptor potential canonical (TRPC) channels, especially TRPC3/6, were proposed to be essential therapeutic targets for kidney injury. Huangkui capsule (HKC), an important adjuvant therapy for CKD, showed superior efficacy for CKD at stages 1–2 in clinical practice. However, its anti-fibrotic effect and the underlying mechanisms remain to be investigated. In the present study, we evaluated the efficacy of HKC on renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and explored the potential underlying mechanism. Administration of HKC by intragastric gavage dose-dependently suppressed UUO-induced kidney injury and tubulointerstitial fibrosis. Similarly, HKC suppressed the expression level of α-smooth muscle actin (α-SMA), increased the expression of E-cadherin, and suppressed the mRNA expression of a plethora of proinflammatory mediators that are necessary for the progression of renal fibrosis. Mechanistically, HKC suppressed both canonical and non-canonical TGF-β signaling pathways in UUO mice as well as the TRPC6/calcineurin A (CnA)/nuclear factor of activated T cells (NFAT) signaling axis. In addition, TRPC6 knockout mice and HKC treated wild type mice displayed comparable protection on UUO-triggered kidney tubulointerstitial injury, interstitial fibrosis, and α-SMA expression. More importantly, HKC had no additional protective effect on UUO-triggered kidney tubulointerstitial injury and interstitial fibrosis in TRPC6 knockout mouse. Further investigation demonstrated that HKC could directly suppress TRPC3/6 channel activities. Considered together, these data demonstrated that the protective effect of HKC on renal injury and interstitial fibrosis is dependent on TRPC6, possibly through direct inhibition of TRPC6 channel activity and indirect suppression of TRPC6 expression.


INTRODUCTION
Renal fibrosis is the common final outcome of almost all progressive chronic kidney diseases (CKD) which affects approximately 10% population for limited options of treatment (Djudjaj and Boor, 2019). Currently, consensus has been reached that tubular cell differentiation and interstitial fibroblast activation play important roles in the process of renal fibrogenesis (Liu, 2011). Calcium ion (Ca 2+ ) which is an important signaling molecule is tightly associated with cell differentiation and activation (Clapham, 2007). Transient receptor potential canonical (TRPC) channels are a group of Ca 2+ -permeable, nonselective cation channels. TRPC channels, especially TRPC3/ 5/6, play critical roles in the development of kidney diseases. Gainof-function mutations of TRPC6 lead to focal and segmental glomerulosclerosis (FSGS), a common and increasing cause of the end-stage renal disease (Winn et al., 2005;Riehle et al., 2016). Conversely, genetic ablation or pharmacological inhibition of TRPC6 suppresses unilateral ureteral obstruction (UUO)induced interstitial fibrosis in mice (Wu et al., 2017;Lin et al., 2019). In addition, TRPC6 has also been shown to contribute to the development of experimental pulmonary fibrosis (Hofmann et al., 2017), myofibroblast transdifferentiation and wound healing (Davis et al., 2012). Pharmacological blockade of TRPC6 ortholog, TRPC3, inhibits fibroblast proliferation and myofibroblast differentiation (Saliba et al., 2015) and genetic ablation of TRPC3 attenuates UUO-induced renal fibrosis in mice (Wu et al., 2017). Although TRPC5 has been shown to alter podocyte function, inconsistent findings were reported. Genetic ablation or pharmacologic inhibition of TRPC5 has been reported to benefit kidney filter dynamics by balancing podocyte cytoskeletal remodeling (Schaldecker et al., 2013;Zhou et al., 2017). However, another group showed that the overexpression or activation of the TRPC5 ion channels cannot cause kidney barrier injury or aggravate such injury under pathologic conditions (Wang et al., 2017). In addition, there is no evidence that TRPC5 channels play roles in the progression of renal interstitial fibrosis.
Abelmoschus manihot (L.) Medik. (A. manihot), which belongs to Malvaceae family, is a medical plant widely used to treat inflammatory disease since ancient China (Rubiang-Yalambing et al., 2016). In 1999, based on the effectiveness on CKD in clinical practice, a formulation (Huangkui Capsule, HKC) of ethanol extract of A. manihot flowers was approved by China's State Food and Drug Administration under category III of traditional Chinese medicine for chronic nephritis treatment (Chen et al., 2016). A multicenter, randomized, controlled clinical trial demonstrates that HKC displays superior anti-proteinuria efficacy than losartan in patients with CKD at stages 1-2 (Carney, 2014;Zhang et al., 2014). In type II diabetic patients, HKC significantly decreases the levels of proteinuria and serum creatinine (Scr) . Currently, HKC has been used as an important adjuvant therapy for CKD (Zhang et al., 2014). Pharmacological studies have reported the protective effect of HKC against renal injury in diabetic nephropathy and adriamycin-induced renal injury animal models. HKC decreases albuminuria, attenuates early glomerular pathology and renal tubular epithelial-mesenchymal transition in the diabetic nephropathy animal model (Mao et al., 2015;Ge et al., 2016;Kim et al., 2018;Wu et al., 2018;Han et al., 2019). Similarly, in an adriamycin-induced renal injury murine model, HKC attenuates kidney inflammation and glomerular injury, likely through inhibition of reactive oxygen species (ROS)-mitogen-activated protein kinase (MAPK) signaling pathway (Tu et al., 2013;Mao et al., 2015;Li et al., 2019).
Chemical and pharmacological investigation has revealed that flavonoids are the main bioactive chemical constitutes of HKC to improve diabetic nephropathy (Lai et al., 2009). Pharmacokinetic studies demonstrate that the flavonoids are the main compounds detected in the blood and kidney tissue suggesting that the flavonoids are the potential active components (Lai et al., 2007;Xue et al., 2011). In human kidney-2 cells, the flavonoids in HKC, including quercetin, isoquercitrin, hyperoside, gossypetin-8-O-b-D-glucuronide and quercetin-3′-O-glucoside, prevents epithelial to mesenchymal transition (Cai et al., 2017). However, the molecular mechanism of anti-fibrotic effects of HKC remains to be established.
UUO surgery is the widely used renal fibrosis model, which displays characteristic tubular cell injury, interstitial inflammation, and fibrosis, recapitulating the pathology observed from both irreversible acute kidney injury and CKD (Chevalier et al., 2009;Ucero et al., 2014). In the present study, we evaluated the efficacy of HKC against renal fibrosis in UUO mice and the possible involvement of TRPC channels on HKC renal protection and anti-fibrotic effects. We demonstrate that HKC displays anti-fibrotic effect in a UUO mouse model with better efficacy than losartan. Further mechanistic studies demonstrate that the anti-fibrotic effect of HKC is through TRPC6 dependent pathway.

HPLC Analysis of the Chemical Constitutes of HKC
The chemical constitutes of HKC were analyzed on a ZORBAX SB-C18 reverse-phase column (4.6 mm × 250 mm, 5 mm, Agilent Technologies Inc., Santa Clara, CA, USA) performed on an Agilent 1260 Series HPLC system and the absorptions were monitored by a VW detector at the wavelength of 254 nm. The column was eluted with a gradient concentration of acetonitrile with trifluoroacetic acid (TFA) at a flow rate of 1 mL/min using the following program. Solvent A: 0.1% TFA in ddH 2 O, solvent B: acetonitrile, 0-5 min, 6% B; 5-30 min, 6-17% B; 30-40 min, 17% B; 40-60 min, 17-40% B.

Generation of TRPC6 Knockout Mice
TRPC6 knockout (KO) mice were generated in Animal Core Facility of Nanjing Medical University based on CRISPR-Cas9 approach combined with a double-nicking strategy (Ran et al., 2013). Exon 7 of Trpc6, located on chromosome 9, was chosen to disrupt the Trpc6 gene. Trpc6 gene disruption was confirmed by genotyping using nested PCR analysis with genomic DNA as the template and two sets of primers as follows: 5′-TCCC CTTATTCAAGTCAGAATATACTACA-3′, and 5′-GGGA GGTATTTGTCATGTAATCTGACTC-3′ for the first step; 5′-ATACTACACACACTTGAGAAGTTCTTCAGA-3′, and 5′-TTGGGAAGGTTCCTTTATGCTAGT-3′ for the second step. Predicted PCR products were 827 bp for wild type (WT) mice and 590 bp for the TRPC6 KO mice.

Preparation of HKC Solution
HKC solutions were prepared as described previously (Mao et al., 2015). Animal dose (1.5 g/kg/day) was obtained according to the daily dose of HKC (7.5 g/day) in clinical practice. Two additional lower doses (0.5 and 0.15g/kg/day) were also selected to explore the dose-dependency of HKC protective effects on UUO-induced kidney injury. For animal experiments, HKC content was dissolved in ddH 2 O to obtain 0.015, 0.05, and 0.15 g/mL HKC solutions. For patch clam experiment, HKC content was dissolved in DMSO to obtain a stock solution of 300 mg/mL. The stock solution was then diluted with bath solution (see patch clamp section) to final concentrations of 300, 100, 30, and 10 mg/mL.

Cell Culture and Treatment
HEK-293 cells stably expressing mouse TRPC3 or TRPC6 channels were the same lines as described previously . Cells were plated in 35-mm dishes at a density of 1,000 cells/dish and cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 200 mg/mL G418 at 37°C in an incubator with 5% CO 2 and 95% humidity. Cells were cultured for 6-12 h before patch clamp experiments.

UUO Model and Treatment
All the animal care and experimental protocols were approved by the Animal Ethics Committee of China Pharmaceutical University according to Guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals (Protocol No. 220182328). UUO surgery was performed as described previously (Gu et al., 2018). To evaluate the efficacy of HKC on renal fibrosis, a total of 70 male C57BL/6 mice (20-23 g) were randomly divided into 7 groups: (1) sham group, (2) sham plus HKC (1.5 g/kg), (3) UUO mice receiving ddH 2 O, (4)-(6) UUO mice receiving low (0.15 g/kg), medium (0.5 g/kg), and high dose (1.5 g/kg) of HKC, respectively, and (7) UUO mice receiving losartan (10 mg/kg/day). All the drugs were administrated by intragastric gavage (i.g., 10 mL/kg) for 7 consecutive days right after surgery. The HKC dose (1.5 g/kg/day) was equal to the clinical dosage. To verify whether HKC protective effect was through TRPC6 channel, HKC (1.5 g/kg) was administered i.g. for 7 consecutive days in wild type (WT) and TRPC6 KO mice after UUO. Blood samples were collected from ophthalmic veins on day 8. Left kidneys were dissected and half of each left kidney was kept in 10% formalin solution and sectioned for histological examination while the remaining part was frozen into liquid nitrogen for protein and total RNA extraction.

Serum Creatinine, Blood Urea Nitrogen and Hydroxyproline Assay
Scr and BUN levels were quantitatively examined using Scr and BUN assay kits according to the manufacture's instruction. Serum hydroxyproline was determined by an ELISA assay using mouse hydroxyproline quantification kit according to the manufacture's instruction.

Hematoxylin and Eosin and Masson's Trichrome Staining
Four kidneys were randomly chosen in each group and were fixed with 10% formalin, dehydrated, embedded in paraffin, and sliced to a thickness of 5 µm. Sections were stained with hematoxylin and eosin (H & E) or Masson's trichrome and the pictures were digitized using Nanozoomer whole slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). The kidney injury score was assessed by determining the tubular atrophy and degeneration, renal papillary necrosis, interstitial inflammation, and fibrous hyperplasia according to the scoring criteria described previously (Debelle et al., 2002).

Immunofluorescence Staining
Kidney sections were blocked with 5% bovine serum albumin, followed by incubation with primary antibodies against a-SMA (1:200), or E-cadherin (1:200) overnight at 4°C. After washing with PBS for 3 ×, an Alexa Fluor ® 488 conjugated goat anti-rabbit secondary antibody (1:300) was added and incubated for 1.5 h at RT. After washing with PBS for 3 ×, a concentration of 2 mg/mL DAPI was added and incubated for 10 min to visualize the nuclei. All sections were digitized using a digital CMOS camera (Hamamatsu photonics) attached to a Leica DMI8 invert fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Data Analysis
All data points represent the mean ± SEM. The concentrationresponse curves were generated using GraphPad software fitted by a non-linear logistic equation (Ver 7.0, GraphPad Software Inc., San Diego, CA, USA). Statistical significance between groups was calculated using one-way ANOVA followed by post-hoc Dunnett's multiple comparisons. A P value below 0.05 was considered to be statistically significant.
Given the protection of HKC on the renal fibrosis, we investigated the expression level of the fibrotic marker, a-SMA in UUO mice with or without HKC oral administration. Immunofluorescence and western blotting analysis showed that UUO mice displayed dramatic increase in the protein expression of a-SMA in the tubular interstitium compared to sham control (Figures 2A, C). HKC dose-dependently decreased the protein expression and mRNA expression of a-SMA (Figures 2A, C, E). Renal fibrosis is also characterized by the loss of epithelial adhesion which can be indexed by the decrease of antifibrotic protein, E-cadherin (Boutet et al., 2006). As expected, the kidneys of UUO mice displayed dramatic decrease on the protein level of E-cadherin ( Figures 2B, D). HKC dosedependently increased the protein expression of E-cadherin to a level comparable to that of losartan ( Figures 2B, D). Consistent with the response in protein level, HKC dose-dependently increased the mRNA expression of E-cadherin ( Figure 2F).

HKC Inhibited the Expressions of Proinflammatory Mediators in UUO Mice
The inflammatory response is a major contributor to the progression of renal fibrosis (Meng et al., 2014). We therefore further quantitatively accessed the influence of HKC on the expression levels of a plethora of cytokines that have been reported to be involved in the renal fibrosis using qPCR analysis. UUO mice displayed higher mRNA expression levels of cytokines including interleukins (Il-1b, Il-12, Il-6), chemokine Representative western blots and quantification of E-cadherin expression in kidneys from sham and UUO mice with different treatments. b-Tubulin was used as an internal control. (E) mRNA levels of a-Sma in kidneys from sham and UUO mice with different treatments. (F) mRNA levels of E-cadherin in kidneys from sham and UUO mice with different treatments. ## P < 0.01, UUO group vs. sham group; *P < 0.05, **P < 0.01, UUO + HKC group or UUO + losartan group vs. UUO group. Bar graphs represent the mean ± SEM (n = 3).
significantly reduced mRNA levels of these cytokines in UUO mice (Figure 3).
Deletion of Trpc6 Decreased smad2/3 Expression and MAPK Phosphorylation in UUO Mice Recent studies have shown that TRPC6 is a pivotal player in the progression of renal fibrosis. In UUO mice, the expression level of TRPC6 is increased and genetic ablation or pharmacological inhibition of TRPC6 suppresses UUO-induced interstitial fibrosis (Wu et al., 2017). We therefore first investigated whether deletion of Trpc6 suppressed the TGF-b signaling pathway that mimics the effect of HKC. TRPC6 KO mice displayed significantly decreased UUO-induced protein expressions of smad2 and smad3 by 74.77% and 77.50%, respectively ( Figures 5A, B). Deletion of TRPC6 also significantly decreased UUO-induced phosphorylation levels of p38, JNK1/2 and ERK1/2 by 75.53, 84.22, and 67.22%, respectively ( Figures 5C-E) suggesting that TRPC6 also regulates both canonical and non-canonical TGF-b signaling pathways.

HKC Suppressed TRPC6 Expression in the Kidney of UUO Mouse
Given the inhibitory effect on TGF-b signaling of HKC treatment and deletion of Trpc6, we therefore investigated HKC effect on TRPC6 expression in UUO mice. Consistent with previous demonstration (Wu et al., 2017), UUO mice displayed increased protein expression of TRPC6 ( Figures 6A, B). HKC had no effect on basal level of TRPC6 expression. However, administration of HKC suppressed the TRPC6 expression in the kidneys of UUO mice ( Figures 6A, B). TRPC6 mediated Ca 2+ influx induces CnA/NFAT activation that is necessary in the progression of pulmonary fibrosis and myofibroblast transformation (Davis et al., 2012;Hofmann et al., 2017). UUO mice displayed higher expression levels of CnA and NFAT that were inhibited by HKC administration ( Figures  6C-F).

HKC Directly Suppressed TRPC3/6 Activities in Heterologous Expression System
Research has proposed that TRPC6 and NFAT form mutually positive feedback loop that exacerbates the renal fibrosis (Nijenhuis et al., 2011). We therefore investigated whether HKC, as a whole, was able to directly suppress TRPC6 activity in heterologous expression system. GSK1702934A is a potent A B C D E FIGURE 4 | HKC treatment attenuates canonical and noncanonical TGF-b signaling pathways in UUO mice. Representative western blots and quantification of the expression levels of smad2 (A), smad3 (B), p-p38 (C), p-JNK1/2 (D), and p-ERK1/2 (E) in sham and UUO mice with different treatments. GAPDH was used as an internal control. ## P < 0.01, UUO group vs. sham group; *P < 0.05, **P < 0.01, UUO + HKC group vs. UUO group. Bar graphs represent the mean ± SEM (n = 3).

HKC Inhibited UUO Renal Fibrosis Through TRPC6 Channels
Given the direct inhibition of TRPC6 activity of HKC, we next compared the HKC protective effect in both WT and TRPC6 KO mice that were subjected to UUO surgery. In TRPC6 KO mice, tubular damage as well as extracellular matrix deposition was alleviated to a level comparable to that observed in WT UUO mice with HKC administration at a dose of 1.5 g/kg/day ( Figures 8A-C). Administration of HKC in TRPC6 KO mice with UUO surgery didn't display any protection on the renal damage ( Figures 8A, B). Similarly, the degree of HKC (1.5 g/kg/ day) suppressed renal fibrosis in WT UUO mice is comparable to that observed in TRPC6 KO mice after UUO ( Figures 8A, C). HKC administration didn't suppress UUO-induced fibrosis in TRPC6 KO mice with UUO surgery (Figures 8A, C). In WT mice, the a-SMA level was 3.42-fold higher than sham control. Deletion of Trpc6 decreased a-SMA expression by 74.12%, a reduction comparable by HKC treatment in WT UUO mice. Administration of HKC didn't decrease the a-SMA level in TRPC6 KO mice with UUO surgery (Figures 8D, E).

DISCUSSION
Renal fibrosis is the final common pathological manifestation of CKD (Boor et al., 2010;Eddy, 2014). Increased expression of a-SMA and decreased expression of E-cadherin are considered to be the major hallmarks of tubular epithelial-mesenchymal transition (EMT) that lead to the destruction of renal parenchyma (Liu, 2011). Consistent with previous study (Gu et al., 2018), we demonstrated that UUO mice displayed increased expression of a-SMA and decreased expression of E-cadherin suggesting the loss of epithelial and gain of mesenchymal features. HKC treatment significantly decreased the expression of a-SMA and increased the expression of E-cadherin. Consistent with biochemical investigation, HKC also ameliorates the UUO-induced kidney tubulointerstitial injury and interstitial fibrosis.
Although inflammation is an evolutionary defense mechanism following injury and infectious agents, excessive inflammatory cells infiltration promotes the progression of renal fibrosis (Nathan and Ding, 2010;Liu, 2011). We demonstrated that HKC significantly suppressed the expression levels of MCP-1 (chemokine produced by tubular cells) (Vieira et al., 2005), VCAM-1 (monocyte marker) (Chuang et al., 2015), inflammation mediators produced by M1 macrophages such as IL-1b, IL-12, IL-6 and MMP-12 (Shen et al., 2014) and TGF-b, an indicator of M2 macrophages (Meng et al., 2014;Shen et al., 2014). Considered together, these data demonstrated that HKC suppressed the recruitments of monocytes/macrophages that drive the progression of renal fibrosis.
TGF-b, one of the central fibrotic factors, plays a major role in the trans-differentiation of interstitial fibroblast to myofibroblast during renal fibrosis by activation both canonic and non-canonic signaling pathways which involve smad2/3 and MAPK proteins, respectively (Geng et al., 2019). We demonstrated that HKC suppressed both smad2/3 expression and phosphorylated protein of p38, JNK1/2, and ERK1/2 suggesting that HKC was capable of suppressing both canonic and non-canonic TGF-b signaling pathways. This is consistent with the report that HKC attenuates renal inflammation and glomerular injury through inhibition of ROS-MAPK signaling pathway (Tu et al., 2013;Mao et al., 2015;Li et al., 2019).
An interesting finding is that HKC protection against renal fibrosis is through TRPC6 channels. TRPC6 is a Ca 2+ permeable, non-selective cation channels that have been shown to be involved in renal fibrosis (Dryer et al., 2019;Lin et al., 2019). Consistent with previous finding, we demonstrated that UUO stimulated TRPC6 expression and the downstream proteins CnA/NFAT that are necessary in the programming myofibroblast transformation (Davis et al., 2012;Hofmann et al., 2017). Overexpression of TRPC6 has been shown to activate myofibroblast transformation, while fibroblasts lacking TRPC6 were insensitive to TGF-b and angiotensin II-induced trans-differentiation (Davis et al., 2012). HKC significantly suppressed UUO-induced expressions of TRPC6, CnA, and NFAT suggesting that HKC protection against renal fibrosis is likely through TRPC6/CnA/NFAT signaling pathway. In TRPC6 KO mice, we observed reductions on both the protein expression of smad2/3 and the phosphorylated proteins of MAPK demonstrating that genetic ablation of TRPC6 attenuated both canonic and non-canonic TGF-b signaling pathways, a phenomenon consistent with HKC inhibition on TGF-b signaling pathways. The strongest evidence is that TRPC6 KO mice and HKC treated WT mice displayed comparable reductions on the tubulointerstitial injury score, interstitial fibrosis score, and a-SMA expression. Furthermore, administration of HKC in TRPC6 KO mice subjected to UUO didn't display any protection. Considered together, these data demonstrate that HKC protection against renal fibrosis is through TRPC6 dependent pathway. It should be noted that the expression levels of smad2 and smad3 in HKC-treated are higher than that observed in TRPC6 KO mice subjected to UUO. Such difference is likely due to the fact that HKC decreased UUO-induced TRPC6 expression is not complete (56% reduction) while TRPC6 KO is completely absence of TRPC6 expression.
Research has demonstrated that TRPC6 and NFAT form mutually positive feedback loop that exacerbates the renal fibrosis (Nijenhuis et al., 2011). Therefore, it is likely that direct suppression of TRPC6 activity contributes to HKC inhibited TRPC6/CnA/NFAT expression, which is responsible for TGF-b signaling critical for the progression of renal fibrosis. We demonstrate that TRPC6 is a direct target of HKC suggesting that suppression of TRPC6 activity by HKC may contributes to its renal protection. However, whether the direct inhibition of TRPC6 is exclusively responsible for HKC protection against renal fibrosis is arguable since flavonoids, the main active compounds in HKC, have been reported to modulate inflammation (Lu et al., 2018), a critical factor driving renal fibrosis. In addition, we also observed a direct inhibition of TRPC3 by HKC, a structural highly homologous protein to TRPC6 (Tang et al., 2018). Genetic ablation of TRPC3 has been shown to attenuate renal fibrosis in UUO mouse model. However, double TRPC3/6 KO confers the same degree of protection against kidney fibrosis as single TRPC6 deletion (Wu et al., 2017) suggesting that TRPC3 and TRPC6 work in the same pathway, possibly by formation of TRPC3/ TRPC6 heteromultimers.
In summary, we demonstrate that HKC, a clinically used antiproteinuria drug, attenuates tubulointerstitial injury and kidney interstitial fibrosis in a UUO mouse model. We further demonstrate that HKC protection of renal fibrosis is dependent on TRPC6 pathway, possibly through direct inhibition of TRPC6 channel activity and indirect suppression of TRPC6 expression.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/ Supplementary Material.

ETHICS STATEMENT
All the animal care and experimental protocols were approved by the Animal Ethics Committee of China Pharmaceutical University according to Guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals.

AUTHOR CONTRIBUTIONS
All authors contributed to the article and approved the submitted version. L-FG, LZ, H-TG, and Y-JW performed the experiments and analyzed data. L-FG drafted the manuscript. FZ, Z-YC and H-TT participated in data analysis. B-YY, Z-YC, and C-ZC designed the experiments and proofed the paper.

FUNDING
This work was supported by the National Natural Science Foundation of China (81972960, 21777192) and "Double First-Class" University project (CPU2018GF06).