Trilobatin Protects Against Aβ25–35-Induced Hippocampal HT22 Cells Apoptosis Through Mediating ROS/p38/Caspase 3-Dependent Pathway

Emerging evidence reveals that an aberrant accumulation of β-amyloid (Aβ) is the main reason of Alzheimer’s disease (AD) pathogenesis. Thus, inhibition of Aβ-induced neurotoxicity may be promising therapeutic tactics to mitigate AD onset and advance. The development of agent candidates by cultured neurons against Aβ-induced cytotoxicity is widely accepted to be an efficient strategy to explore the drug for AD patients. Previously, we have revealed that trilobatin (TLB), a small molecule monomer, derives from Lithocarpus polystachyus Rehd, possessed antioxidative activities on hydrogen peroxide-induced oxidative injury in PC12 cells. The present study was designed to investigate the effects and the underlying mechanism of TLB on Aβ-induced injury in hippocampal HT22 cells. The results demonstrated that TLB attenuated Aβ25–35-induced HT22 cell death, as evidenced by MTT assay and LDH release. Furthermore, TLB dramatically mitigated cell death after Aβ25–35 insulted via decreasing the intracellular and mitochondrial ROS overproduction and restoring antioxidant enzyme activities, as well as suppressing apoptosis. Of note, Aβ25–35 triggered increase in ratio of Bax/Bcl-2, activation of caspase-3, phosphorylation of tau, JNK, p38 MAPK, and decrease in Sirt3 expression, whereas TLB reversed these changes. Intriguingly, TLB could directly bind to p38, as evidenced by molecular docking and p38 inhibitor. Taken together, the results reveal that TLB effectively protects against Aβ25–35-induced neuronal cell death via activating ROS/p38/caspase 3-dependent pathway. Our findings afford evidence for the potential development of TLB to hinder neuronal death during AD.


INTRODUCTION
Alzheimer's disease (AD), the extremely common type of senile dementia, which is accompanied by progressive learning and memory impairment, as well as cognitive deficits (Deng et al., 2017;Suh et al., 2019). Thus, AD imposes financial, social, and health care burdens around the world. AD is characterized by the aggregation of b-amyloid (Ab) plaques, loss of neurofibrillary tangles (NFTs) and neurons (Hashimoto et al., 2019). In normal physiological conditions, Ab does not exhibit any pathological activity. Whereas, it is believed that Ab deposition is the major element of senile plaques discovered in the brains of AD patients (Weiner et al., 2017). Notably, the aberrant accumulation of Ab in neurons involving complex detrimental mechanisms, including oxidative stress injury, mitochondrial dysfunction, which further results in neuronal loss, even death (Cioffi et al., 2019). Therefore, inhibition of Ab-induced neurotoxicity may be a promising therapeutic strategy to mitigate AD onset and advance. Nevertheless, to date, desired approaches or drugs are still unavailable due to the low efficacy or severe adverse drug reaction such as acetyl cholinesterase inhibitors or antagonists of N-methyl-D-aspartate receptor (Arvanitakis et al., 2019). Hence, the exploit of safe and efficient treatment strategies for AD are still an urgent clinical need. It is worth noting that a lot of neuroprotectants for AD failed in clinical trials, mainly due to an encounter with the hardness in affording therapeutic proteins or drugs to the central nervous system (Yuan et al., 2019). Therefore, it is reasonably assumed that small molecule monomer, primarily derive from the natural product, will be an alternative to affect neurons.
TLB was not only used as a natural sweetener, but also exerted multiple pharmacological activities, including, antioxidative properties (Yang et al., 2004;Li et al., 2018), anti-diabetes mellitus (Wang et al., 2016), anti-inflammation (Fan et al., 2015), anti-hepatoblastoma cells proliferation as a novel SGLT1/2 inhibitor , anti-HIV-1 activity (Yin S. et al., 2018). Intriguingly, our previous study has revealed that TLB significantly protected against hydrogen peroxide (H 2 O 2 )-induced oxidative injury in a neuron-like PC12 cell (Gao et al., 2018). Moreover, since oxidative injury plays a vital role during neuronal damage induced by Ab (Zhou et al., 2016), it is assumed that TLB could also mitigate Ab-induced toxicity in neurons. Thus, based on the above scenario, the current study was designed to evaluate the hypothesis whether TLB could protect against Ab 25-35 -induced injury in the murine hippocampal neuron HT22 cells, and further to explore its detailed mechanism.

Cell Culture and Drug Treatment
The cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin at 37°C in a humidity chamber with 5% (v/v) CO 2 . Ab 25-35 was diluted to 1 mM with sterilized saline water and then incubated at 37°C for 7 days before use (Ghasemi et al., 2014). Thereafter, the HT22 cells were treated with or without 12.5, 25, and 50 mmol/L TLB or 10 mmol/L Z-VAD-FMK (a caspase-3 inhibitor), or 20 mmol/L SB203580 (a p38 inhibitor) and 20 mmol/L Ab 25-35 for 48 h.

MTT Assay
At the end of treatment as described above. The HT22cells were cultured with MTT (5 mg/ml) for another 4 h, then, the medium was carefully removed, and 150 ml DMSO was added into each well. Thereafter, the optical density value of the cells was measured by a microplate reader at a wavelength of 490 nm. Cell viability was expressed as the percentage relative to the absorbance of the untreated control cells.

LDH Release Assay
Neurotoxicity was determined by the release of LDH, an indicator of cytotoxicity. In brief, the HT22 cells were treated as described above, then the supernatant were collected by centrifugation (2,000g, 20 min), and the LDH release was evaluated using an LDH assay kit according to the protocol instruction. Thereafter, absorbance was detected at wavelength of 490 nm, and all values of % LDH released were normalized to the untreated control group. Additionally, changes in cellular morphology were simultaneously observed by phase contrast microscopy.

TUNEL Assay
The apoptosis of HT22 cells was detected using the TUNEL assay by the In Situ Cell Death Detection Kit according to the manufacturer's instructions. Briefly, the HT22 cells were treated as described above, then the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and then incubated with 0.3% Triton X-100 in PBS for 10 min. Thereafter, the HT22 cells were incubated in TUNEL test solution at 37°C for 1 h in the dark, while, the cell nuclei were labeled using DAPI staining. Thereafter, TUNEL-positive cells were observed using a fluorescence microscope (Olympus IX73, Olympus, Tokyo, Japan) with excitation/emission (450/515 nm) wavelengths.

Measurement of Caspase 3/7 Activity
The HT22 cells were treated as described above, and the Caspase-Glo ® 3/7 Assay was used to evaluate the caspase 3/7 activity according to the manufacturer's protocols. The cells were lysed in lysis buffer, and the supernatants were collected with centrifugation (12,500 × g for 5 min at 4°C), then the supernatants were co-incubated with working solution in 96well plates for 30 min at room temperature. Thereafter, multimode reader with excitation/emission (490 nm/520 nm) wavelengths was used to detect the fluorescence intensity. All values of caspase 3/7 activities were expressed as the percentage relative to the absorbance of the untreated control cells.

Measurement of Intracellular ROS and Mitochondrial ROS (mtROS)
The HT22 cells were treated as described above. Intracellular ROS generation was evaluated by DCFH-DA fluorescence probe. Briefly, the cells were loaded with 20 mM DCFH-DA for 30 min in the dark, thereafter, washed twice with PBS and then the fluorescence was observed and imaged under a fluorescent microscope (Olympus IX73; Olympus, Tokyo, Japan) with excitation/emission (485/530 nm) wavelengths. Image Pro-Plus software was used to calculate intracellular ROS, and all values of ROS level were expressed as a percentage compared with the control. Furthermore, the Mito-SOX Red fluorescent probe, a fluorescent indicator for mitochondrial O 2 • − , was used to evaluate mtROS. In brief, the cells were incubated in 5 mM Mito-SOX Red fluorescent probe at 37°C in dark for 30 min, while, the cell nuclei were labeled using DAPI staining. Afterwards, the cells were imaged by fluorescence microscopy (Olympus IX73; Olympus, Tokyo, Japan) with excitation/ emission (510/580 nm) wavelengths.

Measurement of MDA
The level of MDA, a product of lipid peroxidation, was measured using MDA ELISA kit. In brief, the HT22 cells were treated as described above, then culture supernatant was collected by 0.25% trypsin, centrifuge at 3000 × g for 3 min. The level of MDA in the supernatant was detected according to the manufacturer's recommendations.

Measurement of Antioxidant Enzyme Activities
Evaluation of antioxidantive enzyme activities were measured using SOD, GSH-Px, and GSH assay kits. Briefly, the HT22 cells were treated as described above, then the supernatants were collected with centrifugation (3000 × g for 10 min at 4°C). Thereafter, the activities of SOD, GSH-Px, and GSH content were determined according to the manufacturer's instructions.

Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted with the Trizol Reagent, which was reverse transcribed to cDNA with the PrimeScript ™ RT Reagent Kit. qRT-PCR was performed on the CFX96 real-time PCR detection system (Bio-Rad Laboratories Ltd, Hertfordshire, UK) with specific primers, and their sequences were listed as follows: mouse p38e, forward 5′-TGTGAACGAAGACTGT GAGC-3′ and reverse 5′-GCATCCAATTCAGCATGATCTC-3′; mouse p38e, forward 5′-CGCCAGAAGGTGGCTGTAAA-3′ and reverse 5′-TGTCCTCCTCGCGTGGAT-3′; mouse GAPDH forward 5′-ACAACTTTGGCATTGTGGAA-3′ and reverse 5′-GATGCAGGGATGATGTTCTG-3′. In the reaction, 1 ml cDNA of each sample was mixed with SYBR ® GREEN PCR Master Mix according to the protocol of manufacture. And the PCR conditions: 30 s at 95°C, 40 cycles at 95°C for 5 s, followed by 56°C for 60 s. Relative gene expression was evaluated by the 2 −DDCT method. The mRNA levels of p38a and p38b in WT neurons were normalized to 1.0.

Molecular Docking
The affinity between TLB and p38a, p38b, and JNK were performed by Autodock 4.2 and AutodockTools (ADT). In brief, the PDB format file for the p38a (Protein Data Bank ID: 1ZZL), p38b (Protein Data Bank ID: 3GP0), and JNK (Protein Data Bank ID: 4IZY) protein were obtained from the Protein Data Bank database. A three-dimensional structure of TLB was established using the ChemBio3D Ultra 14.0 (PerkinElmer Informatics, USA), which was further proceeded using ADT. Thereafter, the molecular docking of the TLB and p38 proteins was performed using Autodock 4.2. The area of grid box contained the promising binding sites between TLB and JNK or p38 as previous study report .

Statistical Analysis
All data were expressed as mean ± standard deviation (SD). A one-way analysis of variance followed by Bonferroni's test was carried out to test for any differences between individual differences. A P value < 0.05 was considered statistically significant. All experiments were performed in triplicate.

TLB Concentration-Dependently Inhibited Ab 25-35 -Induced Cytotoxicity in HT22 Cells
The effect of Ab 25-35 on HT22 cells were determined, and it was found Ab 25-35 decreased cell viability and increased LDH release in a dose-and time-dependent manner. Since 48 h of Ab 25-35 (20 mM) incubation apparently induced cytotoxicity to about 50% ( Figure 1A), the concentration and incubation period were applied for the following experiments. Thereafter, the protective effects of TLB on Ab 25-35 -induced injury in HT22 cells were determined. The results showed that TLB significantly increased cell viability and decreased LDH release than those of Ab 25-35 group, as evidenced by MTT and LDH release assay, respectively ( Figures 1B, C). Moreover, the beneficial effect of TLB was also verified by morphologic observations. The results revealed that Ab 25-35 -treated cells led to the decrease in the number of cells and floatation than those of the control group; while, TLB reversed these change than those of Ab 25-35 group ( Figure 1D).

TLB Suppressed Ab 25-35 -induced ROS Generation and MDA Content in HT22 Cells
To investigate whether TLB could suppress Ab 25-35 -induced oxidative injury in HT22 cells, the generation of intracellular ROS, mtROS, MDA was detected. The results showed that the generation of intracellular ROS, mtROS, as well as MDA was significantly increased after Ab 25-35 insulted, consisting with the previous study (Angeloni et al., 2019). Whereas, TLB markedly reversed these change of ROS generation both in intracellular ( Figure 2A) and mitochondria ( Figure 2B), as well as MDA content ( Figure 2C) as evidenced by DCFH-DA staining, Mito-SOX staining, and MDA ELISA kit, respectively. These findings suggested that TLB effectively suppressed Ab 25-35 -induced oxidative injury in HT22 cells.

TLB Increased Antioxidant Enzyme Activities in Ab 25-35 -Induced Injury in HT22 Cells
To evaluate the effect of TLB on antioxidant enzyme activities in HT22 cells after Ab 25-35 insulted, the activities of SOD, GSH-Px, and the content of GSH were determined. The results showed that Ab 25-35 significantly decreased SOD and GSH-Px activities, and GSH content, in keeping with the previous study (Campolo et al., 2018). Whereas, TLB significantly reversed these change of SOD ( Figure 3A) and GSH-Px ( Figure 3B), as well as GSH content ( Figure 3C), which indicated that TLB effectively promoted antioxidant enzyme activities in Ab 25-35 -induced injury in HT22 cells.

TLB Mitigated Ab 25-35 -Induced Injury in HT22 Cells Through Mediating Caspase-3-Dependent Apoptosis Pathway
The effect of TLB on Ab 25-35 -induced apoptosis in HT22 cells were detected using TUNEL staining. Ab 25-35 triggered apoptosis in HT22 cells as evidenced by the increasing amounts of TUNEL-positive cells, and this apoptotic effect was significantly attenuated by TLB, which suggested that TLB could inhibit Ab 25-35 -induced apoptosis in HT22 cells ( Figure 4A). Furthermore, Bax/Bcl-2 ratio, cleaved-3 level, and caspase-3 activity were increased upon Ab 2 5-3 5 , whereas TLB significantly reversed these change of Bax/Bcl-2 ratio ( Figures  4B, C), cleaved-caspase-3 level ( Figures 4D, E), and caspase-3 activity ( Figure 4F) in HT22 cell injury induced by Ab 25-35. Intriguingly, to further confirmed the role of caspase activation involved in the attenuation effect of TLB on Ab 25-35 -induced apoptosis, HT22 cells were pre-treated with Z-VAD-FMK, a pan-caspase inhibitor, prior 1 h to TLB treatment. The results revealed that Z-VAD-FMK significantly abolished the cytotoxicity induced by Ab 25-35 in HT22 cells, as evidenced by MTT assay and LDH release ( Figures 4G, H). Collectively, these findings indicated that TLB suppressed Ab 25-35 -induced injury in HT22 cells, at least partially, via mediating caspase-3dependent apoptosis pathway.

p38/Sirt3 Pathway Mediated the Inhibitory Effects of TLB on HT22 Cells Injury Induced by Ab 25-35
The detailed neuroprotective mechanism of TLB on Ab 25-35induced HT22 cell injury was explored. The results showed that the phosphorylation of tau, JNK and p38 was significantly increased, while the expression and activity of Sirt3 were markedly decreased after Ab 25-35 insulted. However, TLB effectively decreased the phosphorylation of tau ( Figures 5A,  B), JNK ( Figures 5C, D), and p38 ( Figures 5E, F) and increased both the Sirt3 expression ( Figures 5G, H) and its activity ( Figure  5I). Furthermore, the neuroprotective effect of TLB on Ab 25-35induced HT22 cell injury was significantly promoted by p38 inhibitor SB203580. The results revealed that SB203580 significantly abolished the cytotoxicity induced by Ab 25-35 in HT22 cells, and promoted the beneficial effect of TLB, as evidenced by MTT assay and LDH release ( Figures 5J, K).
These findings suggested that the role of p38/Sirt3 pathway was involved in the inhibitory effects of TLB on HT22 cells injury induced by Ab 25-35 .

Prediction of Drug Targets of TLB Against Ab 25-35 -Induced Injury in HT22 Cells
To further dissect which isoform of p38 is involved in the beneficial effect of TLB on Ab 25-35 -induced injury, the p38a and p38b mRNA levels were determined using qRT-PCR. The results showed that Ab 25-35 significantly increased mRNA level of p38a isoform, but not affect p38b isoform; whereas, TLB effectively decreased mRNA level of p38a, but not affect p38b ( Figures 6A, B). Furthermore, the molecular docking was used to explore the potential target of TLB during the Ab 25-35induced HT22 cell injury. Interestingly, it was found that the binding energy of TLB with JNK, p38a and p38b was −4.21, −6.05, and −4.45 kcal/mol, respectively. The suppositive binding modes and interaction within the amino acid pocket of TLB with p38a, including LYS53, HIS107, GLY110, ALA111, TYR35, and LEU167, which further confirmed that (I) The Sirt3 activity was measured using Sirt3 activity assay kit. (n = 6). HT22 cells were pre-treated with SB203580, a p38 inhibitor, prior 1 h to TLB treatment. (J) The cell viability was determined by MTT assay. (K) LDH release from HT22 cells was measured using an LDH assay kit. Data are presented as mean ± SD. *P < 0.05; **P < 0.01 versus control group; ## P < 0.01 versus Ab 25-35 group.
TLB bound to the hydrophobic pocket of p38a, in keeping with the results in vitro ( Figure 6C). These findings indicated that the TLB might directly bound to p38a to develop its pharmacological activities.

DISCUSSION
The current study disclosed that: (1) TLB, a naturally-small molecule monomer, derived from herbal Lithocarpus polystachyus Rehd. protected against Ab 25-35 -induced injury in HT22 cells; (2) The beneficial effects of TLB were due to suppression of caspase-3-dependent apoptosis pathway, as well as inhibition of oxidative injury via mediating p38/Sirt3 pathway; (3) p38a might be the potential target of TLB as evidenced by molecular docking (Figure 7).
Emerging evidence suggests that the extracellular deposition of Ab plaques and the accumulation of intracellular NFTs are deemed to be the most vital pathophysiology of AD. Ab exposure could exacerbate tau-mediated neurotoxicity via facilitating tau phosphorylation (Holubova et al., 2019). In the present study, we found that Ab 25-35 apparently injured HT22 cells and promoted tau protein phosphorylation, consistenting with previous report (Lee et al., 2016). TLB not only effectively attenuated Ab 25-35induced injury in HT22 cells, but also inhibited tau protein phosphorylation, which suggested that TLB could suppress Abinduced neurotoxicity by restraining tau phosphorylation.
Recent studies reveal that oxidative stress injury plays an important role in pathogenesis of AD with the markers of ROS and MDA overproduction and antioxidant enzyme activities reduction (Dong et al., 2018;Ji et al., 2019). The results showed that Ab 25-35 significantly induced ROS overproduction overwhelmed antioxidant enzyme system, leading to cellular redox balance disequilibrium. Whereas TLB dramatically abolished Ab 25-35 -induced oxidative stress injury through reducing ROS overproduction and augmenting antioxidant enzyme activities, which suggested that the beneficial effect of TLB on Ab 25-35 -induced hippocampal neuron injury was attributed to its antioxidant properties. Furthermore, Bcl-2 and Bax are the vital members of Bcl-2 family proteins, and these proteins mediate multiple effectors thereby activating caspases in apoptosis (Sun et al., 2016). Of note, caspases, especially caspase-3, an eventual protein in the apoptotic cascade, which catalyzes the specific cleavage of multiple vital cellular proteins . In this study, Ab 25-35 -induced hippocampal neuron apoptosis by facilitating upregulationg of Bax/Bcl-2 ratio and cleaved-caspase-3 level. While TLB effectively reversed these change, suggesting that TLB mitigated Ab 25-35 -induced oxidative injury in HT22 cells, at least partly, through mediating caspase-3-dependent apoptosis pathway.
Notably, the silent information regulator 2 (Sir2) family is mechanically crucial in neurons upon oxidative stress . Sirtuin3 (Sirt3) is a vital deacetylase among the seven subtypes of SIR2 family, which has ability of scavenging ROS and elevating antioxidant enzyme activities (Qin et al., 2018). Interestingly, a recent study has shown that Sirt3 is abundant in the brain, and it might be a mediator that connects Ab . Thus, we focus on the role of Sirt3 during the beneficial effect of TLB on HT22 cell injury challenged by Ab [25][26][27][28][29][30][31][32][33][34][35] . Our findings demonstrated that Ab 25-35 led to Sirt3 expression and activity decrease, in keeping with the previous studies Liu et al., 2019). Whereas TLB reversed the decline in Sirt3 expression and activity after Ab 25-35 insults, which indicated that Sirt3 contributed to the attenuation effects of TLB on Ab 25-35 -induced oxidative injury in hippocampal neuron. It is worth noting that Sirt3 will be downregulated via activating mitogen-activated protein kinase (MAPK) (Shen et al., 2019), which is a family of serine-threonine kinases and has been FIGURE 7 | Presentation of a proposed mechanism for the neuroprotective role of TLB against Ab 25-35 -induced oxidative injury in hippocampal HT22 cells. TLB is thought to direct bind p38 and inhibit p38 phosphorylation, thereby increasing Sirt3 expression and then reducing overproduction of ROS both in intracellular and mitochondria, and elevating antioxidant enzyme activities. The beneficial effects of TLB on Ab 25-35 -induced neuronal apoptosis, at least partly, through mediating ROS/p38/caspase 3-dependent pathway. considered to play a crucial role in the regulation of oxidative stress and cell death (Kim and Sieburth, 2018). MAPK signaling pathways are related to the pathogenesis of a variety of neurodegenerative diseases, such as AD (Chang et al., 2018). Upon exposure to Ab stimuli, JNK and p38 are activated in neurons (Wang Q. et al., 2019). Our findings found that phosphorylation of JNK and p38 showed a remarkable increment after Ab insults; whereas, TLB significantly reversed these change. Furthermore, the inhibitory effect of TLB on Ab 25-35 -induced hippocampal neuron injury was substantially promoted by p38 inhibitor. Of note, there are four major isoforms of p38 (a, b, g, and d), which are expressed in different tissues and cell types and often functionally distinct (Bachstetter and Van Eldik, 2010). Among them, p38a is expressed in most tissues and cell types, p38b is in brain, p38g in endocrine glands, and p38d in skeletal muscle. Therefore, in attempting to elucidate the role of p38 in the suppressive effect of TLB on Ab 25-35 -induced neuronal injury, it is necessary to dissect between p38a and p38b. The results further indicated that p38a mRNA level was increased, but not affected p38b after Ab insulted, consisting with previous report (Schnoder et al., 2016). TLB mitigated the mRNA level of p38a, but not affect p38b, suggesting that TLB selectively inhibition of p38a to attenuated Ab-induced hippocampal neuron injury. Intriguingly, TLB could directly bind to p38a, but did not bind to p38b, as well as JNK, as evidenced by molecular docking. Thus, it is reasonable to speculate that p38a might be the potential target of TLB. These findings suggested that TLB effectively protected against Ab 25-35 -induced injury in hippocampal neuron, at least partly, through inhibiting the MAPK pathway and then activated Sirt3.

CONCLUSION
In summary, this study highlights a crucial role of the ROS/p38/ caspase 3-dependent pathway in the attenuation effect of TLB on Ab 25-35 -induced apoptosis in hippocampal neurons. Our findings provide evidence for the potential development of TLB to hinder neuronal death during AD, thereof the neuroprotective effect of TLB on AD is priority to further explore in vivo.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors..