Oxymatrine ameliorates agomelatine-induced hepatocyte injury through promoting proteasome-mediated CHOP degradation

Biomedicine & Pharmacotherapy 114 (2019) 108784

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Oxymatrine ameliorates agomelatine-induced hepatocyte injury through promoting proteasome-mediated CHOP degradation

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Yina Jiaa,1, Sen Longc,1, Nan Jiangb, Zhe Shand, Yingmei Lue, Feng Hanb, Jianqiang Yua, , ⁎ Lili Fengb, a

Department of Pharmacology, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, 750004, China Key Laboratory of Cardiovascular & Cerebrovascular Medicine, School of Pharmacy, Nanjing Medical University, Nanjing, 211166, China c Department of Pharmacy, Hangzhou Seventh People’s Hospital, Mental Health Center Zhejiang University School of Medicine, Hangzhou, 310013, China d Institute of Translational Medicine, Zhejiang University, Hangzhou, 310058, China e Department of Neurobiology, Nanjing Medical University, Nanjing, 211166, China b

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A B S T R A C T

Keywords: Oxymatrine CHOP degradation Hepatocyte injury ER stress Agomelatine

Background: The novel antidepressant drug agomelatine has been observed to cause adverse effect of hepatotoxicity in clinical applications. This study was designed to explore protective agents and investigated the underlying mechanism on L02 cells. Method: L02 cells were treated with agomelatine and oxymatrine (OMT) and cell apoptosis were analyzed through flow cytometric analysis, CCK-8 assay and TUNEL assay. In a separate experiment, the expressions of ER stress-related proteins were determined by western blot. In addition, MG132, chloroquine (CQ) and bafilomycinA1(BafA1) were used to investigate the potential pathway participating in CHOP degradation. Results: OMT significantly rescued agomelatine-induced hepatocyte apoptosis. Agomelatine treatment resulted in accumulation of CHOP protein in L02 cells, and this phenomenon could be significantly reduced by OMT, whereas abolished by MG132 treatment. Conclusion: We have demonstrated for the first time that OMT ameliorates the hepatocyte toxicity induced by agomelatine through decreasing CHOP on protein level. The underlying mechanism was proved to involve the molecular events that OMT promotes CHOP degradation via proteasome pathway. Overall, these results suggest that using OMT in combination with agomelatine may provide a safety strategy for clinical depression treatment.

1. Introduction Antidepressant was reported to have potential to cause liver damage even at therapeutic concentration, which led to a major safety concern in clinical applications towards depressed patients. Among all patients, 0.5%–3% individuals who have been treated with antidepressants were reported to suffer from asymptomatic abnormal liver function, including agomelatine [1–4]. Agomelatine is widely used for the therapy of depression and has been proved to be effective in clinical. Patients who need to take agomelatine were recommended to test their liver

function first, with a continuously monitor for serum ALT level during the treatment [5,6]. What’s worse, long-term usage of agomelatine will induce liver damage. The side-effect causes both psychological and mental burden for patients. However, a drug could be rejected by patients and doctors and even withdraw from market because of druginduced liver injury [7]. Considering the contradiction between pharmacotherapy effectiveness and adverse reactions, understanding the cellular mechanisms behind antidepressants-induced hepatotoxicity is essential to optimize outcomes for clinical treatment. Endoplasmic reticulum (ER) stress plays a critical role in

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATF6, activating transcription factor 6; BafA1, bafilomycinA1; CCK-8, cell counting kit-8; CETSA, cellular thermal shift assay; CQ, chloroquine; ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α; ISRIB, integrated stress response inhibitor; MAO, monoamine oxidase; OMT, oxymatrine; PERK, protein kinase RNA-like ER kinase; SEM, standard error of the mean; SNRIs, serotonin-norepinephrine reuptake inhibitors; SSRIs, selective serotonin reuptake inhibitors; TUNEL, TdT-mediated dUTP Nick-End labeling assay; DMEM, dulbecco’s modified eagle’s medium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; one-way ANOVA, one-way analysis of variance ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Yu), [email protected] (L. Feng). 1 The authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.108784 Received 28 January 2019; Received in revised form 3 March 2019; Accepted 13 March 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Fig. 1. Agomelatine induced hepatocyte toxicity in L02 cells. (A) L02 cells were exposed to agomelatine from 10 to 500 μM for 24 h, and cell viability was measured by using CCK-8 assays. The data was representative of five independent experiments. (B) L02 cells were treated with different concentration agomelatine from 10 to 500 μM for 12, 24, and 48 h, and cell viability was measured by using CCK-8 assays. The data was representative of three to five independent experiments. (C) L02 cells were stimulated with 20, 50, 100 μM agomelatine for 24 h. Representative graph of flow cytometric was shown that cells were doublestained with PI and Annexin-V and analyzed by using flow cytometry assay. (D) The statistical graph of flow cytometric was representative of three independent experiments. (E) L02 cells were exposed to different concentrations from 20 to 200 μM agomelatine for 24 and 48 h. The change of absolute value of ALT was evaluated by ALT kit. (F) The change of absolute value of AST was assessed by AST kit. The data was representative of three to seven independent experiments. All results were shown mean ± SEM. *p < 0.05; **p < 0.01; *** p < 0.001 versus control. AMT means the abbreviation of agomelatine.

poisons induced liver damage [22,23]. Therefore, in this study, we sought a combination therapy that would restrain agomelatine-induced hepatotoxicity in L02 cells and further explored the underlying mechanism of OMT. Our aim was to provide candidate strategy of clinical practice for relieving antidepressant-induced liver injury.

mammalian cells as maintaining protein-quality-control and recovering cellular homeostasis under disturbance state. During the pathological processes of chronic or acute liver disease, ER stress response was found activated in response to the disorder of protein secretion [8–10]. Over activated or persistent ER stress results in high expression of CHOP protein [11], and the excessive accumulation of CHOP protein brings about cell damage or cell apoptosis through switching cellular signaling from pro-survival pathway to pro-death [12–14]. Thus, restoring the ER stress homeostasis might be an effective therapeutic strategy for patients with liver injury [15,16]. OMT, a quinolizidine alkaloid extracted from Sophora flavescens Ait, has been indicated for possessing several pharmacological characteristics such as antiviral hepatitis, anti-inflammatory, antitumor and ischemia damage [17–21]. Experiments on rodents have designated a protective effect of OMT towards ethanol, high-glucose or chemical

2. Methods and materials 2.1. Reagents Agomelatine was obtained from Hansoh Pharma. OMT was purchased from Luye Pharma. In addition, all other chemical agents were bought from Sigma-Aldrich, unless otherwise mention.

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Fig. 2. Agomelatine induced hepatocyte damage through triggering ER stress. (A) Representative western blot outcomes shown that the gray image of BIP, p-IRE1α, ATF6, CHOP in L02 cells after treatment with 20 μM–200 μM agomelatine for 24 h. (B) The presented statistical bar graph was the fold change of BIP, p-IRE1α, ATF6, CHOP. (C) Representative western blot results shown that the gray image of BIP, pIRE1α, ATF6, CHOP in L02 cells after being exposed to 50 μM agomelatine for 3 h–48 h. (D) The displayed statistical bar graph was the fold change of BIP, p-IRE1α, ATF6, CHOP. Three results were showed with almost same results. All results were shown mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus control. AMT means the abbreviation of agomelatine.

using a multimode reader (Beckman Coulter; DTX880), measured the optical absorbance at 450 nm.

2.4. Flow cytometry assay Flow cytometric assays were double stained by annexinV-FITC and propidium iodide (PI) (BioVision) to analyze cell apoptosis, following the manufacturer’s instruction. L02 cells were treated with agomelatine (20, 50, 100 μM) for 24 h to evaluate the damage of hepatocytes. In a separate experiment, another group of L02 cells were pretreated with OMT (0.5, 1, 2 μM) for 18 h and then exposed to 50 μM agomelatine for 24 h and 48 h to evaluate the protective function of OMT. Briefly, L02 cells were collected, washed twice with PBS, added to 5 μl Annexin VFITC and 5 μl PI for the cell suspension, and incubated at room temperature in the dark for 15 min. For each sample, at least 1 × 106 cells were detected by using a FACS-Calibur flow cytometer (BD Biosciences).

2.2. Cell line and cell culture The immortalized normal human hepatic cell line L02 (HL-7702) was purchased from the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; C11995500) with supplemented 20% fetal bovine serum (10099-141; Gibco, Waltham, MA, USA) and cultured at 37 °C in a humidified condition of 5% CO2. Cells were digested and passaged when growing into 80% confluence.

2.5. ALT and AST assays 2.3. Cell counting kit-8 (CCK-8) assays L02 cells were seeded in 6-well plate at a density of 2.0 × 105 cells per well, and cultured overnight to allow cells growing to 70–80% density. L02 cells were treated with different concentration agomelatine (20, 50, 100, 200 μM) for 24 h and 48 h to evaluate the hepatotoxicity of agomelatine. In a separate experiment, in order to assess hepatocyte protective effect of OMT, L02 cells were pre-incubated with OMT (0.5, 1, 2 μM) for 18 h, then treated with 50 μM agomelatine for 24 h and 48 h. L02 cell supernatant was collected, then the damage degree was evaluated by commercially available assay kits supplied by Nanjing Jiancheng Bioengineering Institute with catalog C009-2 and C010-2. The absolutely value of ALT and AST was detected on a multimode reader (Beckman Coulter; DTX880) through measuring the optical absorbance at 510 nm.

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L02 cells were planted in 96-well plate by 8.0 × 10 cells per well and cultured overnight to allow growing to 70–80% density before treating with agomelatine in dosage dependent or time dependent to detect hepatocyte toxicity. In addition, L02 cells were pretreated liver protectants with different concentration of OMT, silymarin, tiopronin or schisandrin B before treating with 50 μM agomelatine for 24 h and 48 h to investigate the hepatocyte protective effect of liver protectants. In a separate experiment, L02 cells were treated with ISRIB (2.5, 5, 10 nM) for 1 h and then stimulated with 50 μM agomelatine for 24 h. Finally, L02 cells were incubated with 200 μl complete medium containing 20 μl CCK-8 reagent per well for 1 h. CCK-8 assay (Dojindo; CK04) was used to estimate cell viability which was determined by 3

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Fig. 3. Agomelatine-induced hepatocyte toxicity was ameliorated by OMT. (A) L02 cells were treated with 0.5, 1, 2 μM OMT for 18 h before being exposed to 50 μM agomelatine for 24 h. Representative graph of flow cytometric was shown that cells were double-stained with PI and Annexin-V and analyzed by flow cytometry assay. (B) The statistical graph of flow cytometric was representative of three independent experiments. (C) L02 cells were pretreated with 0.5, 1, 2 μM OMT for 18 h, then exposed to 50 μM agomelatine for 24 h, and cell viability was assessed by using CCK-8 assays. The data was representative of four independent experiments. (D) L02 cells were treated with 0.5, 1, 2 μM OMT for 18 h then stimulated with 50 μM agomelatine for 24 h, and the change of absolute value of ALT was estimated by ALT kit. (E) The change of absolute value of AST was estimated by AST kit. The data was representative of three to four independent experiments. (F) After treatment with OMT and agomelatine, apoptotic cells were analyzed by TUNEL assay. Representative images were shown that double staining with DAPI (blue) and TUNEL (green). (G) Representative statistical graph was the percentage of TUNEL-positive apoptotic cells. The data was representative of five independent experiments. All data were expressed mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 versus control. #p < 0.05, ##p < 0.01, ###p < 0.001 versus agomelatine. AMT means the abbreviation of agomelatine. OMT means the abbreviation of oxymatrine.

then treated with 50 μM agomelatine for 24 h. In situ DNA fragmentation were assayed by using TUNEL staining kit (Roche; 11684795910), according to the manufacturer's instruction. Briefly, L02 cells were fixed with 4% paraformaldehyde for 15 min and treated with 0.1% Triton X-100 for 5 min, then incubated with TUNEL reaction mixture

2.6. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay L02 cells were seeded in 24-well plate at the number of 3.0 × 104 cells per well, and cultured overnight to allow cells growing to 70–80% density. L02 cells were pre-incubated with 0.5, 1, 2 μM OMT for 18 h, 4

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Fig. 4. OMT attenuated hepatocyte injury through promoting the degradation of CHOP. (A) L02 cells were pretreated with 0.5, 1, 2 μM OMT for 18 h and then treated with 50 μM agomelatine for 24 h. Representative western blot outcomes showed that the gray image of BIP, p-IRE1α, ATF6, CHOP. (B) The displayed statistical bar graph was the fold change of BIP, p-IRE1α, ATF6, CHOP. Three results were showed with almost same results. All results were shown mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus control. #p < 0.05, ##p < 0.01, ###p < 0.001 versus agomelatine. AMT means the abbreviation of agomelatine. OMT means the abbreviation of oxymatrine.

Fig. 5. OMT promoted the degradation of CHOP mediated by proteasome. (A) L02 cells were respectively treated with 1 μM MG132, 10 μM CQ, or 50 nM BafA1 for 1 h. Representative western blot results showed that the gray image of CHOP. (B) The presented statistical bar graph was the fold change of CHOP. (C) L02 cells were treated with 2 μM OMT 18 h followed by 1 μM MG132 for 1 h before being exposed to 50 μM agomelatine for 24 h. Representative western blot outcomes showed that the gray image of CHOP. (D) The displayed bar graph was the fold change of CHOP. Three results were showed with almost same results. All results were shown mean ± SEM, *p < 0.05, **p < 0.01, *** p < 0.001 versus control. AMT means the abbreviation of agomelatine. OMT means the abbreviation of oxymatrine.

and cultured overnight to allow growing to 70–80% density. Then cells exposed to agomelatine in dosage dependent or time dependent to evaluate the injury mechanism of agomelatine. In addition, in order to estimate hepatocyte protective mechanism of OMT, L02 cells were preincubated with OMT (0.5, 1, 2 μM) for 18 h, then treated with 50 μM agomelatine for 24 h. In a separate experiment, one group L02 cells were treated with 1 μM MG132, 10 μM CQ, or 50 nM BafA1 for 1 h, referring to the method reported in other article [24–27]. Meanwhile, another group of L02 cells were treated with OMT (0.5, 1, 2 μM) for 18 h, then exposed to 1 μM MG132, 10 μM CQ, or 50 nM BafA1 for 1 h,

for 1 h at 37℃ in the dark. The images were captured by the confocal microscope (Zeiss; LSM710) by using 10× objectives. A total number of L02 cells were recorded after counterstaining with 4′,6-diamidino-2phenylindole dihydrochloride (DAPI) (nuclei marker), and the apoptotic cells which was showed as the percentage of TUNEL positive L02 cells/the total number of nuclei was analyzed by Image J software. 2.7. Western blotting L02 cells were planted in 6-well plate by 2.0 × 105 cells per well 5

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Fig. 6. OMT did not activate AMPKα and AKT to regulate CHOP degradation. (A) Molecular docking of OMT, A-769662 and PKA with AMPK was performed by using SYBYL-X 1.3 software. (B) L02 cells were pretreated with 0.5, 1, 2 μM OMT for 18 h before treatment with 50 μM agomelatine for 24 h. Representative western blot outcomes showed that the gray image of p-AMPKα. (C) The presented bar graph was the fold change of p-AMPKα. Three results were showed with almost same results. (D) L02 cells were pretreated with 0.5, 1, 2 μM OMT for 18 h before treatment with 50 μM agomelatine for 24 h. Representative western blot outcomes showed that the gray image of p-AKT as dosage dependent. (E) Representative western blot results showed that the gray image of p-AKT as time dependent. (F, G) The presented statistical bar graph was the fold change of p-AKT. Three results were showed with almost same results. (H) Representative western blot results showed that the gray image of AKT as temp dependent. All results were shown mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus control. AMT means the abbreviation of agomelatine. OMT means the abbreviation of oxymatrine.

next, stimulated with 50 μM agomelatine for 24 h. Finally, the protein level of CHOP were detected by western blot. L02 cells were lysed in lysis buffer consisting of 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), 50 mM NaF, 30 mM sodium pyrophosphate, 10 mM EDTA, 4 mM EGTA, 1 mM Na3VO4, 1 mM dithiothreitol, 100 nM calyculin A, 50 μg/ml leupeptin, 50 μg/ml trypsin inhibitor and 25 μg/ml pepstatin A. Twenty micrograms of protein was loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before electrophoresis and transferred to an immune-blot PVDF membrane. Protein samples were then incubated with primary antibodies: Phospho-AKT (Ser473) (1:1000; Cell Signaling Technology, 9271), AKT (pan) (11E7) (1:2000; Cell Signaling Technology, 4685), PhosphoAMPKα (Thr172) (1:1000; Cell Signaling Technology, 2531), BIP (C50B12) (1:1000; Cell Signaling Technology, 3177), ATF6 (1:2000; Abcam, 37149), Phospho-IRE1α (1:2000; Abcam, 48187), PhosphoeIF2α (1:2000; Cell Signaling technology, 3398), eIF2α (1:2000; Cell Signaling technology, 9722), CHOP (1:2000; Cell Signaling Technology,

5554), or ACTB (1:5000; Sigma Aldrich, A5441) overnight at 4℃ before applied to the secondary antibodies conjugated with anti-rabbit or antimouse IgG-horseradish peroxidases. Finally, all signal bands were compared and analyzed by Image J and standardized through the ACTB in the matched experiments. 2.8. Cellular thermal shift assay (CETSA) L02 cells were respectively exposed to 50 μM agomelatine and 2 μM OMT for 3 h in culture dish before washed and harvested with 1 × PBS. The cell precipitation was divided into nine aliquots within each group. Each sample was individually heated at corresponding temperature (37, 40, 43, 46, 49, 52, 55, 58, 61℃) for 3 min. In order to detach the soluble protein from cell precipitates, all cell lysates were centrifuged at 17,500 × g for 30 min at 4 °C. Finally, all cell supernatants were quickly freezethawed three times at -80℃ and quantitated to same protein level before analysis by using western blot. 6

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were significantly decreased when combined use of OMT and agomelatine (Fig. 3D, E). Moreover, the efficacy of OMT on alleviating hepatocyte injury was also estimated by TUNEL staining. Extensively enhancement of positive cell of TUNEL staining was observed in the agomelatine-treated group, as compared to 0.1% DMSO-treated group, while the cellular apoptosis was dramatically reduced when L02 cells were treated with different concentration of OMT combined with agomelatine (Fig. 3F, G). To comprehensively evaluate the protection of OMT, we further preformed the same detection when cells were treated with agomelatine for 48 h, but we found that OMT significantly rescued agomelatine-induced cell injury, even better than in 24 h (Supplementary Fig. S2. AeE). In addition, we also evaluated other liver protectants in vitro. Unfortunately, silymarin and tiopronin have little effective on protecting cells from agomelatine induced cytotoxicity (Supplementary Fig. 3S. A, B). Surprisingly, combination therapy of schisandrin B and agomelatine exaggerated cell apoptosis (Supplementary Fig. 3S. C). These results indicated that combination therapy of OMT and agomelatine could alleviate the cytotoxicity caused by agomelatine in L02 cells.

2.9. Statistical analysis Statistical analyses were carried out by GraphPad Prism 6 Software. For comparison between the two groups, the data were analyzed by ttests. For multiple groups comparison, the data were determined by one-way analysis of variance (one-way ANOVA) followed by Tukey’s post hoc test. Experimental results were given mean ± standard error of the mean (SEM), and a value of p < 0.05 was considered to be significant. 3. Results 3.1. Agomelatine induced cytotoxicity in L02 cells In order to determine the hepatotoxic character of agomelatine and establish the appropriate cell model for further study, we treated L02 cells with various dose of agomelatine under multiple time periods and monitored the cell viability by using CCK-8 assay. Results indicated that the cell survival rate was significantly inhibited by agomelatine in a concentration and time dependent way (Fig. 1A, B). To further confirm the optimistic effect of agomelatine on cell apoptosis, L02 cells were double stained with Annexin V/PI and analyzed by using flow cytometry. Compared with control group (8.81%), the quantity of apoptosis cells was significantly increased as dose-dependent when treated with different concentration of agomelatine for 24 h (24.56%, 40.02%, 46.44%) (Fig. 1C, D). Furthermore, agomelatine-induced hepatocyte injury was estimated by using ALT and AST kit. L02 cells were exposed to agomelatine from 20 to 200 μM for 24 or 48 h, and the results showed that the absolute value of ALT and AST significantly elevated as dose- and time-dependent (Fig. 1E, F). All these observations confirmed the hepatocyte injury of agomelatine in L02 cells. Considering the minor difference of cell toxicity between 50 and 100 μM agomelatine, we chose 50 μM treatment for 24 h in the further experiments.

3.4. OMT attenuated hepatocyte injury through promoting the degradation of CHOP Combination therapy of OMT and agomelatine in L02 cells, compared with the agomelatine alone given group, protein level of CHOP was significantly suppressed, while the protein level of BIP, p-IRE1α and ATF6 did not change (Fig. 4A, B). These observations suggested that reduced CHOP level by OMT treatment contributes to its protective effect against the agomelatine-induced hepatocyte injury. 3.5. OMT triggered the degradation of CHOP mediated by proteasome In mammalian cells, proteins are degraded mainly via proteasome, lysosome or autophagosome [29]. Therefore, we further investigated the underlying mechanisms in order to address how OMT decreased CHOP level. To achieve this, we pretreated L02 cells with the various inhibitors to block the corresponding protein degradation pathway. The results shown that only MG132 (proteasome inhibitor), rather than CQ (autophagy inhibitor) and BafA1 (lysosome inhibitor), maintained the protein level of CHOP in L02 cells (Fig. 5A, B). Furthermore, significantly reduction of CHOP protein was reserved when MG132 was added to the medium containing with OMT and agomelatine (Fig. 5C, D). Because CHOP ubiquitination and degradation on proteasome is via the phospho-AMPKα1 pathway [29]. We examined that the binding force of OMT and AMPK (score: 3.9771) was much lower to the agonist A-769662 (score: 7.4704) and inhibitor PKA (score: 9.7824) (Fig. 6A). Consistently, we investigated whether AMPK signaling were involved in OMT and agomelatine combination therapy. We found that OMT did not influence the activation of AMPK (Fig. 6B, C), providing firm evidence that AMPK pathway is unrelated to the present context. AKT inhibition was reported to induce CHOP expression [30]. In line with the studies, agomelatine also displayed obvious suppression on phospho-AKT and enhancement on CHOP (Fig. 6D–G). We further preformed the CETSA assay to examine whether OMT and agomelatine targeted AKT directly. The result shown that either OMT or agomelatine failed to maintain the thermal stability of AKT (Fig. 6H). Together, our observations indicated a key role of proteasome in the protection of OMT on agomelatine-induced ER stress.

3.2. Agomelatine induced hepatocyte injury through activating ER stress Emerging evidences implied that ER stress participates in pathological process of viral hepatitis, alcoholic or nonalcoholic liver disease and hepatic ischemia or reperfusion injury [28]. Here, our results suggested that the expression of BIP, p-IRE1α, ATF6 and CHOP was significantly increased as dose-dependent and time-dependent manner in agomelatine treated L02 cells, compared with 0.1% DMSO control group (Fig. 2A–D). However, the activation of eIF2α was barely disturbed by agomelatine (Supplementary Fig. S1. AeD). Notably, Integrated Stress Response inhibitor (ISRIB), the antagonist of PERK-related pathway which inhibited phosphorylation of eIF2α, failed to rescue the apoptotic cells treated with agomelatine (Supplementary Fig. S1. E). 3.3. OMT treatment ameliorated agomelatine-induced hepatocyte toxicity One crucial aim of this study is to determine whether the combination of OMT and agomelatine can alter agomelatine-induced hepatocyte injury. Our results suggested in comparison to control group (3.68%), the Annexin V/PI-positive cells (23.76%) was significantly elevated when treated with 50 μM agomelatine for 24 h. On the other hand, the percentage of apoptosis cells were remarkably reduced (17.74%, 13.19%, 9.87%) when treated with OMT (Fig. 3A, B). The present result suggested that OMT has an anti-apoptotic function towards agomelatine treated L02 cells. In addition, CCK-8 assay result confirmed that 0.5, 1 and 2 μM OMT could attenuate the hepatocyte toxicity after treatment with 50 μM agomelatine for 24 h (Fig. 3C). Furthermore, this result was further agreed by the ALT and AST assay. L02 cells were pretreated with 0.5, 1 and 2 μM OMT for 18 h before being exposed to 50 μM agomelatine for 24 h. The results indicated that ALT and AST absolute value, which represented hepatocyte damage,

4. Discussion Clinical cases reports have confirmed the hepatic injury induced by antidepressants for psychiatric patients [31–34]. As the first study to explore the protective strategy for agomelatine administrating patients, we proved that combination of OMT with agomelatine was an effective 7

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degraded in a proteasome-dependent way in agomelatine and OMT simultaneously treated cell. Our study might provide a safety therapeutic strategy for patients who administrated agomelatine to restrain the potential liver injury.

method and further investigated the underlying mechanism. We successfully established a cell model using L02 cells to mimic the agomelatine-induced hepatocyte injury. Taking advantage of this artificial cellular evaluation system, we screened some liver protectants and found that combining usage of OMT and agomelatine significantly rescued the cell injury. We screened various dose of agomelatine to measure its hepatotoxicity. Unfortunately, cell apoptosis was not detected until 50 μM in vitro. For the purpose of searching protective compounds, we chose 50 μM as working dose to artificially mimic agomelatine’s liver toxicity, although it was much higher than the max blood concentration of agomelatine administrating patients [35]. Liver damage and hepatotoxicity induced by agomelatine was reported to be a dose-dependent event [36], in spite of plasma half-life of agomelatine is about 2 h [37]. According to reported data, after treatment with agomelatine, the drug can be quickly distributed in gastrointestinal, liver, brain, kidney, spleen or uterus [38,39]. However, whether agomelatine accumulated in liver was still unclear. We will further design experiments to explore the underlying mechanisms. We screened some compound using cell injury model induced by the high dose agomelatine. We found that OMT shown protective effect against cell injury, indicating by significantly reduced percentage of apoptosis cell and markedly decreased absolute value of ALT/AST. At the same time, as the treatment time of agomelatine increasing, we found that the protective effect of OMT became more and more obvious. Further proved that OMT as a great liver protectant can be applied to ameliorate agomelatine-induced hepatocyte injury. However, combination of agomelatine and silymarin, tiopronin or schisandrin B did not ameliorate cell viability. This observation strongly suggested the specific pharmacological effect of OMT. Therefore, the molecular mechanism of OMT against agomelatine-induced hepatic cell injury were further examined. As highly metabolic cells, hepatocytes were responsible for protein synthesis and secretory [40]. It was greatly necessary for hepatocytes to maintain ER homeostasis and capability. ER stress has been reported involved in progression of liver related diseases [41], which was mediated by three classical signaling pathways, including inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6 (ATF6) and protein kinase RNA-like ER kinase (PERK) [35,42]. The optimum evaluated marker of ER stress, CHOP protein, was significantly increased in agomelatine treated cells. Indeed, our results found that agomelatine triggered ER stress was mediated by IRE1α and ATF6 pathway, rather than PERK pathway. These observations indicated the mechanism of agomelatine, which might provide potential targets for us to interrupt to reduce its cytotoxicity. Unexpectedly, we found that combination of OMT and agomelatine failed to suppress the increased p-IRE1α and ATF6, while protein level of CHOP was significantly inhibited. These data revealed a previously unrecognized mechanism of OMT in attenuating hepatocyte toxicity in agomelatine treated L02 cells. Xiaoyan Dai and colleagues proved that AMPKα1 promoted CHOP degradation mediated by proteasome in macrophages, however, AMPKα was not activated by OMT [29]. Naoya Mimura and colleagues demonstrated that inhibited AKT triggers expression of CHOP [30], but both OMT and agomelatine didn’t target to AKT directly. Thus, we turned our attention to the degradation of CHOP. Proteasome pathway inhibitor, MG132, rather than lysosome and autophagy inhibitors, reserved the protein level of CHOP in OMT and agomelatine treated cells. Therefore, lysosome pathway and the autophagy pathway did not involve in the inhibitory effect of OMT on CHOP expression. These data demonstrated that combination of OMT and agomelatine mediated the proteasome-dependent CHOP degradation to alleviate ER stress induced cell apoptosis. However, there are still alternative mechanisms should be explored in future. In summary, our data illustrated that combination of agomelatine and OMT protected cells from ER stress induced apoptosis. And we proved the detailed molecular mechanisms was that CHOP was

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