Resveratrol inhibits hepatic stellate cell activation by regulating autophagy and apoptosis through the SIRT1 and JNK signaling pathways

Abstract

Resveratrol, a natural polyphenol found in grapes, berries, peanuts, and medicinal plants, has previously been reported to have several biological activities, including inhibiting hepatic brosis. Hepatic stellate cell (HSC) activation is the major cellular source of matrix protein-secreting myo broblasts, which are the major drivers of liver brogenesis. Numerous studies on the protective effects of resveratrol against liver brosis have focused on the inhibition of HSC activation. Although the underlying mechanisms remain to be fully elucidated, autophagy and apoptosis regulation might be intimately related. The mouse HSC line JS1 was stimulated with resveratrol to assess the mechanism and relationship between autophagy and apoptosis. Resveratrol dose-dependently modulated JS1 cell viability. Moreover, resveratrol inhibited JS1 cell activation and induced autophagy and apoptosis. This anti brotic effect was attenuated when autophagy was inhibited using chloroquine (CQ) or 3-methyladenine (3-MA) or when apoptosis was inhibited using Z-VAD-FMK. Furthermore, whether the Sirtuin1 (SIRT1) and c-Jun N-terminal kinase (JNK) signaling pathways were associated with resveratrol-induced autophagy and apoptosis in JS1 cells was examined. The SIRT1 inhibitor EX527 reversed autophagy, and the JNK inhibitor SP600125 was able to reverse both autophagy and apoptosis induced by resveratrol. These ndings suggest that the SIRT1 and JNK signaling pathways may be involved in resveratrol-mediated inhibition of HSC activation by regulating autophagy and apoptosis. Materials And Methods Cells and cell cultureThe immortalized mouse HSC line JS1 was kindly provided by Professor Scott L. Friedman (Mt. Sinai School of Medicine, USA). JS1 cells were cultured in DMEM (Gibco; Thermo Fisher Scienti c, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scienti c, Inc.) and 1% penicillin/streptomycin at 5% CO 2 and 37°C. The medium was replaced with media supplemented with 0.5% FBS for 12 h prior to treatment. Different concentrations of resveratrol (cat. no. R5010; Sigma) was added to the medium and an incubated for different durations, according to the experimental design. JS1 cells were pretreated with 5 mM 3-methyladenine (3-MA; cat. no. M9281; Sigma-Aldrich; Merck KGaA), 30 µM chloroquine (CQ) diphosphate salt (cat. no. C6628; Sigma-Aldrich; Merck KGaA) or 20 µM Z-VAD-FMK (cat. no. C1202; Beyotime Institute of Biotechnology) for 2 h and 10 µM EX527 (cat. no. ab141506; Abcam) or 10 µM SP600125 (cat. no. ab120065; Abcam) for 1 h, followed by treatment with or without 50 µM resveratrol for 24 h. Each in vitro experiment was repeated three times. Measurement of JS1 cell viabilityCells were plated in plastic 96-well plates and incubated with 0.1, 1, 10, 50 or 100 µM resveratrol for 24 h. Subsequently, cell viability was assessed using an MTT Cell Proliferation and Cytotoxicity Assay kit (cat.