The Involvement Of Nrf2-keap1-ARE Pathway In Autophagy-regulated Ethanol-induced HSCs Activation

Objective: To explore the effect of autophagy inhibitor3-methyladenine on theactivation of hepatic stellate cells, and further investigate whether autophagyinhibition results in decreased HSCs activation induced by ethanol viaNrf2-keap1-ARE signaling pathway.Methods: In this study, the in vitro model of alcohol-induced hepatic stellatecells activation is set up by culturing the cells with alcohol. We used3-methyladenine(3-MA) as autophagy inhibitor to block autophagy in HSC-T6cells. Toupregulate or downregulate the expression of Nrf2in HSC-T6cells, pEGFP-Nrf2orsi-Nrf2were transfected into cells by lipofectin. RT-PCR and western blotting wereused to detect the expression of Nrf2and the activation markers of hepatic stellatecells, including α-SMA and collagenⅠ. Microtubule-associated protein light chain3beta, the marker protein of autophagy, and P62, the selective degradation substrate ofautophagy, was detected by western blotting,as well as MDC staining, to determinethe autophagy level in HSC-T6cells. Cell proliferation rate was assessed by MTTassay and cell cycle was detected by flow cytometry.Results: A significant elevated autophagy level was observed during HSC-T6activation induced by ethanol. Treatment of HSC-T6cells with3-MA resulted insignificant decreased autophagy and activation level of HSC-T6cells. In addition, thelevel of Nrf2activation increased accompanied by3-MA-caused decrease ofautophagy level. Nrf2overexpression markedly inhibited ethanol-induced HSCsactivation. Conversely, knockdown of Nrf2significantly abolished the inhibitoryeffect of3-MAon HSCs activation.Conclusion: Ethanol could promote autophagy which contributes to HSCsactivation. Reduction of autophagy level by3-MA could suppress activation of HSCsand down-regulate the expressions of α-SMA and collagenⅠthrough Nrf2. Takentogether, these results indicate that selective interruption of autophagy in HSCs mayprovide a therapeutical strategy for alcoholic liver fibrosis.