Objective:To investigate effects of berberine (BBR) on cholesterol synthesis in HepG2 cells with free fatty acid (FFA)-induced steatosis and to explore the underlying mechanisms.Methods:A steatosis cell model was induced in HepG2 cell line fed with FFA (0.5 mmol/L,oleic acid:-palmitic acid =2:1),and then treated with three concentrations of BBR;cell viability was assessed with cell counting kit-8 assays.Lipid accumulation in cells was observed through oil red O staining and total cholesterol (TC) content was detected by TC assay.The effects of BBR on cholesterol synthesis mediators were assessed by Western blotting and quantitative polymerase chain reaction.In addition,both silent information regulator 1 (SIRT1) and forkhead box transcription factor O1 (FoxO1) inhibitors were employed for validation.Results:FFA-induced steatosis was successfully established in HepG2 cells.Lipid accumulation and TC content in BBR groups were significantly lower (P ＜ 0.05,P ＜ 0.01),associated with significantly higher mRNA and protein levels of SIRT1(P ＜ 0.05,P ＜ 0.01),significantly lower sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy 3-methylglutaryl-CoA reductase levels (P ＜ 0.05,P ＜ 0.01),as well as higher Acetyl-FoxO1 protein level (P ＜ 0.05,P ＜ 0.01) compared to the FFA only group.Both SIRT1 inhibitor SIRT1-IN-1 and FoxO1 inhibitor AS1842856 blocked the BBR-mediated therapeutic effects.Immunofluorescence showed that the increased SIRT1 expression increased FoxO1 deacetylation,and promoted its nuclear translocation.Conclusion:BBR can mitigate FFA-induced steatosis in HepG2 cells by activating SIRT1-FoxO1-SREBP2 signal pathway.BBR may emerge as a potential drug candidate for treating nonalcoholic hepatic steatosis.