Abstract: To investigate the effects of costunolide on ethanol-induced hepatocyte injury and steatosis in vitro and the underlying mechanism.Ethanol-induced LO2 hepatocyte cell injury model was established in vitro.We examined the effects of costunolide on the release of alanine aminotransferase (ALT) and aspartate aminotransferase (AST),the intracellular contents of total cholesterol (TG) and triglyceride (TC),the mRNA and protein expression of lipogenesis-related transcription factors and the activity of AMP-activated protein kinase (AMPK) in ethanol-stimulated LO2 cells.The results showed that ethanol at concentrations higher than 100 mM significantly suppressed the viability of LO2 cells,and ethanol at 100 mM was selected for establishing hepatocyte injury model in vitro.Costunolide restored the ethanol-induced inhibition of LO2 cell viability,and costunolide at 20 μM exerted significant effects.Costunolide also reduced the supernatant levels of ALT and AST in ethanol-stimulated LO2 cells.Furthermore,costunolide concentration-dependently decreased the accumulation of lipid components in ethanol-stimulated LO2 cells,and reduced the intracellular levels of TG and TC.Molecular examinations showed that ethanol significantly upregulated the mRNA and protein expression of SREBP-1c and downregulated the mRNA and protein expression of PPARα in LO2 cells.However,costunolide concentration-dependently reduced SREBP-1c expression and restored PPARα expression at both mRNA and protein levels in ethanol-stimulated LO2 cells.Furthermore,costunolide significantly promoted AMPK phosphorylation,and AMPK specific inhibitor BML-275 remarkably abrogated the regulatory effects of costunolide on SREBP-1c and PPARα.In conclusion,costunolide could significantly attenuate ethanol-induced hepatocyte injury and steatosis in vitro.These effects were associated with activation of AMPK and modulation of lipid metabolism-related transcription factors.These data provided experimental evidence for developing costunolide as a therapeutic option for alcohol fatty liver disease.

Key words: costunolide, alcohol fatty liver disease, hepatocyte, steatosis, AMPK