Restoration of autophagy by puerarin in ethanol-treated hepatocytes via the activation of AMP-activated protein kinase

► Ethanol reduces autophagy activity by suppressing AMPK activity in hepatocytes. ► Puerarin recovers autophagy activity by stimulating AMPK and suppressing mTOR. ► Puerarin plays hepatoprotective role in ethanol-treated hepatocytes. ► Puerarin might be used for treating alcoholic liver damage. We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100mM) for 24h reduced cell viability by 20% and increased the cellular concentrations of cholesterol and triglycerides by 40% and 20%, respectively. Puerarin stimulation significantly recovered cell viability and reduced cellular lipid accumulation to a level comparable to that in untreated control cells. Ethanol incubation reduced autophagy significantly as assessed by microtubule-associated protein1 light chain 3 (LC3) expression using immunohistochemistry and immunoblot analysis. The reduced expression of LC3 was restored by puerarin in a dose-dependent manner in ethanol-treated cells. The effect of puerarin on mammalian targets of rapamycin (mTOR), a key regulator of autophagy, was examined in ethanol-treated hepatocytes. Immunoblotting revealed that puerarin significantly induced the phosphorylation of 5′AMP-activated protein kinase (AMPK), thereby suppressing the mTOR target proteins S6 ribosomal protein and 4E-binding protein 1. These data suggest that puerarin restored the viability of cells and reduced lipid accumulation in ethanol-treated hepatocytes by activating autophagy via AMPK/mTOR-mediated signaling.