Dihydromyricetin attenuates palmitic acid-induced oxidative stress by promoting autophagy via SIRT3-ATG4B signaling in hepatocytes

Mantian Mi,Hedong Lang,Xianglong Zeng,Bo Li,Min Zhou,Li Huang,Cong Wang,Long Yi

doi:10.1186/s12986-021-00612-w

Abstract

BackgroundOxidative stress in hepatocytes was important pathogenesis of nonalcoholic steatohepatitis (NASH). Autophagy was a cellular process that can remove damaged organelles under oxidative stress, and thus presented a potential therapeutic target against NASH. This work aimed to investigate whether autophagy was participated in the protective effects of dihydromyricetin (DHM) on palmitic acid (PA)-induced oxidative stress in hepatocytes and the underlying mechanism.MethodsHepG2 and HHL-5 cell lines were pretreated with DHM (20 μM) for 2 h, followed by PA (0.2 mM) treatment for 16 h. The oxidative stress was assessed by the quantification of intracellular reactive oxygen species (ROS), mitochondrial ROS (mtROS), mitochondrial membrane potential (MMP) and mitochondrial ultrastructural analyses. The protein expressions of SIRT3, LC3I/II, P62 and ATG4B, as well as the acetylation of AGT4B were determined by western blotting using HepG2 and HepG2/ATG4B± cells with heterozygous knockout of ATG4B.ResultsExposure to PA resulted in increased intracellular ROS and mtROS, decreased MMP and aggravated mitochondrial injury in HepG2 cells, which were notably attenuated by DHM treatment. DHM-induced inhibition of oxidative stress was associated with the induction of autophagy, characterized by upregulated ATG4B and LC3 II as well as downregulated P62 levels. Furthermore, the inhibitory effects of DHM on PA-induced autophagy arrest and oxidative stress were eliminated when pretreated with a SIRT3 inhibitor 3-TYP or conducted in HepG2/ATG4B± cells, suggesting that SIRT3 and ATG4B were involved in DHM-induced benefits. Moreover, DHM treatment increased the protein expression of SIRT3 and SIRT3-dependent deacetylation of ATG4B in HepG2 cells.ConclusionOur results demonstrated that DHM attenuated PA-induced oxidative stress in hepatocytes through induction of autophagy, which was mediated through the increased expression of SIRT3 and SIRT3-mediated ATG4B deacetylation following DHM treatment.

Highlights

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver disease which is becoming the main cause of chronic liver disease
DHM inhibits palmitic acid (PA)‐induced oxidative stress in hepatocytes To clarify the effects of DHM on PA-induced oxidative stress in hepatocytes, Human hepatocellular cancer cell line (HepG2) cells were labeled with DCFH-DA probe and qualified by flow cytometry (FCM) to determine the intracellular reactive oxygen species (ROS) level
DHM treatment suppressed the increase of intracellular ROS (Fig. 3D, E) and mitochondrial ROS (mtROS) (Fig. 3F, G) as well as decreased membrane potential (MMP) (Fig. 3H, I) in PA-induced hepatocytes

Summary

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver disease which is becoming the main cause of chronic liver disease. According to the “two-hit theory”, liver steatosis (the “first hit”) induces fat accumulation, followed by oxidative stress (the “second hit”) which leads to proinflammatory molecules release, mitochondrial damage and lipid peroxidation [3]. Palmitic Acid (PA), which was commonly employed for NAFLD induces oxidative stress by endoplasmic reticulum (ER) stress, ER calcium depletion, intracellular calcium dyshomeostasis and mitochondrial dysfunction, respectively [6,7,8]. Protecting hepatocytes against oxidative stress and maintaining redox homeostasis is important to reverse or retard the progression of NASH. Oxidative stress in hepatocytes was important pathogenesis of nonalcoholic steatohepatitis (NASH). This work aimed to investigate whether autophagy was participated in the protective effects of dihydromyricetin (DHM) on palmitic acid (PA)-induced oxidative stress in hepatocytes and the underlying mechanism

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