RSV Regulates Pink1/Parkin Associated With Mitophagy In Alcoholic Fatty Liver

Alcoholic fatty liver(AFL)is a major cause of liver injury,which is associated with accumulation of lipid in the liver by chronic alcohol consumption.The pathologic features of alcoholic fatty liver develop over prolonged periods.Alcohol is widely used around the world,which can lead to serious damage to body by excessive drinking,whether in a binge or chronic fashion.At the cellular level,excessive drinking results in mitochondria damage and fatty acid synthesis increased.Ethanol pathogenesis is contributed by some complicated factors,such as disrupts mitochondrial ?-oxidation of fatty acids,decreased fatty acid oxidation,increased transportation of fatty acidsanf and blunted export of fatty acids.However,the defense mechanisms against the detrimental effect of AFL is not well understood.Autophagy is a process that is activated under many stress conditions and widely involved in the pathogenesis of various diseases.As autophagy is an effective defense system against complicated pathological insults,we investigated whether autophagy is activated in response to injury of alcoholic fatty liver and whether it plays an important role in mitigating ethanol-induced pathology,meanwhile,we investigated whether just as some studies indicated that autophagy protects against alcohol liver injury by removing damaged mitochondria via mitophag,which is a selective form of autophagy specific for degradation of damaged mitochondria.Autophagy can be both selective and non-selective.However,the mechanism of how these damaged mitochondria be removed by mitophagy was not well understood.Resveratrol(RSV)is found in red wines,grapes,peanuts and other plants which is a dietary polyphenol.It has many pharmacological effects,some studies have shown that RSV has an anticancer effect on glioma cells by inducing autophagy.Thus,whether the RSV could alleviate AFL by inducing autophagy is the purpose of our experimental study.Pink1 protein exists in the normal mitochondrial outer membrane that is a Parkinson's disease associated protein,Pink1 protein is rapidly degradation by proteolytic in normal mitochondria.However,due to mitochondrial damage,proteolytic enzymes were inhibited,resulting in a large accumulation of Pink1 protein on the mitochondrial membrane.Therefore,changed Pink1 protein level can reflect the degree of mitophagy in intracellular.Parkin is well known for the induction of mitophagy which is widely distributed in the brain tissue,skeletal muscle,heart and liver,and it plays an important role through the Pink1-Parkin-mediated mitophagy pathway in damaged mitochondria.When Pink1 is steady accumulated in the mitochondria,Parkin will be transferred from the cytoplasm to the mitochondrial,which may play a protective role in the Pink1-Parkin-mediated mitophagy pathway agains alcoholic liver injury.Furthermore,it is hypothesized that RSV might remove of damaged mitochondria,lipid droplets,and other damaged organelles in the liver by induced Pink1-Parkin-mediated mitophagy.Objective:To investigate the effects of resveratrol on mitochondrial autophagy in the liver of alcoholic fatty liver,based on the Pink1 / Parkin signaling pathway.Methods:C57BL / 6J mice(male,6-8 weeks old)were randomly divided into 7 groups: normal group,model group,RSV(10,30,100 mg / kg)group,chloroquine(3mg / kg)group,Chloroquine + resveratrol(3mg / kg + 30 mg / kg)group,using L-D classic alcohol fatty liver modeling method.The serum biochemical correlations were measured.Liver fat deposition was observed by HE staining.Liver lipid droplets and mitochondrial autophagosomes were measured by transmission electron microscopy(DEM).Western blot was used to detect the expression of mitophagy LC3,Pink1 and Parkin protein.LO2 cells were divided into normal group,model group,resveratrol(45 ?mol)group,chloroquine(10 ?mol)group,resveratrol + chloroquine(45 ?mol + 10 ?mol),respectively,and ethanol and sodium oleate were cultured with LO2 cells for 48 hours to prepare alcohol fatty liver model.The resveratrol or chloroquine was administered for 24 hours.The contents of TC and TG,the levers of ROS and the levers of mitochondrial membrane potential in the cells were detected.Western blot was used to detect the expression of autophagy LC3,Pink1 and Parkin protein in mitochondria.Results:1.The levels of ALT,AST,TG and TC in the serum of the model group were significantly higher than those in the control group,and the levels of ALT,AST,TG and TC in the liver of the model group were significantly higher than those in the control group.HE staining showed that the size and number of lipid droplets in the liver of the mice were significantly reduced in the resveratrol group.2.The number of autophagosomes in the liver of the resveratrol group was significantly higher than that in the model group,and the number of autophagy bodies in the chloroquine blocker group was significantly decreased.Western blot analysis showed that the expression of mitochondrial autophagy-related protein(LC3,Pink1,Parkin)in the liver of resveratrol group was also increased compared with the model group.3.The results showed that the contents of TC and TG in the model group were significantly higher than those in the normal group,and the content of TC and TG in the model group were significantly higher than those in the normal group.After adding the chloroquine blocker,the mitochondrial membrane potential of the model group was significantly decreased.Given RSV,the mitochondrial membrane potential was significantly increased,but after adding CQ this phenomenon has been reversed.4.Western blot analysis showed that the expression of mitochondrial autophagy-related protein(LC3,Pink1,Parkin)were increased and the protein expression of in chloroquine was significantly decreased.Conclusion:Resveratrol can alleviate chronic alcoholic fatty liver and increase the number of autophagy bodies in the liver.The mechanism may be related to improving mitochondrial autophagy through the Pink1 / Parkin signaling pathway.