The Effect Of ISGylation Of β-catenin On Liver Lipid Metabolism And Hepatocyte Apoptosis With Alcoholic Fatty Liver And Its Mechanisms Of Action

Alcoholic liver disease(ALD)is the most common chronic liver disease in the world and one of the main causes of morbidity and mortality.ALD is a complex process that includes a wide range of liver lesions,including hepatitis,fatty liver,liver fibrosis,and cirrhosis.Among them,Alcohol fatty liver(AFL)is the earliest stage of alcoholic liver disease.As a reversible stage in alcoholic liver disease,it is of great significance for the clinical treatment of alcoholic liver disease.Recent studies have indicated that Wnt/β-catenin play an important role in AFL.And remodelling this pathway to alleviate AFL progression is expected to be a new strategy for ALD treatment.Wnt pathway is a highly conserved signal transduction pathway,which plays an important role in many aspects of liver physiology.The Wnt pathway is divided into classical and non-classical modes,and consists of secreted glycoproteins,cell surface receptors,co-receptors,and complex intracellular regulatory mechanisms,which mediate and regulate a large number of cellular functions.β-catenin is the main effector of the classical Wnt pathway,and its deletion in hepatocyte-specific β-catenin leads to increased susceptibility to alcoholic fatty liver disease.Hepatocytes with β-catenin disorder exhibit mitochondrial dysfunction,oxidative phosphorylation defects,and increased oxidative stress.ISG15 can covalently bind with β-catenin protein by HERC5,leading to ISG degradation and further regulating the expression of β-catenin protein.However,the relationship between β-catenin and the ISG15 system in alcoholic fatty liver disease remains unclear.Therefore,the main research is divided into the following parts:(1)The expression of HERC5、ISG15 and β-catenin in AFLIn vivo experiments,this experiment mainly used the Binge model to replicate the alcoholic fatty liver model,and human liver tissue L-02 and mouse AML-12 cells were used in vitro experiment.The results of Western blot and qPCR showed that the expression of HERC5 and ISG15 was significantly increased with ethanol stimuli,while the expression of β-catenin was significantly decreased.It is suggested that ISGylation may exist in AFL,and ISG15 modification system may be involved in the regulation of expression of β-catenin(2)Regulate the effect of HERC5 on β-cateninIn L-02 cells,the expression of β-catenin was detected by silencing the expression of HERC5 by small interfering RNA(siRNA)and transfected with pEGFPHERC5.The expression of β-catenin was detected by Western blot and qPCR.The results showed that the silenced HERC5 increased β-Catenin Expression;overexpression of HERC5 reduces the expression of β-Catenin.It is suggested that in AFL,HERC5 can regulate the ISGylation level of β-Catenin in ethanol-stimulated L-02 cells.(3)Regulation of HERC5 on lipid metabolism in alcoholic fatty liverIn L-02 cells,silencing and overexpression models were constructed by transfection of HERC5-siRNA and pEGFP-HERC5.Western blot,qPCR and TG showed that HERC5-siRNA alleviated alcohol-induced steatosis SREBP-1,PPAR-α and TG abnormal expression;pEGFP-HERC5 promotes abnormal expression of alcohol-induced steatosis indicators SREBP-1,PPAR-α and TG.It was suggested that HERC5 could regulate lipid metabolism in alcoholic fatty liver.(4)Regulation of HERC5 on apoptosisAs above,a silent and overexpressing HERC5 cell model was established in L-02 cells,apoptosis protein was detected by Western blot and apoptosis of hepatocytes was detected by flow cytometry.The results showed that HERC5-siRNA inhibited hepatocyte apoptosis;pEGFP-HERC5 promotes hepatocyte apoptosis.It was suggested that HERC5 could regulate the apoptosis in ethanol-stimulated L-02 cells.(5)N-acetylcysteine(NAC)(ROS inhibitor)reverses β-catenin inhibition by HERC5 overexpression and restores its activity.In the L-02 cells,the over-expression model was constructed with pEGFP-HERC5 plasmid and NAC was added simultaneously.The expression of ROS was detected by flow cytometry.The results showed that the overexpression of HERC5 increased the production of ROS;while NAC significantly reduced the production of ROS;in addition,Western blot,qPCR and TG showed that NAC reduced the abnormal expression of alcohol-induced steatosis index;Western blot and flow cytometry detected apoptosis changes,and the results showed that NAC reduced apoptosis;therefore,the above results indicate the use of N-Acetylcysteine reverses the inhibition of β-catenin caused by overexpression of HERC5 and restores its activity.It is suggested that the ISGylation of β-catenin can affect the lipid metabolism and apoptosis by regulating the level of ROS.In summary,we preliminarily demonstrated that ISG15 and HERC5 can promote ISG degradation of β-catenin in alcoholic fatty liver.And ISGylation of β-catenin can affect cell lipid metabolism and apoptosis by regulating the level of ROS.