Chronic-plus-binge Alcohol Intake Induces Production Of Proinflammatory MtDNA-enriched Extracellular Vesicles And Hepatitis Via ASK1 And P38MAPK?-dependent Mechanisms

Background:ALD encompasses a broad spectrum of hepatic pathologies,ranging from simple steatosis(fatty liver)to severe forms of liver injury such as alcoholic steatohepatitis(ASH),progressive fibrosis,end-stage cirrhosis,complications from portal hypertension,and superimposed hepatocellular carcinoma(HCC).At present,the incidence of AH is increasing year by year in our country,and the burden of disease is increasing.In particular,the short-term(3 months)mortality rate of severe alcoholic hepatitis(SAH)can be as high as 35%-50%.There is no effective treatment for SAH.Although a series of important scientific advances have been made for the disease,the pathogenesis of ALD is still not fully understood,and it is urgent to explore new drug targets.Objective:The aim of this study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1(ASK1)and p38 mitogen-activated protein kinase(p38 MAPK)in chronic-plus-binge ethanol-induced hepatitis and mtDNA-enriched EV release.Method:In this study,we first analyzed oxidative stress-related genes in liver tissues of different alcoholic hepatitis mouse models through microarrays,and found the most significant oxidative stress-related genes in this model.Next,we determined the signaling pathway ASK1-p38a,activated by oxidative stress,that may be involved in the pathogenesis and progression of alcoholic hepatitis,and further we used the NIAAA alcoholic hepatitis mouse model to explore how the signaling pathway ASK1-p38a participate in the pathogenesis of alcoholic hepatitis,and finally conducted preliminary research on the mechanism through in vivo and in vitro experiments.Result:Microarray analysis revealed that hepatic expression of metallothionein 1/2(Mt1/2),encoding two most potent antioxidant proteins,showed the highest upregulation after chronic-plus-binge ethanol challenge.Genetic deletion of the Mt1/2 aggravated ethanol-induced liver injury,as evidenced by increased serum ALT,increased hepatic neutrophil infiltration,and elevated oxidative stress and activation of stress kinases including ASK1 and p38 MAPK in the liver.Inhibition or genetic deletion of ASK1 or p38MAPKa ameliorated ethanol-induced liver injury,infiltration of neutrophils and macrophages,inflammation,reactive oxygen species(ROS)levels,and hepatic expression of phagocytic oxidase as well as ER stress markers.Mechanistic studies revealed that genetic deletion of the Ask1 or p38Mapka gene or treatment with their inhibitors attenuated ethanol-induced EV secretion and mtDNA content in EVs from hepatocytes in vivo and in vitro.Taken together,these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38MAPK,thereby promoting alcoholic hepatitis.