Role And Mechanism Of TNFRSF12A In Cholestatic Liver Injury

Background&AimsCholestasis is a common clinical syndrome caused by the lack of bile flow from the liver to the small intestine,the decrease of bile flow and the excessive accumulation of bile acids and other toxic compounds in the liver.Cholestasis can be induced by drug intake,mutation of hereditary bile acid transport protein,congenital biliary atresia,intrahepatic and extrahepatic biliary obstruction,virus infection,abnormal hormone levels and so on.Chronic cholestasis can easily lead to liver injury,inflammation,liver fibrosis,liver cirrhosis,and even liver failure.Liver transplantation is the main treatment for end-stage liver disease,and there is a lack of effective drug treatment,seriously affecting the survival and prognosis of patients.At present,the molecular mechanism of cholestatic liver injury is still unclear.Tumor necrosis factor receptor superfamily 12A(TNFRSF12A),also known as fibroblast growth factor-inducible 14(Fn14),is involved in tissue response to acute and chronic injury.TNFRSF12A was highly expressed in acute and chronic tissue injury of skeletal muscle injury,multiple sclerosis,ischemic heart disease,stroke and glomerular disease.Recent studies have confirmed that TNFRSF12A is up-regulated in liver tissues of chronic liver diseases such as nonalcoholic steatohepatitis,alcoholic liver disease,chronic hepatitis C and hepatocellular carcinoma.However,the function and molecular mechanism of TNFRSF 12A in cholestatic liver injury are not clear.Therefore,in this paper,through the analysis of clinical cholestatic liver tissue samples,mouse cholestasis model and in vitro bile acid uptake model,to explore the expression,function and molecular mechanism of TNFRSF 12A in cholestatic liver,so as to provide a basis for revealing the pathogenesis and therapeutic targets of cholestasis.Methods1.The expression of TNFRSF 12A mRNA and protein in clinical obstructive cholestasis liver tissues,bile duct ligation mouse liver tissues and bile acid uptake hepatocellular carcinoma PLC/PRF/5-ASBT cell were detected by real-time quantitative PCR(RT-qPCR)and Western blotting(WB).2.Firstly,we successfully constructed and identified Tnfrsfl2a knockout(Tnfrsfl2a-KO)mice by transcription activator-like effector nuclease method(TALEN).Secondly,we performed bile duct ligation(BDL)and sham operation in wild type(WT)mice and Tnfrsf12a-KO mice to establish cholestasis mice model.Finally,the mice were killed and their serum and liver tissues were collected.The biochemical indexes of serum and liver of mice were analyzed by automatic biochemical analyzer.Hematoxylin and eosin(H&E)staining,real-time quantitative PCR,Western blotting and immunohistochemistry were used to detect the function and expression of key signal molecules after the loss of TNFRSF12A expression in cholestatic liver tissue of mice.The morphological changes of liver injury in transgenic mice and control mice were observed by transmission electron microscope(TEM).3.Bile acid uptake model of hepatocellular carcinoma PLC/PRF/5-ASBT cell line(hepatoma cell line PLC/PRF/5 stably transformed Apical sodium-dependent bile acid transport)were used for in vitro studies.Change the gene expression of TNFRSF12A and analyze the molecular mechanism of TNFRSF12A involved in the regulation of cholestatic liver injury.Results1.Hepatic levels of TNFRSF12A mRNA and protein were markedly increased in liver tissues from clinical obstructive cholestasis and bile duct ligation mice.The expression of TNFRSF12A was up-regulated by conjugated bile acid,and it's dependent on the time of bile acid treatment.2.Functional studies in Tnfrsfl2a-KO mice with BDL indicated that Tnfrsfl2a deficiency markedly reduced serum levels of alanine aminotransferase(ALT),alkaline phosphatase(ALP),and aspartate aminotransferase(AST)in bile duct ligation mice,and TNFRSF12A deletion alleviate liver injury in bile duct ligation mice.The results of liver histology showed that there was no significant change in the morphology of necrosis and apoptosis between WT and Tnfrsfl2a-KO mice.Further transmission electron microscopic study of liver histomorphology confirmed that cholestasis could induce hepatocyte pyroptosis and the deletion of TNFRSF12A reduced hepatocyte pyroptosis in bile duct ligation mice.3.The molecular mechanism study in vitro hepatoma cell line PLC/PRF/5 revealed that TNFRSF12A induces hepatocyte pyroptosis and aggravates liver injury by activating NF-?B/GSDMD signal pathway in cholestasis.ConclusionHepatic TNFRSF12A is induced in cholestasis and its up-regulation aggravates cholestatic liver injury via promoting hepatocyte pyroptosis.TNFRSF12A is expected to be a molecular target for the treatment of cholestatic liver injury.