The Effect Of Mitophagy On Liver Fibrosis

Liver fibrosis is a common pathological process of various liver diseases,some of which will progress to cirrhosis and / or hepatocellular carcinoma(HCC).With high mortality and poor prognosis,liver fibrosis is a major threat to human health.As a reversible injury-repair process,excessive deposition of extracellular matrix(ECM)plays an important role in promoting the progression of liver fibrosis.Because the ECM mainly comes from the activation and secretion of HSCs,inhibiting the activation of HSCs is expected to reverse liver fibrosis.As the major organelles to produce ATP,mitochondria are involved in a variety of metabolic processes,so to ensure the mitochondrial homeostasis is essential for the maintenance of normal cell functions.Mitophagy,first proposed by Lemasters in 2005,is defined as through the degradation of damaged or redundant mitochondria to maintain quality and quantity homeostasis of mitochondria,thereby,to keep regular cell functions.One study has shown that mitophagy may exist in HSCs activated by resveratrol,and that mitophagy are related to the cell toxicity of resveratrol.However,the relationship between mitophagy and liver fibrosis is not clear.Therefore,this study intends to make C57BL/6 male mice and HSCs cell line LX-2 as the research object to investigate the effects of mitophagy on liver fibrosis from animal and cellular levels.Objective: To study the effects of mitophagy on liver fibrosis,and to provide new insights for the treatment of liver fibrosis.Methods: This experiment was composed of two levels to detect the mitophagy changes in the course of liver fibrosis formation: carbon tetrachloride induced liver fibrosis in mice and TGF-? activated HSCs cell lines.Then the mitophay inhibitor Cyclosporine A and the inducer Valinomycin was applied to further verify the relationship between mitophagy and liver fibrosis.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)levels and HE staining of liver tissue was used to observe liver inflammation.Sirius red staining and Immunohistochemistry(IHC)were performed to determine collagens and the expression of a-SMA.Western blot(WB)was used to analyze the expression of ?-SMA,Collagen I,HSP60,TOM20,PINK1.Results:(1)Mitophagy was inhibited in the process of live fibrosis: In vivo,carbon tetrachloride induced liver fibrosis and oil control mice's hepatic tissues were collected,respectively.Compared with the oil group,the serum ALT and AST levels were higher and the hepatic inflammation was more serious in carbon tetrachloride induced liver fibrosis.Besides,Sirius red staining,IHC and WB all showed that the expressions of collagen and ?-SMA were increased in fibrosis group.All above indicated that carbon tetrachloride succeeded in inducing liver fibrosis.WB showed that TOM20 and HSP60,representative of the remaining amount of mitochondria,were also increased.In vitro,during HSC further activation,the levels of ?-SMA,Collagen I,TOM20 and HSP60 were all increased,and the mitophagy related protein PINK1 was decreased.(2)Liver fibrosis aggravated when mitophagy inhibitor was used.In vivo,the expression of Tom20 and Hsp60 were all increased when mitophagy inhibitor was used,indicating that Cyclosporine A may effectively inhibit the mitophagy.Compared with the carbon tetrachloride group,the ALT and AST levels were higher,the inflammation was more serious,and the expression of collagen and ?-SMA were also increased when Cyclosporine A was added.In vitro,compared with the TGF-? group,the mitophagy related protein PINK1 was decreased,TOM20 and HSP60 were all increased in CsA and TGF-? combined group.The expression of ?-SMA and Collagen I were also increased.(3)The activation of HSCs was inhibited when mitophagy inducer was used: Compared with the TGF-? only group,the expression of ?-SMA,Collagen I,TOM20 and HSP60 were all decreased when VaL added,and the mitophagy related protein PINK1 was increased indicating that mitophagy inducer may reverse liver fibrosis in a certain degree.Conclusion: The mitophagy was inhibited during the formation of liver fibrosis and HSCs further activation.In some extent,mitophagy inhibitors can aggravate liver fibrosis,and mitophagy inducer can reverse liver fibrosis.Mitophagy intervention is expected to become a new target for the treatment of liver fibrosis.