Role Of Mitochondria Damage Mediated By Abnormal Accumulation Of TDP-43 In CCl₄-induced Liver Fibrosis

ObjectiveLiver fibrosis is a pathophysiological process in which liver cells are repeatedly destroyed and then regenerated,and extracellular matrix is diffusely deposited in the liver during the development of chronic liver disease.The liver is one of the organs rich in mitochondria,and a healthy mitochondrial network is essential for normal liver function.Mitochondrial damage caused by pathological factors leads to the leakage of mtDNA,which triggers inflammatory responses through the cGAS-STING pathway and promotes the progression of liver fibrosis.Recent studies have reported that abnormal accumulation of TDP-43 on mitochondria can interfere with the normal function of nerve cell mitochondria,induce neuroinflammatory responses,and participate in the occurrence of neurodegenerative diseases.However,there is no report that TDP-43 aggregation-induced mitochondrial damage is involved in the process of liver fibrosis.Therefore,in this study,CCl4 exposure was used to establish a mouse model of liver fibrosis.Thus,the changes of mitochondrial morphology and function,the expression and distribution of TDP-43 and the related protein expression of cGAS-STING pathway in liver tissue were detected.On this basis,an autophagy intervention model was established with rapamycin to observe the effect of drug intervention on CCl4-induced liver fibrosis and mitochondrial damage,so as to explore the role and possible mechanism of mitochondrial damage caused by abnormal accumulation of TDP-43 in liver fibrosis,and provide new therapeutic targets for liver fibrosis.MethodIn this study,male C57BL/6 mice were selected and chronically exposed to CCl4 to establish a mouse model of liver fibrosis and a rapamycin intervention model to explore the role of mitochondrial damage mediated by abnormal accumulation of TDP-43 in liver fibrosis and its effects molecular mechanism.The levels of ALT and AST in serum were determined by automatic biochemical analyzer;the pathological changes of liver fibrosis were detected by H&E staining,Sirius Red staining and Masson staining;the damage of mitochondrial ultrastructure was observed by transmission electron microscope;Western Blot and immunofluorescence staining were used to detect The expression of fibrosis marker proteins,inflammation-related proteins,key molecules of cGAS-STING signaling pathway,and the expression and localization of TDP-43 in the cytoplasm and mitochondria;RT-PCR technology was used to determine the content of cytoplasmic mtDNA..Results(1)Chronic CCl4 exposure induced a significant increase in the expression levels ofα-SMA,Col-I and TGF-β1 in liver tissue of mice.Transmission electron microscopy showed abnormal mitochondrial morphology,Western Blot showed that the levels of PINK1 and Parkin were increased,and qPCR showed that the mtDNA content in the cytoplasm was increased.(2)CCl4 exposure induces activation of hepatic inflammatory signaling pathway.The expression levels of NLRP3,caspasel and IL-1β were significantly increased in the liver,and the expressions of key molecules cGAS,STING,IRF3 and downstream NF-κB in the cGAS-STING signaling pathway were significantly increased.(3)The expression of TDP-43 in hepatocytes increased during CCl4 exposure-induced liver fibrosis,and the co-localization with mitochondria was significantly increased,suggesting that TDP-43 was deposited in mitochondria.(4)Rapamycin treatment can effectively promote the regression of liver fibrosis and alleviate mitochondrial damage.(5)Rapamycin can effectively promote the clearance of abnormal accumulation of TD43 in mitochondria induced by CCl4.At the same time,RAPA can inhibit the expression of NLRP3 inflammasome and key molecules of cGAS-STING signaling pathway.Conclusion(1)CCl4 exposure induces the accumulation of TDP-43 in the mitochondria of hepatocytes and induces mitochondrial damage.(2)Mitochondrial damage can activate the NLRP3 inflammasome,and mtDNA released from mitochondrial damage can activate the cGAS-STING signaling pathway,which together induce inflammation and promote the formation of liver fibrosis.(3)RAPA intervention can eliminate damaged mitochondria and abnormally accumulated TDP-43 by activating mitophagy,thereby alleviating inflammatory response and promoting fibrosis regression.