The Role And Mechanism Of Aberrant Mitochondrial Aggregation Of TDP-43 By Calpain In Acetaminophen Induced Acute Liver Injury

ObjectiveSince it went public,acetaminophen(APAP)has become the most commonly used overthe-counter(OTC)antipyretic and analgesic drug in the world.It is safe and effective within the therapeutic does range,but excessive intake of APAP will lead to acute liver injury(ALF),even liver failure.Mitochondrial dysfunction is the key pathological event in APAP-induced acute liver injury.In recent years,mitochondrial damage caused by toxic exposure induces mitochondrial stress and activates a cascade of signaling responses to cope with damage.Mitochondrial unfolded protein response(UPRmt)is commonly activated in the context of mitochondrial damage and contributes to maintenance mitochondrial quality control by regulating a wide range of gene expression.However,whether it is involved in APAP hepatotoxicity remains unclear.Recent studies have reported that abnormal expression of DNA-binding protein TDP-43 in mitochondria causes serious mitochondrial damage of nerve cells,thereby inducing the activation of UPRmt,and the activation of UPRmt participates in the development of neurodegenerative diseases.Although recent studies have reported that TDP-43 was also detected in liver tissue and involved in the occurrence and development of liver disease,such as fatty liver and hepatic fibrosis.However,there is no evidence to show whether the mitochondrial injury induced by TDP-43 aggregation is involved in APAP-induced acute liver injury.Calpain is a calcium-dependent neutral cysteine protease.It has shown that Calpain activation can lead to mitochondrial dysfunction in liver injury induced by APAP.More importantly,Calpain can specifically cleaved TDP-43 and produce the fragments which are prone to aggregate,forming inclusion body.In order to explore the mechanism of Calpain and TDP-43 in the regulation of mitochondrial dysfunction in APAP-induced liver injury,this study established the animal model of APAP acute liver injury to explore the relationship among TDP-43,UPRmt and APAP-induced liver injury.On this basis,Calpain-specific inhibitor was used to established the Calpeptin intervention model to detected changes in mitochondrial Calpain degradation system,TDP-43 accumulation and UPRmt signaling pathway in the liver,and then to provide new therapeutic targets for APAP-induced acute liver injury.Method1.The establishment of animal models:male C57BL/6 mice were selected in this study to established the time-model and Calpeptin intervention model.2.Biochemical index and liver pathology detection:ALT and AST from all mice were detected.Then paraffin sections were made for H&E staining,and pathological changes of acute liver injury were observed under microscope.3.Observation of mitochondrial damage by transmission electron microscope:take 1mm3 of liver tissue from mouse,and immerse it in 3%glutaraldehyde fixing solution 2 h,then 1%OsO4 fixed for 1h,dehydrated by ethanol gradient.Then embedded with Epon812,prepared ultrathin sections.Then ultrathin sections were double-stained with uranyl acetate and lead citrate,and finally mitochondrial morphology was observed by transmission electron microscopy and the field of view was selected for photography.4.Detection of protein expression by Western blot:the mitochondrial protein,cytoplasmic protein and nuclear protein were extracted after the liver tissue homogenate,then the protein samples were prepared to detected the expression of Calpain-related proteins,mitochondrial dynamic proteins,TDP-43,UPRmt signaling pathway related proteins.5.Immunofluorescence staining of liver tissue:mouse paraffin sections were prepared routinely,then paraffin sections were dewaxed and hydrated.After antigen retrieval,the paraffin sections were immunofluorescence staining.Finally,the conjugation between endoplasmic reticulum and mitochondria,the localization of chaperone protein mtHSP70 and mitochondria,and the localization of UPRmt transcription factor CHOP and nucleus were detected by fluorescent antibody staining.Result1.APAP overdose caused acute liver injury and mitochondrial dysfunction.After APAP,the ALT and AST continued to increase within 0-24 h,and decreased at 48h,but still higher than the control group(0h).The pathological results showed that the degree of liver injury was consistent with the serological results.The large area of hepatocyte necrosis was appeared and the structure of hepatic lobule was blurred at 24 h,but the liver injury decreased at 48 h.In addition,the expression of mitochondrial dynamic related proteins Drp1 increased and Mfn2 decreased.At the same time,the cleavage fragment of Drpl and Mfn2 both increased.More importantly,the electron microscopic results showed that APAP overdose resulted in abnormal changes in mitochondrial morphology,mitochondria are swollen,ridges disappear and mitochondrial stroma is loose2.Overdosing APAP resulted to the activation of Calpain and the cleavage and mislocation of TDP-43 in mitochondria:Western blot detected that the level of m-Calpain,μ-Calpain and Calpastatin in mitochondria increased significantly.At the same time,the expression of TDP-43 in mitochondria increased and the small molecular fragments were detected,suggesting that TDP-43 was aggregated in mitochondria.3.The activation of UPRmt promoted the recovery of liver injury:the expression of chaperone proteins HSP60,mtHSP70 and protease ClpP increased in mitochondria,and the highest level was found at 48 h.At the same time,the bZIP transcription factors ATF5 and CHOP both increased,and the nuclear translocation of CHOP also increased significantly.In addition,the intracellular eif2α and p-eif2α both increased.All these results suggested that UPRmt is activated in APAP-induced liver injury,which promoted the recovery of liver injury.4.Calpepetin effectively inhibited APAP-induced liver injury and mitochondrial dysfunction:after Calpeptin intervention,the ALT and AST in mice basically returned to normal level.H&E staining showed that the APAP-induced hepatocyte necrosis in mice basically disappeared,and the structure of liver lobule returned to normal.Meantime,Calpeptin inhibited the expression of Drpl and cleavage of Drpl and Mfn2 induced by APAP.5.Calpepitin inhibited the localization and cleavage of TDP-43 in mitochondria:the level of TDP-43 was significantly reduced after Calpeptin intervention,especially the level of cleavage fragments of 35 kDa,the co-localization of TDP-43 and mitochondria also decreased.6.Calpeptin inhibited the activation of UPRmt:the expression of HSP60,mtHSP70 and ClpP decreased,and the level of ATF5 and CHOP returned to normal.Furthermore,cytoplasm eif2α and p-eif2α decreased significantly.This suggests that the translation and transcription of intracellular proteins also return to normal after Calpeptin intervention,Conclusion1.Overdosing APAP caused the activation of Calpain,resulting severe acute liver injury and the impairment of mitochondria.2.Activated calpain promoted TDP-43 cleavage and mislocalization to the mitochondria.3.UPRmt was activated by accumulation of TDP-43 in mitochondria and mitochondrial damage.4.The activation of UPRmt promoted the recovery of acute liver injury induced by APAP.5.Calpeptin inhibited APAP-induced acute liver injury and mitochondrial damage.