The Role And Mechanism Of M~6A Demethylase ALKBH5 In Regulating Drp1-mediated Mitochondrial Fission In Liver Fibrosis

BackgroundMitochondrial Dynamin-related protein 1(Drp1)mediated mitochondrial fission plays a key role in various cellular processes,such as cell proliferation,migration and apoptosis,as well as diseases,such as fibrosis and tumor.The activation of hepatic stellate cells(HSCs)is a main drive step in the progress of liver fibrosis,and its activation can affect the outcome of liver fibrosis.However,the mechanism of mitochondrial fission in liver fibrosis and activated HSCs remains poorly understood.At present,more and more evidence has confirmed that Drp1 is regulated by epigenetic modification in the process of regulating mitochondrial fission,and regulates the occurrence of fibrosis by affecting the expression of Drp1.However,whether m ~6A modification is involved in mitochondrial fission and liver fibrosis has not been studied.Thereby,we report that Alk B homolog 5(ALKBH5)inhibits mitochondrial fission and HSCs activation,and improves liver fibrosis.Our study shows that mitochondrial fission is the key event in HSCs activation,which depends on the decrease of ALKBH5expression.ObjectiveThis study investigates the role and mechanism of m~6A demethylase ALKBH5 in regulating Drp1-mediated mitochondrial fission in liver fibrosis through in vivo and in vitro experiments.The findings provide new insights and potential methods for preventing and treating liver fibrosis.MethodsTo investigate the role of mitochondrial metabolism in liver fibrosis,we induced liver fibrosis in C57BL/6J mice by intraperitoneally injecting them with an olive oil solution containing 10%carbon tetrachloride(CCl4)twice a week for 12 weeks.In the final week,we injected the ALKBH5 lentivirus into the tail vein to establish an overexpression model.After the animal model is completed,HE,Sirius red and Masson staining are used to detect the pathological changes of the liver histopathology,as well as the changes of the liver function by the serum Alanine transaminase(ALT)and aspartate aminotransferase(Aspartate aminotransferase,AST);Observe the changes in mitochondrial morphology;detect m6A levels and sites by Dot blot and Me RIP;detect liver fibrosis markers type I collagen(Collagen I)and smooth muscle actin(Alpha-smooth muscle actin,α-SMA),ALKBH5,YTHDF1 and Drp1 levels.To create an in vitro model,we stimulated HSCs with 5 ng/ml transforming growth factor-β1(TGF-β1)for 24 hours.The study then observed the morphological changes of mitochondria in HSCs using a confocal microscope.The proliferation and migration ability of HSCs were detected using MTT,CCK-8,flow cytometry,scratch,and Trans-well migration.To determine the correlation between m6A level and HSCs,Dot blot and Me RIP were used.The expression of ALKBH5,YTHDF1,Drp1,Collagen I andα-SMA was detected using RT-q PCR and Western blot.To construct in vitro models of Drp1 silencing and ALKBH5 overexpression,we treated HSCs with 4μg of si RNA-Drp1 and LV5-ALKBH5 with a titer of 30 MOI,respectively.We observed the morphological changes of mitochondria using confocal microscopy and performed cell function experiments.The expression of related indicators was detected by RT-q PCR and Western blot.Results1.Increased mitochondrial fission in liver fibrosis tissue and activated HSCs,accompanied by increased expression of Drp1.2.Mitochondrial fission promotes the proliferation and migration of HSCs,and inhibiting the expression of Drp1 will inhibit the occurrence of mitochondrial fission,and inhibit HSCs activation and degree of liver fibrosis.3.ALKBH5 was significantly down-regulated both in vivo and in vitro,and its low expression was related to the increase of the m~6A level.4.Overexpression of ALKBH5 significantly reduces the occurrence of mitochondrial fission,inhibits the proliferation and migration of HSCs,and alleviates the process of liver fibrosis.5.Mechanistically,ALKBH5 regulates the m~6A demethylation of Drp1,up-regulates the expression of YTHDF1,and increases the expression level of Drp1.ConclusionOur results highlight a novel mechanism whereby ALKBH5 plays an essential role in HSCs activation and liver fibrosis.ALKBH5 inhibits mitochondrial fission and attenuates liver fibrosis by reducing Drp1 methylation modification in an m~6A-YTHDF1 dependent manner.Our observations indicate a novel m~6A regulatory mechanism that attenuates mitochondrial fission as a viable intervention and may provide a promising diagnostic and therapeutic target for liver fibrosis.