Study On ASIC1a Promoting Alcoholic Liver Injury By Up-regulating Ferritinophagy Through PERK/elF2α/ATF4

Objective Alcoholic liver injury(ALI)is liver damage caused by excessive alcohol intake for a long time.Chronic alcohol consumption can cause a series of pathological changes in liver,such as oxidative stress,production of reactive oxygen species(ROS),unstable iron accumulation,mitochondrial damage,endoplasmic reticulum stress(ERS),etc.ASIC1 a is a pH receptor that can be activated by extracellular acidic substances,which mediates calcium influx to trigger ERS.Persistent ERS can trigger the activation of PERK,IRE1 and ATF6 pathways,and activate ferritinophagy and autophagy.Ferritinophagy is autophagy-dependent cell death.The degradation of intracellular ferritin leads to iron overload and ROS accumulation,which mediates the activation of PERK-eIF2α-ATF6.In ALI,alcohol can induce ERS and ferritinophagy in the liver,as well as hypoxia and glycolysis activation in the liver,so there may be a high expression of ASIC1 a,but the study of ferritinophagy and ALI has not been reported.Therefore,we will study the expression and possible mechanism of ASIC1 a,ERS and ferritinophagy in the liver of ALI mice.Methods In this study,Western blot,immunohistochemistry and qRT-PCR were used to observe the expression of ASIC1 a in vivo and in vitro in mice.In vivo,ALI mouse model was established by Gaobin method.C56BL/6 mice were randomly divided into four groups: CD-fed,EtOH-fed,EtOH-fed+rAAV8-Vector,EtOH-fed+rAAV8-shASIC1 a.Mouse serum and liver were collected to explore the role and possible mechanism of ASIC1 a,PERK-elF2α-ATF4 pathway and ferritinophagy in ALI from the aspects of serology,pathology,protein and mRNA.In vitro,mouse hepatocyte AML12 cells were stimulated with alcohol at 200 m M concentration for 9 hours to establish an in vitro model.ASIC1 a specific blocker and small RNA technology were used to block or knock down the expression of ASIC1 a in AML12 cells.HE staining,oil red O staining,Western blot staining,immunohistochemistry and qRT-PCR were used to observe the histopathological changes,lipid deposition,endoplasmic reticulum stress PERK-elF2α-ATF4 pathway and the expression of ferritinophagy related proteins in ALI.In vitro,AML12 cells were given ATF4 si RNA and PERK specific inhibitor ISRIB to inhibit endoplasmic reticulum stress PERK-elF2α-ATF4 pathway.The changes of ferritinophagy expression and lipid deposition were detected by Western blot,immunofluorescence,qRT-PCR,electron microscopy and oil red O staining.Results In vivo,alcohol induced liver injury and steatosis,accompanied by activation of ASIC1 a and ferritinophagy marker ACSL4,ferritinophagy and PERK-eIF2α-ATF4 expression in endoplasmic reticulum stress pathway.In vitro,the expression of ASIC1 a and ferritinophagy increased in alcohol-induced AML12 cells,which was consistent with the treatment of ferritinophagy inducer erastin.The results of HE staining and Oil Red O staining showed that silencing ASICa in vivo improved alcohol-induced liver injury and steatosis.The results of immunohistochemistry,Western blot and qRT-PCR showed that silencing ASIC1 a could reduce alcohol-induced ferritinophagy,ferritinophagy and ERS.The results of Western blot,qRT-PCR,immunofluorescence and oil red O staining showed that knocking down ASIC1 a in AML12 cells could reduce alcohol-induced PERK-eIF2α-ATF4 pathway and ferritinophagy,and improve lipid accumulation in hepatocytes.The results of Western blot,electron microscope,immunofluorescence and oil red O staining showed that PcTX-1 blocking ASIC1 a in AML12 cells could reduce alcohol-induced PERK-eIF2α-ATF4 pathway,ferritinophagy,GSH depletion,MDA accumulation and autophagy lysosome number,and improve hepatocyte lipid accumulation,mitochondrial swelling and destruction and autophagy vesicle formation.In addition,endoplasmic reticulum stress and steatosis can be reduced by knocking down the ATF4 in AML12 cells.Finally,specific blocking of ASIC1 a in AML12 cells with ISRIB can reduce alcohol-induced ferritinophagy,oxidative damage and steatosis.Conclusion We confirmed that ASIC1 a accelerates the progression of ALI and explored the possible mechanisms involved in PERK/elF2α/ATF4 and ferritinophagy.We found that ASIC1 a expression was elevated in ALI mice and ethanol-induced AML12 cells.In vivo and in vitro,reduction of ASIC1 a attenuated inflammation and lipid accumulation in ALI,as well as reduced PERK/elF2α/ATF4 expression levels in ERS,along with reduced levels of ferritinophagy.In addition,we found that inhibition of PERK/elF2α/ATF4 axis and knockdown of ATF4 inhibited ferritinophagy and attenuated mitochondrial damage and lipid accumulation.In conclusion,our experimental results demonstrate that ASIC1 a promotes ferritinophagy as an important process in the pathogenesis of ALI.All results identify a novel regulatory mechanism,PERK/elF2α/ATF4-ferritinophagy,may be a potential target for ALI therapy.