Downregulation Of Citrate Synthase Expression Leads To Endoplasmic Reticulum Stress Mediated Apoptosis In Cochlear Cells

Objective:A/J mouse is a model of age-related hearing loss(AHL)with progressive early onset hearing loss.Mutations in the mitochondrial citrate synthase(Cs)gene play an important role in hearing loss of A/J mice,but the molecular mechanism is not yet clear.To investigate the pathogenesis of damage to cochlear cells in A/J mice caused by Cs mutation in vitro,we downregulated the expression of CS in HEI-OC1 cells through shRNA,observed the changes of related pathways,and administered related drug intervention.Methods:Four shRNA lentivirus vectors were designed to infect HEI-OC1 cells.The infection efficiency was observed by fluorescence microscopy.The expression of CS was detected by qRT-PCR and western blotting.The lowest CS expression was selected as the experimental group and shRNA-NC as the control group for subsequent experiments.The proliferation ability was measured by CCK-8.Mitochondrial superoxide content was detected by MitoSOX kit.Expression level of mitochondrial stress,endoplasmic reticulum stress(ERS)and apoptotic pathway related molecules(CAT,SOD2,BIP,ATF6,pATF6,CHOP,Cleaved Caspasel2,Bcl-2,Bax)were detected by western blotting.4-(2-aminoethyl)benzene sulfonyl fluoridehydrochloride(AEBSF),an ATF6 inhibitor,was used to interfere with cells in the group with low CS expression.The proliferation ability was measured by CCK-8.ERS and apoptosis pathway related molecules(pATF6,CHOP,Cleaved Caspasel2,Bax)were observed by western blotting.Antioxidant α-lipoic acid(ALA)was used to interfere with cells in the group with low CS expression.The proliferation ability was measured by CCK-8.Mitochondrial superoxide content was detected by MitoSOX kit.Expression level of apoptotic pathway related molecules(Cleaved Caspasel2,Bcl-2,Bax)were detected by western blotting.Results:The expression level of CS was the lowest in the shRNA-Csl429 group.Compared with the shRNA-NC group,the proliferation ability of CCK-8 was decreased,and the superoxide level was increased in shRNA-Cs1429 group.Key molecules of mitochondrial stress(CAT,SOD2),endoplasmic reticulum stress(BIP,pATF6,CHOP)and apoptosis(Cleaved Caspasel2,Bax)were increased.Expression of anti-apoptotic protein(Bcl-2)was decreased.AEBSF,an ATF6 inhibitor,was added into shRNA-Cs1429 cells.The proliferation ability of shRNA-Cs1429+AEBSF treated cells was the significantly higher than that of the shRNA-Cs1429 group.Key molecules of endoplasmic reticulum stress and apoptosis(pATF6,Cleaved Caspasel2,Bax)were decreased.Moreover,Antioxidant ALA was added to the shRNA-Cs1429 group.The proliferation ability of shRNA-Cs1429+ALA treated cells was the significantly higher than that of the shRNA-Cs1429 group.The expression levels of Cleaved Caspase12 and Bax,markers of apoptosis were downregulated.The expression level of anti-apoptotic protein(Bcl-2)was increased.Conclusion:The low expression model of CS was successfully constructed in HEI-OC1 cochlear cells.Low CS expression in HEI-OC1 cells led to the decrease of cell proliferation ability.Oxidative damage and ATF6 mediated ERS contributes to the cell pathology.AEBSF can improve cell proliferation and inhibit endoplasmic reticulum stress and apoptosis mediated by ATF6.The antioxidant ALA can improve cell proliferation,reduce the ROS level and alleviate the cell apoptotic signals.This study may provide a new theoretical basis for understanding and the treatment of AHL.