Low Expression Of Citrate Synthase Gene Leads To Increased Cell Superoxide Production And Cell Apoptosis

Objective:A/J mice are a mouse model of age-related hearing loss.It has been demonstrated that a mutation in citrate synthase(CS)gene contributes to the early onset of hearing loss in A/J mice at about one month of age.To understand the effects of a decreased activity of CS that results from the mutation in Cs gene on hearing loss in A/J mice,human kidney cell line(293T)and mouse cochlear cells(HEI-OC1)were transiently transfected with human or mouse short hairpin RNAs of Cs(shRNACs)to give insufficiency expression of CS.To observe the expression of key molecules in endoplasmic reticulum stress and mitochondria-dependent metabolism,peroxidation and apoptosis pathways,to study the changes of key molecules in the signal transduction pathways.A/J mice were supplented with a-lipoic acid,which is an antioxidantand,and then to observe the effect of hearing.Methods:In this study,cochlea HEI-OC1 cells and human 293T cells were choosed.(1)Selection of Plasmid Expression Vectors:One of the four plasmids was selected by quantitative PCR with high inhibition efficiency.(2)The ATP assay was used to detect ATP production difference between Cs knockdown group and control groups.(3)The changes of oxygen consumption rate and the concentration of oxygen in the closed trough in Cs knockdown group and control groups were detected by mitochondrial respiration assay.(4)The mitochondrial oxidative stress,endoplasmic reticulum stress and the key molecules of apoptosis pathway(SOD2,UCP2,BIP,CHOP,Caspase-3)were detected by Western blot.(5)MitoSOX kit was used to detect the changes of superoxide anion production in Cs knockdown group and control groups.(6)The changes of mitochondrial membrane potential in Cs knockdown group and control groups were detected by JC-1 kit.(7)ABR and DPOAE were measured at various intervals at 3,4,6,and 8 weeks for A/J mice supplemented with a-lipoic acid and controls.Results:In comparison with the negative control,the consumption rate of oxygen was decreased and the ATP production was retarded in Cs knockdown groups.Meanwhile,oxidative stress in 293T and HEI-OC1 cells was induced as determined by SOD2 levels and mitochondrial superoxide formation assay(MitoSOX),whereas the uncoupling protein2(UCP2)was downregulated.Moreover,the expression levels of Bip and CHOP,markers of endoplasmic reticulum stress were upregulated.The expression levels of apoptosis related genes,especially caspase-3 were upregulated and mitochondrial membrane potential was reduced.Furthermore,we found that the free radical scavenger a-lipoic acid could delay the onset of AHL in A/J mice.Conclusion:In conclusion,our data indicated that knockdown of Cs induced the disturbance of energy metabolism,oxidative damage,ERS and finally cell apoptosis.Supplement of a-lipoic acid can delay the onset of AHL in A/J mice.