UBA52调控GPX4蛋白稳定性对H2O2诱导的RPE细胞氧化应激损伤的影响OA

Objective To investigate the effect of ubiquitin A-52 residue ribosomal protein fusion prod-uct 1(UBA52)that regulates the protein stability of glutathione peroxidase 4(GPX4)on oxidative stress in-jury in retinal pigment epithelial(RPE)cells.Methods UBA52-knockdown lentivirus was constructed and experimental grouping was performed.An oxidative stress injury model of RPE cells was established via hy-drogen peroxide(H2O2)treatment.The expression levels of UBA52 mRNA and protein were detected by RT-qPCR and Western blot respectively,cell viability was measured by Cell Counting Kit-8(CCK-8),and cell ap-optosis level was detected by flow cytometry.The expression levels of selenoprotein glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and acyl-CoA synthetase long-chain family member 4(ACSL4)were detected by Western blot;intracellular iron ion level was detected by iron ion assay kit;the in-teraction between UBA52 and GPX4 was verified by co-immunoprecipitation.After treatment with protea-some inhibitor MG132 and protein synthesis inhibitor cycloheximide(CHX),the changes of GPX4 protein ex-pression level were detected by Western blot.Results Compared with the NC-shCtrl group,the mRNA ex-pression level of UBA52 in the NC-shCtrl+H2O2 group was increased(P<0.05),and the mRNA expression level of UBA52 in the shUBA52+H2O2 group was decreased compared with that in the NC-shCtrl+H2O2 group(P<0.05).The protein expression level of UBA52 in the NC-shCtrl+H2O2 group was higher than that in the NC-shCtrl group,and the protein expression level of UBA52 in the shUBA52+H2O2 group was lower than that in the NC-shCtrl+H2O2 group(P<0.05).On the 2nd,3rd,4th and 5th days after H2O2 treatment,cell viability in the NC-shCtrl+H2O2 group was lower than that in the NC-shCtrl group(P<0.05),and cell viability in the shUBA52+H2O2 group was higher than that in the NC-shCtrl+H2O2 group(P<0.05).The total cell apoptosis rate in the NC-shCtrl+H2O2 group was increased compared with that in the NC-shCtrl group(P<0.05),and the total cell apoptosis rate in the shUBA52+H2O2 group was decreased compared with that in the NC-shCtrl+H2O2 group(P<0.05).Compared with the NC-shCtrl group,the pro-tein expression levels of GPX4 and SLC7A11 in the NC-shCtrl+H2O2 group were decreased,and the protein expression level of ACSL4 was increased(P<0.05);compared with the NC-shCtrl+H2O2 group,the protein expression levels of GPX4 and SLC7A11 in the shUBA52+H2O2 group were increased,and the protein ex-pression level of ACSL4 in the shUBA52+H2O2 group was decreased(P<0.05).The intracellular iron ion level in the NC-shCtrl+H2O2 group was increased compared with that in the NC-shCtrl group(P<0.05),and the intracellular iron ion level in the shUBA52+H2O2 group was decreased compared with that in the NC-shCtrl+H2O2 group(P<0.05).The UBA52 signal in the precipitates of the shUBA52+CHX group and the shUBA52+MG132 group was decreased compared with that of the shCtrl+CHX group and the shCtrl+MG132 group(P<0.05).The protein expression level of GPX4 in the shUBA52+CHX group and the shU-BA52+MG132 group was increased compared with that in the shCtrl+CHX group and the shCtrl+MG132 group(P<0.05).Conclusion Downregulation of UBA52,by regulating the stability of GPX4 protein,may subsequently affect the process of ferroptosis in RPE cells,and thereby alleviate the damage to RPE cells caused by oxidative stress.