Mechanism of NR4A1 regulating hydrogen peroxide-induced apoptosis in human umbilical vein endothelial cells via the IκBα/NF-κB pathway
Objective To investigate the expression of orphan nuclear receptor 4A1(NR4A1)in human umbilical vein endothelial cells(HUVECs)induced by hydrogen peroxide(H2O2)oxidative stress,and its effects and mechanism in cell apoptosis.Methods After HUVECs were treated with different concentrations and exposure times of H2O2,cell activity,apoptosis and protein expressions of Bcl-2,Bax and NR4A1 were detected with CCK-8,TUNEL and Western blotting,respectively.siRNA was transfected into HUVECs to obtain the knocked down NR4A1 cells(si-NR4A1)and control(si-NC).After H2O2 treatment,the levels of cell apoptosis,and the Bax/Bcl-2 ratio were detected.Lentivirus transfection was used to establish stably overexpressed NR4A1 HUVECs,which were treated with H2O2 and divided in-to empty vector control group(NC),NR4A1 overexpressed group(OV),NC+H2O2 group,and OV+H2O2 group.The cell apoptosis was determined with TUNEL,and the protein expressions of NR4A1,Bcl-2,Bax,total Iκ Bα,and the nuclear/cytoplasmic localization of P65 protein in each group were determined with Western blotting.Results Af-ter the cells were treated with 200 μmol/L H2O2 for 6 hours,the vitality of HUVECs significantly decreased,the rate of apoptosis significantly increased,the Bax/Bcl-2 ratio increased(P＜0.001),and the protein expression of NR4A1 in-creased.Compared with the si-NC group,the si-NR4A1 group had decreased Bax/Bcl-2 ratio and cell apoptosis after H2O2 treatment(P＜0.001).Compared with the NC+H2O2 group,the OV+H2O2 group had significantly decreased cell apoptosis rate and Bax/Bcl-2 ratio(P＜0.05).Compared with the NC+H2O2 group,the OV+H2O2 group had signifi-cantly decreased P65 protein expression in the nucleus(P＜0.05),but significantly increased expression in the cyto-plasm(P＜0.001).Compared with the NC+H2O2 group,the OV+H2O2 group had significantly upregulated total Iκ Bαexpression(P＜0.001).Conclusion H2O2 induces apoptosis in HUVECs;NR4A1 inhibits cell apoptosis by regulating the expression of IκBα and inhibiting the nuclear translocation of NF-κB in HUVECs.
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