NR4A1通过IκBα/NF-κB通路调控过氧化氢诱导人脐静脉内皮细胞凋亡的机制

Mechanism of NR4A1 regulating hydrogen peroxide-induced apoptosis in human umbilical vein endothelial cells via the IκBα/NF-κB pathway

目的探讨过氧化氢(H_(2)O_(2))诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)氧化应激中孤核受体NR4A1表达变化,以及其对细胞凋亡的影响及机制。方法使用不同浓度及时间梯度H_(2)O_(2)处理HUVECs,采用CCK-8法检测细胞活性,TUNEL法检测细胞凋亡,Western blotting法检测Bcl-2、Bax与NR4A1蛋白表达;采用siRNA转染建立敲低NR4A1与对照的HUVECs,分为si-NC组及si-NR4A1组,H_(2)O_(2)处理后检测各组细胞抑凋亡蛋白Bcl-2、凋亡蛋白Bax表达,TUNEL法检测细胞凋亡;采用慢病毒感染建立稳定过表达NR4A1与对照的HUVECs,并进行H_(2)O_(2)处理,分为空载对照组(NC组)、过表达组(OV组)、NC+H_(2)O_(2)组及OV+H_(2)O_(2)组。TUNEL法检测各组细胞凋亡、Western blotting法检测各组中NR4A1、Bcl-2、Bax、总IκBα的蛋白表达、P65蛋白在细胞核/质定位。结果200μmol/L H_(2)O_(2)处理细胞6 h,HUVECs活力显著下降,凋亡水平显著上升,Bax/Bcl-2比值升高(P<0.001);NR4A1蛋白表达量增加;与si-NC组相比,si-NR4A1组在H_(2)O_(2)处理后细胞凋亡率、Bax/Bcl-2比值下降(P<0.001);OV+H_(2)O_(2)组细胞凋亡率、Bax/Bcl-2比值较NC+H_(2)O_(2)组显著降低(P<0.001);相较于NC+H_(2)O_(2)组,P65蛋白在OV+H_(2)O_(2)组中细胞核内表达显著降低(P<0.05),细胞质内表达显著上调(P<0.001);与NC+H_(2)O_(2)组相比,OV+H_(2)O_(2)组总IκBα表达上调(P<0.001)。结论H_(2)O_(2)诱导HUVECs发生细胞凋亡;HUVECs内NR4A1蛋白过表达可抑制细胞凋亡,其作用机制可能与调控IκBα的表达与抑制NF-κB的核转位有关。

Objective To investigate the expression of orphan nuclear receptor 4A1(NR4A1)in human umbilical vein endothelial cells(HUVECs)induced by hydrogen peroxide(H_(2)O_(2))oxidative stress,and its effects and mechanism in cell apoptosis.Methods After HUVECs were treated with different concentrations and exposure times of H_(2)O_(2),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 H_(2)O_(2) 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 H_(2)O_(2) and divided into empty vector control group(NC),NR4A1 overexpressed group(OV),NC+H_(2)O_(2) group,and OV+H_(2)O_(2) group.The cell apoptosis was determined with TUNEL,and the protein expressions of NR4A1,Bcl-2,Bax,total IκBα,and the nuclear/cytoplasmic localization of P65 protein in each group were determined with Western blotting.Results After the cells were treated with 200μmol/L H_(2)O_(2) 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 increased.Compared with the si-NC group,the si-NR4A1 group had decreased Bax/Bcl-2 ratio and cell apoptosis after H_(2)O_(2) treatment(P<0.001).Compared with the NC+H_(2)O_(2) group,the OV+H_(2)O_(2) group had significantly decreased cell apoptosis rate and Bax/Bcl-2 ratio(P<0.05).Compared with the NC+H_(2)O_(2) group,the OV+H_(2)O_(2) group had significantly decreased P65 protein expression in the nucleus(P<0.05),but significantly increased expression in the cytoplasm(P<0.001).Compared with the NC+H_(2)O_(2) group,the OV+H_(2)O_(2) group had significantly upregulated total IκBαexpression(P<0.001).Conclusion H_(2)O_(2) induces apoptosis in HUVECs;NR4A1 inhibits cell apoptosis by regulating the expression of IκBαand inhibiting the nuclear translocation of NF-κB in HUVECs.