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

作　　者：姜子晗 芦兴晨 孙露 赵蕙琛 左丹 马小莉[3] 刘元涛 张玉超[4] JIANG Zihan;LU Xingchen;SUN Lu;ZHAO Huichen;ZUO Dan;MA Xiaoli;LIU Yuantao;ZHANG Yuchao(Qingdao Clinical Medical School of Qingdao University,Qingdao Municipal Hospital,Qingdao 266011,Shandong,China;Department of Clinical Nutrition,Qilu Hospital of Shandong University(Qingdao),Qingdao 266011,Shandong,China;Department of Endocrinology,Qingdao Municipal Hospital,Qingdao 266011,Shandong,China;Editorial Office of Medical Journal,Qingdao Municipal Hospital,Qingdao 266011,Shandong,China;Department of Endocrinology,Qilu Hospital of Shandong University(Qingdao),Qingdao 266011,Shandong,China)

机构地区：[1]青岛大学附属青岛临床医学院青岛市市立医院,山东青岛266011 [2]山东大学齐鲁医院(青岛)临床营养科,山东青岛266011 [3]青岛市市立医院内分泌科,山东青岛266011 [4]青岛市市立医院医学杂志编辑部,山东青岛266011 [5]山东大学齐鲁医院(青岛)内分泌科,山东青岛266011

出　　处：《山东大学学报（医学版）》2024年第3期11-19,共9页Journal of Shandong University:Health Sciences

基　　金：山东省自然科学基金面上项目(ZR2022MH086);山东省医药卫生科技发展计划(202103060651);2021年度青岛市医药卫生科研计划(2021-WJZD005)。

摘　　要：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 regul

关 键 词：孤核受体NR4A1 人脐静脉内皮细胞 IκBα蛋白 NF-κB核转位 细胞凋亡 

分 类 号：R587[医药卫生—内分泌]