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山东大学学报 (医学版) ›› 2024, Vol. 62 ›› Issue (3): 11-19.doi: 10.6040/j.issn.1671-7554.0.2023.1124

• 基础医学 • 上一篇    下一篇

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

姜子晗1,芦兴晨1,孙露2,赵蕙琛3,左丹4,马小莉3,刘元涛5,张玉超4   

  1. 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
  • 发布日期:2024-05-06
  • 通讯作者: 张玉超. E-mail:yc.zhang@hotmail.com
  • 基金资助:
    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)

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

JIANG Zihan1, LU Xingchen1, SUN Lu2, ZHAO Huichen3, ZUO Dan4, MA Xiaoli3, LIU Yuantao5, ZHANG Yuchao4   

  1. 1. Qingdao Clinical Medical School of Qingdao University, Qingdao Municipal Hospital, Qingdao 266011, Shandong, China;
    2. Department of Clinical Nutrition, Qilu Hospital of Shandong University(Qingdao), Qingdao 266011, Shandong, China;
    3. Department of Endocrinology, Qingdao Municipal Hospital, Qingdao 266011, Shandong, China;
    4. Editorial Office of Medical Journal, Qingdao Municipal Hospital, Qingdao 266011, Shandong, China;
    5. Department of Endocrinology, Qilu Hospital of Shandong University(Qingdao), Qingdao 266011, Shandong, China
  • Published:2024-05-06

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摘要： 目的 探讨过氧化氢(H2O2)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H2O2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,H2O2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H2O2处理,分为空载对照组(NC组)、过表达组(OV组)、NC+H2O2组及OV+H2O2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 200 μmol/L H2O2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H2O2处理后细胞凋亡率、Bax/Bcl-2比值下降(P<0.001);OV+H2O2组细胞凋亡率、Bax/Bcl-2比值较NC+H2O2组显著降低(P<0.001);相较于NC+H2O2组,P65蛋白在OV+H2O2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+H2O2组相比,OV+H2O2组总IκBα表达上调(P<0.001)。 结论 H2O2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。

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

Abstract: 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 into 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 IκBα, and the nuclear/cytoplasmic localization of P65 protein in each group were determined with Western blotting. Results After 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 increased. 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 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+ H2O2 group, the OV+ H2O2 group had significantly upregulated total IκBα expression(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.

Key words: Orphan nuclear receptor NR4A1, Human umbilical vein endothelial cells, IκBα protein, NF-κB nuclear translocation, Apoptosis

中图分类号: 

  • R587
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