目的 探讨核因子E2相关因子(Nrf2)/血红素氧合酶-1(HO-1)是否参与黄连素(BBR)介导人脐静脉内皮细胞(HUVECs)抗过氧化氢(H2O2)引起的凋亡以及潜在的机制.方法 不同浓度BBR(5和10μmol/L)预处理细胞12 h后,应用H2O2(200μmol/L,4 h)构建细胞损伤模型,CCK-8法检测细胞活性; TUNEL标记凋亡细胞; DCFH-DA检测细胞活性氧(ROS)水平;免疫荧光法标记Nrf2细胞内的分布情况; Western blot法检测Nrf2/HO-1通路和凋亡相关蛋白含量.此外,应用干扰核糖核酸(siRNA)特异性阻断Nrf2/HO-1后,重复上述检测.结果 相对于对照组,H2O2降低细胞活性,增加细胞凋亡及ROS水平(P ＜0.05).不同浓度的BBR有效抑制上述变化,且呈"剂量依赖性"(P ＜0.05).同时,BBR进一步促进H2O2导致的Nrf2核内移位及HO-1表达增加(P ＜0.05).应用siRNA不仅可以显著抑制Nrf2/HO-1的活化,而且明显逆转BBR对HUVECs的保护作用(P ＜0.05).结论 BBR通过激活Nrf2/HO-1通路,增强HUVECs清除ROS的能力,从而发挥抗H2O2损伤的作用.%Objective To investigate whether berberine (BBR) could protect human umbilical vein endothelial cells (HUVECs) against H2O2-induced injury by activating nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway. Methods After pretreated with BBR (5 and 10 μmol/L) for 12 h, HUVECs cells were then insulted by H2O2 (200μmol/L) for additional 4 h. The cell viability was evaluated by CCK-8 analysis. The apoptotic cells were measured with TUNEL stainning. The cellular reactive oxygen species (ROS) was detected by DCFH-DA. The distribution of Nrf2 in cells was marked by immunofluorescence. Western blotting was used to analyze the proteins expression of Nrf2/HO-1 signaling pathway. Additionally, the detections above were repeated after Nrf2 siRNA treatment. Results Compared with control group, H2O2 decreased cell activity and increased apoptosis and ROS levels in HUVECs (P ＜0.05). Different concentrations of BBR (5 and 10 μmol/L) effectively inhibited the changes above in a dose-dependent manner (P ＜ 0.05). Meanwhile, BBR further enhanced the translocation of Nrf2 from cytoplasm to nucleus and the expression of HO-1, which was induced by H2O2 injury (P ＜0.05). Nrf2 siRNA not only significantly inhibited the activation of Nrf2/HO-1, but also obviously reversed the protective effects of BBR on HUVECs (P ＜ 0.05). Conclusion BBR promotes the ability of HUVECs to scavenge ROS by activating the Nrf2/HO-1 pathway in H2O2-induced injury.
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