二苯乙烯苷上调HO-1表达负向调控H_2O_2诱导人脐静脉内皮细胞凋亡的研究

Study on Two Stilbene Glycoside Up-regulating HO-1 Expression Negative Regulation H_2O_2 Inducing Human Umbilical Vein Endothelial Apoptosis

目的探讨二苯乙烯苷(TSG)上调HO-1表达负向调控H_2O_2诱导人脐静脉内皮细胞凋亡的作用。方法对人脐静脉内皮细胞进行体外培养,将实验分成对照组(空白组,无过氧化氢)、过氧化氢组(过氧化氢浓度300μmol/L)和二苯乙烯苷组(二苯乙烯苷浓度10μmol/L+过氧化氢浓度300μmol/),采用显微镜观察细胞形态,MTT法检测细胞增殖率,流式细胞术检测细胞凋亡率,Westernblot检测HO-1蛋白的表达及Nrf2核转位。结果与对照组比较,过氧化氢组内皮细胞增殖率明显降低(P<0.05);与过氧化氢组比较,二苯乙烯苷组内皮细胞增殖率明显增加(P<0.05)。与对照组比较,过氧化氢组内皮细胞凋亡率上升(P<0.05);二苯乙烯苷组细胞凋亡率明显低于过氧化氢组(P<0.05)。与对照组比较,过氧化氢组内皮细胞12 h后HO-1蛋白的表达减少;与过氧化氢组比较,二苯乙烯苷组可明显升高HO-1蛋白的表达(P<0.05)。二苯乙烯苷组处理内皮细胞后,胞浆中的核转录因子Nrf2(NF-E2-related factor 2)水平降低,胞核中Nrf2水平逐渐增加;转染了Nrf2小干扰RNA片段(si RNA,Small interfering RNA)的内皮细胞中HO-1蛋白表达明显减少。结论二苯乙烯苷(TSG)可能通过核转录因子Nrf2诱导人脐静脉内皮细胞表达HO-1,从而发挥对内皮细胞凋亡的抑制作用。

Objective to investigate effect of two stilbene glucoside(TSG) up-regulating HO-1 expression negative regulation H2O2 inducing human umbilical vein endothelial apoptosis. Methods culture human umbilical vein endothelial cells in vitro, divide experiment subjects into control group(blank group, no hydrogen peroxide), hydrogen peroxide group(with hydrogen peroxide concentration of 300 μmol/L) and two stilbene glucoside group(with two stilbene glycoside concentration of 10μmol/L+ hydrogen peroxide concentration of 300μmol/), observe cell morphology with microscope, detected cell proliferation rate by MTT method, and cell apoptosis rate by flow cytometry, detect HO-1 protein expression and Nrf2 nuclear translocation by Westernblot. Results compared with control group, endothelial proliferation rate of hydrogen peroxide group was significantly decreased(P<0.05); compared with hydrogen peroxide group, endothelial proliferation rate of two stilbene glycoside group was significantly increased(P<0.05). Compared with control group, endothelial apoptosis rate of hydrogen peroxide group was increased(P<0.05); apoptosis rate of two stilbene glucoside group was significantly lower than hydrogen peroxide group(P<0.05). Compared with control group, HO-1 protein expression of endothelial cells after 12 h of hydrogen peroxide group decreased; compared with hydrogen peroxide group, HO-1 protein expression of two stilbene glycoside group was significantly increased(P<0.05). In two styrene glycosides group, after process of endotheliocytes, nuclear transcription factor Nrf2 level(NF-E2-related factor 2) in cytoplasm decreased, Nrf2 level in karyon increased gradually; HO-1 protein expression in endothelial cells of transfected Nrf2 small interfering RNA fragment(si RNA, small interfering RNA) was decreased significantly. Conclusion two stilbene glucoside(TSG) may induce HO-1 expression in human umbilical vein endothelial cells by nuclear transcription factor Nrf2, thereby take effect of inhibiting apoptosis of endothelial cells.