NADPH氧化酶/ROS信号通路在维生素E改善氧化性低密度脂蛋白致人脐静脉内皮细胞氧化损伤中的作用

Role of NADPH oxidase/ROS signaling pathway in the protective effect of vitamin E on oxLDL induced human umbilical vein endothelial cells injury

目的：建立氧化性低密度脂蛋白（oxLDL）诱导损伤人脐静脉内皮细胞（HUVEC）损伤模型，研究维生素 E 对 oxLDL 诱导的 HUVEC 损伤的保护作用及其作用机制。方法体外培养 HUVEC，以不同浓度维生素 E （50~150μmol/ L）预孵育细胞24 h，再加入200μg/ ml 的 oxLDL 继续培养24 h；采用 CCK-8检测细胞生存率， DCFH-DA 检测细胞内活性氧（ROS）水平，RT-PCR 检测 NADPH 氧化酶亚基（p47phox、p67phox、NOX4）mRNA 表达水平。结果成功建立了 oxLDL 诱导 HUVEC 损伤模型；与正常组比较，oxLDL 组的细胞生存率显著降低，ROS 生成大量增加，差异有统计学意义（P ﹤0.01）；与 oxLDL 组比较，不同浓度维生素 E 组的细胞生存率显著升高，ROS生成大量减少，差异有统计学意义（P ﹤0.05）；oxLDL 组的 p47phox、p67phox、NOX4 mRNA 表达量分别是正常组的2.15±0.19、2.48±0.20、3.01±0.24倍（P ﹤0.01），而经不同浓度维生素 E 预孵育可剂量依赖性的显著降低oxLDL诱导的 p47phox、p67phox、NOX4 mRNA 表达上调，差异有统计学意义（P ﹤0.05）。结论 oxLDL 可引起 HUVEC 细胞生存率降低，NADPH 氧化酶亚基表达上调及 ROS 的大量产生；50~150μmol/ L 维生素 E 可剂量依赖性地改善oxLDL 对内皮细胞的损伤，通过抑制 NADPH 氧化酶/ ROS 信号通路的活化来减少 HUVEC 损伤，从而起到抗动脉粥样硬化的作用，为维生素 E 的抗动脉粥样硬化作用提供了实验依据。

Objective To establish a oxLDL-induced injury model of human umbilical vein en-dothelial cells（HUVECs）and investigate the protective effect and mechanism of vitamin E on HUVEC damaged by oxLDL. Methods HUVEC were cultured in vitro. HUVEC were pretreated with different concentrations of vitamin E（50 - 150 μmol/ L）for 24 h,then were given 200 μg / ml oxLDL for another 24 h;The survival rate of HUVEC was detected by CCK-8;DCFH-DA was used to indicate ROS genera-tion;RT-PCR was used to measure the NADPH oxidase subunits（p47phox,p67phox,NOX4）mRNA expression. Results Compared with the control group,the cell viability of oxLDL group was significantly decreased,while ROS generation was notably increased,there were significant differences（P ﹤ 0. 01）;Compared with oxLDL group,the cell viabilitys of different concentration of vitamin E groups were signifi-cantly increased,while the generations of ROS were notably decreased,there were significant differences （P ﹤ 0. 05）. p47phox,p67phox,NOX4 mRNA level of oxLDL group was respectively 2. 15 ± 0. 19, 2. 48 ± 0. 20,3. 01 ± 0. 24 times of control group（P ﹤ 0. 01）;While pretreated with different concentra-tions of vitamin E significantly inhibited p47phox,p67phox,NOX4 mRNA expression and the effect of vitamin E was in a dose dependent,there were significant differences（P ﹤ 0. 05）. Conclusions oxLDL can decrease the cell viability,increase the expression of NADPH oxidase subunits,increase the forma-tion of ROS;50 - 150 μmol/ L vitamin E can in dose-dependent inhibit oxLDL induced HUVEC injury and the NADPH oxidase / ROS signaling pathway is one of the main mechanisms of the protective effect of vitamin E,which may be responsible for vitamin E to prevent and treat atherosclerosis.