不同水平硒对oxLDL诱导人脐静脉内皮细胞氧化损伤的保护作用

Protective Effects of Different Selenium Levels on oxLDL-induced Human Umbilical Vein Endothelial Cell Injury

目的探讨不同水平硒对氧化型低密度脂蛋白(oxLDL)诱导的人脐静脉内皮细胞(EA.hy926细胞)损伤的保护作用。方法体外培养EA.hy926细胞,用不同浓度(0、0.05和0.5μmol·L^(-1))的亚硒酸钠(Na2SeO3)培养细胞24 h后,将细胞分为6组:缺硒组(0μmol·L^(-1)Se),正常硒组(0.05μmol·L^(-1)Se),补充硒组(0.5μmol·L^(-1)Se),缺硒(0μmol·L^(-1)Se)+oxLDL组,正常硒(0.05μmol·L^(-1)Se)+oxLDL组,补充硒(0.5μmol·L^(-1)Se)+oxLDL组,继续培养16 h后,采用MTT法检测细胞存活率,NADPH法检测谷胱甘肽过氧化物酶(GPx)活力,WST法检测细胞内超氧化物歧化酶(SOD)活力,硫代巴比妥酸法检测丙二醛(MDA)含量,DCFH-DA探针检测细胞内活性氧(ROS)水平,JC-1法检测线粒体损伤情况,蛋白印迹法检测细胞GPx1和GPx4表达水平。结果相同硒水平下,oxLDL能降低细胞存活率(P<0.05),降低细胞GPx和SOD活力,提高ROS水平(P均<0.05),降低线粒体膜电位,下调细胞GPx1和GPx4蛋白的表达水平(P均<0.05);细胞经oxLDL诱导后,与正常硒水平相比,缺硒不能保护oxLDL诱导的细胞氧化损伤,而补充硒可提高细胞存活率(P<0.05),并上调GPx1和GPx4蛋白表达水平(P均<0.05);其次,细胞未经oxLDL诱导时,与正常硒水平相比,缺硒使细胞存活率下降、GPx和SOD活力降低,MDA和ROS含量上升,GPx1和GPx4蛋白表达减少(P均<0.05),而补充硒能够促进GPx1和GPx4蛋白的表达(P均<0.05)。结论补充硒能够减缓oxLDL诱导EA.hy926细胞发生的氧化应激,且GPx1和GPx4参与此过程。

Objective To investigate the protective effects of different selenium levels on ox LDL-induced human umbilical vein endothelial cells(EA.hy926 cells)injury.Methods The cultured EA.hy926 cells in vitro were incubated with different concentrations of sodium selenite(Na2Se O3)(0,0.05,0.5 μmol·L^(-1)) for 24hours,respectively.Then,the cells were divided into 6 groups:selenium deficient group(0 μmol·L^(-1)Se),normal selenium group(0.05 μmol·L^(-1)Se),selenium supplement group(0.5 μmol·L^(-1)Se),selenium deficiency(0 μmol·L^(-1)Se)+ox LDL group,normal selenium(0.05 μmol·L^(-1)Se)+ox LDL group,supplement selenium(0.5 μmol·L^(-1)Se)+ox LDL group.After treatment of selenium and ox LDL for 16 hours,the cell survival rate was detected by MTT assay.Glutathione peroxidase(GPx)activity was analyzed by NAPDH,superoxide dismutase(SOD)activity was evaluated by WST assay and intracellular malondialdehyde(MDA)content was measured by thiobarbituric acid method.ROS levels were determined by DCFH-DA probe.Intracellular and mitochondrial damage was assessed by JC-1 fluorescent analysis.The expression levels of GPx1 and GPx4 were evaluated by Western blot.Results At the same selenium level,ox LDL could significantly inhibit the survival rate of EA.hy926 cells(P<0.05),reduce the activities of GPx and SOD,increase ROS level(P all<0.05),reduce mitochondrial membrane potential and down-regulate the expression levels of GPx1 and GPx4 proteins(P all<0.05)in cells;when cells were treated with ox LDL,compared with the normal selenium level,the deficient of selenium could not protect the ox LDL-induced cell oxidative damage,but the supplementation of selenium could significantly improve the survival rate of ox LDL-induced cells(P<0.05),up-regulate GPx1 and GPx4protein expressions(P all<0.05);in addition,when cells were not treated with ox LDL,compared with the normal selenium level,the deficient of selenium could decrease the survival rate of cells,the activities of GPx and SOD and the expression levels of GPx1 and GPx4,significantly increase MDA and ROS levels(P all<0.05);the supplementation of selenium could significantly improve GPx1 and GPx4 protein expressions(P all<0.05).Conclusion Selenium supplementation(0.5 μmol·L^(-1)) could protect ox LDL-induced oxidative stress in EA.hy 926 cells,and the glutathione peroxidases GPx1 and GPx4 were involved in this process.