氧化应激对内皮细胞NF-κB、iNOS和NO信号表达的影响

Effects of Oxidative Stress on NF-κB,iNOS and NO in Endothelial Cells

目的观察氧化剂第三丁基过氧化氢(t-BHP)对体外培养的内皮细胞存活率及核转录因子κB(NF-κB)、iNOS和一氧化氮(NO)的影响。方法体外培养人脐静脉内皮细胞(HUVEC),用不同浓度的t-BHP处理,具体如下:(1)检测细胞存活率。t-BHP作用浓度分别为12.5,25,50,100μmol/L,作用时间分别为30,60,90,120 min,采用MTT法观察;(2)检测NF-κB p65、iNOS蛋白表达量及细胞内NO水平。t-BHP的作用浓度为50μmol/L,作用时间分别为30,60,90,120 min,采用Western blot测NF-κB p65、iNOS蛋白表达量,Griess化学显色法测细胞内NO水平;(3)iNOS抑制剂左旋硝基精氨酸甲酯(L-NAME,600μmol/L)预处理内皮细胞24 h,用Griess化学显色法检测细胞内NO水平。结果 t-BHP可降低内皮细胞的存活率,且呈时间及剂量依赖性,50μmol/Lt-BHP作用30 min细胞核NF-κB活化片段p65蛋白表达达高峰,作用60 min细胞质iNOS蛋白达高峰;iNOS抑制剂L-NAME可明显抑制内皮细胞NO升高。结论氧化应激可引起内皮细胞损伤,其效应可能通过NF-κB-iNOS-NO途径介导。

Objective To explore the effects of Tert-butyl hydroperoxide（t-BHP） on NF-κB,iNOS and NO in human umbilical vein endothelial cells.Methods Human umbilical vein endothelial cells were cultured in vitro.Cells viability was measured by MTT assay.Nitric oxide levels were measured by Griess assay.Inducible nitric oxide synthase（iNOS） protein and NF-kBP65 fragment were detected by Western blot.Results Cell viability was reduced in a time-and-dose-dependent manner after exposure to t-BHP at a range of 12.5～100 μmol/L for 30～120 min.The levels of cytoplasmic iNOS protein were elevated and reached a peak at 60 min.The expression of nucleus NF-kB p65 fragment reached a peak at 30 min after the treatment with t-BHP.The levels of Nitric oxide were markedly attenuated after the treatment with L-NAME 600 μmol/L for 24 hours.Conclusion NF-κB-iNOS-NO signalling pathway can mediate t-BHP induced damage in human umbilical vein endothelial cells.