不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H2O2刺激的反应

The antioxidant system mediated by Nrf2 in C2C12 cells responding to H2O2 stimulus under different oxygen concentration

目的:探讨不同氧浓度下小鼠骨骼肌卫星细胞系(C2C12细胞)对H2O2刺激反应的变化及其机制。方法:小鼠骨骼肌卫星细胞系(C2C12细胞),经培养复苏后,将细胞分为7组,每组设8个复孔,各组分别加入浓度为0.1mmol/L、0.25mmol/L、0.5mmol/L、0.75mmol/L、1mmol/L、2mmol/L的H2O2,分别作用1h、2h后测细胞活力,选择细胞H2O2刺激的最佳作用时间和浓度;C2C12细胞分为不同氧浓度组:21%O2、12%O2、8%O2、5%O2每组设8个复孔,12h后,H2O2作用1h,收集细胞;检测细胞Nrf2蛋白荧光和蛋白表达量,测定Nrf2和抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1的mRNA表达量及细胞ROS水平。结果:选择H2O2作用时间相对较短的1h和浓度0.5mmol/L作为本实验的H2O2刺激条件。与21%O2组相比,12%O2组细胞Nrf2蛋白荧光增强,Nrf2的mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1的mRNA表达均显著增加(P<0.05或P<0.01),细胞ROS水平明显降低(P<0.01);8%O2组仅GPX-1mRNA显著增加(P<0.05),其他指标变化不大;5%O2组细胞Nrf2mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、NQO-1、GPX-1的mRNA表达均明显降低(P<0.05或P<0.01),细胞ROS水平则明显升高(P<0.01)。结论:不同氧浓度下C2C12细胞中Nrf2介导的抗氧化系统对H2O2刺激反应不同,12h的12%O2浓度可促进C2C12细胞Nrf2的抗氧化作用,而5%O2浓度的严重低氧则作用相反。

Objective: To apply hypoxia of different oxygen concentration on C2C12 cells to study the changes of Nrf2 antioxidant system under H2O2. Methods: The perfect simulative effect time and concentration of H2O2 were chosen. Cell vitality was tested after C2C12 cells cultured in 0. 1 mmol/L,0. 25 mmol/L,0. 5 mmol/L,0. 75 mmol/L,1 mmol/L and 2 mmol/L H2O2 for 1 or 2 h respectively. The C2C12 cells were divided into different oxygen concentration group: 21% O2,12% O2,8% O2,5% O2 respectively. And then cells were treated with H2O2 for 1 h,and collected for determination. Immunofluorescence of Nrf2 and the protein expression of Nrf2 were detected. The expressions of antioxidant enzymes superoxide dismutase 1( SOD1),superoxide dismutase 2( SOD2),catalase( CAT),NADPH quinine oxidoreductase-1( NQO-1),glutathione peroxidase-1( GPX-1),Heme oxygenase-1( HO-1) mRNA and cellular ROS levels were tested by high quality fluorescence assay. Results: 0. 5 mmol/L H2O2 for 1 h was selected as the conditions of H2O2 stimulation. Compared with 21% O2 group,the expressions of Nrf2 mRNA and protein,antioxidant enzymes SOD1,SOD2,CAT,HO-1,NQO-1,GPX-1 mRNA were increased significantly( P<0. 05 or P<0. 01),and ROS level was lower( P<0. 01)in 12% O2 group cells;only the expression of GPX-1 mRNA was increased( P<0. 05) in 8% O2 group;the expressions of Nrf2 mRNA and protein expression,antioxidant enzymes SOD1,SOD2,NQO-1,GPX-1 mRNA were decreased significantly( P < 0. 05 or P <0. 01),and ROS level was higher( P<0. 01) in 5% O2 group. Conclusion: Hypoxia can affect the Nrf2 antioxidant system,and the different oxygen concentrations have different impact. In addition,12% O2 for 12 h could promote the Nrf2 antioxidant system,and5% extremely low oxygen may inhibit it.