不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H2O2刺激的反应*

doi:10.12047/j.cjap.5773.2019.067

摘要/Abstract

摘要： 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0.1 mmol/L、0.25 mmol/L、0.5 mmol/L、0.75 mmol/L、1 mmol/L、2 mmol/L的H₂O₂,分别作用1 h、2 h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,12 h后,H₂O₂作用1 h,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1 h和浓度0.5 mmol/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-1 mRNA显著增加(P＜0.05),其他指标变化不大;5%O₂组细胞 Nrf2 mRNA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,12 h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。

关键词: 氧浓度, 过氧化氢, 小鼠骨骼肌卫星细胞系, 转录因子NF-E2相关因子, 抗氧化

Abstract: 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 H₂O₂stimulation. 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, and 5% extremely low oxygen may inhibit it.

Key words: oxygen concentration, H₂O₂, C2C12 cells, Nrf2, antioxidant

姬卫秀, 张缨. 不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H₂O₂刺激的反应^*[J]. 中国应用生理学杂志, 2019, 35(4): 317-321.

JI Wei-xiu, ZHANG Ying. The antioxidant system mediated by Nrf2 in C2C12 cells responding to H₂O₂stimulus under different oxygen concentration[J]. CJAP, 2019, 35(4): 317-321.

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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 H₂O₂stimulus under different oxygen concentration

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