化学性低氧对H_2O_2损伤C_2C_(12)细胞的保护作用

The Protective Effect of Chemical Hypoxia on Oxidative Damage Induced by H_2O_2 in C_2C_(12) Cell

目的:通过化学模拟(COCl2)低氧处理小鼠骨骼肌卫星细胞(C2C12),探讨合理的体外化学模拟低氧模式及其保护机制,为骨骼肌损伤的修复提供理论依据。方法:体外培养C2C12细胞,利用COCl2模拟低氧状态,MTT法检测C2C12细胞活性,DCF法测定各组细胞内活性氧(ROS)含量,Western blot法检测细胞核内Nrf2蛋白含量。结果:1)COCl2处理后,各实验组吸光度呈剂量依赖性下降,与对照组相比,COCl2浓度≥25μmol/L,差异具有显著意义(P<0.05)。2)COCl2处理后加入H2O2,各实验组吸光度与H2O2组比较,5、10μmol/L COCl2预处理组细胞活性明显改善(P<0.05)。3)与对照组相比,H2O2组相对荧光强度显著上升;与H2O2组比较,COCl2预处理组相对荧光强度则有明显下降(P<0.05)。4)对照组C2C12细胞核内Nrf2蛋白含量极少,H2O2组核内Nrf2含量有所增加,预处理组核内Nrf2蛋白含量增加则更加显著。结论:体外培养C2C12细胞进行化学模拟低氧时,要考虑COCl2的作用浓度,低浓度COCl2预处理可能通过激活Nrf2减轻H2O2诱导的C2C12细胞氧化应激损伤。

Objective： In this study,mouse skeletal muscle satellite cell（C2C12） was used as the cell model for COCl2 to explore reasonable mode for COCl2-simulated hypoxia in vitro,and the mechanism of protective effect by the pretreatment on H2O2-induced oxidative damage of COCl2 in C2C12 cell,for providing references for the reparation of skeletal muscle.Methods： C2C12 cell was cultivated in vitro.The hypoxia condition was simulated by COCl2.Cell activity of C2C12 cell was determined by MTT method.The content of reactive oxygen species（ROS） in C2C12 cell was examined by DCF method and the protein content of Nrf2 in the nucleus of C2C12 cell by Western blot.Results： 1） After COCl2 treatment,comparing with the control group,the absorbance in all experiment groups decreased in dose dependent when COCl2≥25 μmol/L,and the difference was significance（P0.05）.2） After COCl2 treatment,the researcher added H2O2 into C2C12 cell.Comparing with H2O2 group,the cell activity improved significantly in 5 and 10 μmol/L COCl2 groups（P0.05）.3） Comparing with the control group,the relative fluorescence intensity upgraded significantly in H2O2 group.But comparing with the H2O2 group,the relative fluorescence intensity decreased significantly in 5 and 10 μmol/L COCl2 groups（P0.05）.4） The level of Nrf2 protein in the nucleus of C2C12 was very low in the control group.The content of Nrf2 in the nucleus increased in H2O2 group,and it further increased significantly in 5 and 10 μmol/L COCl2 groups.Conclusion： Simulating hypoxia used COCl2 in C2C12 cell in vitro need to consider the concentration of COCl2.Pretreatment of COCl2 at low level may decrease H2O2-induced C2C12 cell oxidative damage through activating Nrf2.