高氧抑制SIRT1和PGC-1α表达引起肺泡上皮细胞线粒体功能障碍

Down-regulation of SIRT1 and PGC-1α expression caused by hyperoxia induces mitochondrial dysfunction in human alveolar epithelial cells

目的探讨沉默配对型信息调节因子2同源物1-过氧化物酶体增殖物激活受体γ共激活因子1α(SIRT1-PGC-1α)信号途径介导高氧对A549人肺泡上皮细胞线粒体功能的影响及其可能机制。方法取对数生长期的人肺泡上皮细胞,随机分为对照组和高氧组。对照组置于37℃、50 mL/L CO2饱和湿度培养箱中培养,高氧组予以950 mL/L O2处理。培养24 h后,Mito SOX^TM染色法检测线粒体活性氧(Mito-ROS)水平,JC-1染色检测线粒体膜电位,实时定量PCR检测线粒体DNA含量及SIRT1、PGC-1α、核呼吸因子1(NRF1)和线粒体转录因子A(TFAM)mRNA水平,Western blot法检测SIRT1、PGC-1α、NRF1和TFAM蛋白水平。结果与对照组相比,高氧组细胞Mito-ROS明显增加,膜电位明显下降;高氧组线粒体DNA含量减少,SIRT1、PGC-1α、NRF1和TFAM的mRNA和蛋白表达均下降。结论高氧诱导SIRT1和PGC-1α表达降低引起人肺泡上皮细胞线粒体功能障碍。

Objective To investigate SIRT1-PGC-1αsignaling pathway-mediated effect of hyperoxia on mitochondrial function in A549 human alveolar epithelial cells and its possible mechanism.Methods Human alveolar epithelial cells in logarithmic growth phase were randomly divided into control group and hyperoxia group.The control group was cultured in a 37℃,50 mL/L CO2 saturated humidity incubator,and the hyperoxia group was treated with 950 mL/L O2.Following 24-hour culture,Mito SOX^TM staining was used to detect the level of mitochondrial reactive oxygen species(Mito-ROS)and JC-1 staining to detect the mitochondrial membrane potential.Real-time quantitative PCR was performed to detect the mitochondrial DNA content and the mRNA levels of SIRT1,PGC-1α,nuclear respiratory factor 1(NRF1)and mitochondrial transcription factor A(TFAM),and Western blotting to detect the protein levels of SIRT1,PGC-1α,NRF1 and TFAM.Results Compared with the control group,the Mito-ROS of the hyperoxia group increased significantly,while the membrane potential decreased obviously;the mitochondrial DNA content of the hyperoxia group went down,and the mRNA and protein expression of SIRT1,PGC-1α,NRF1 and TFAM dropped.Conclusion Hyperoxia induces mitochondrial dysfunction in human alveolar epithelial cells by inhibiting the expression of SIRT1 and PGC-1α.