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目的探讨活性氧(ROS)清除剂N-乙酰半胱氨酸(NAC)能否保护H9c2心肌细胞对抗化学性缺氧引起的损伤。方法应用化学性低氧模拟物氯化钴(CoCl2)处理H9c2心肌细胞,建立化学性缺氧损伤心肌细胞的实验模型。在CoCl2处理H9c2心肌细胞前60min把NAC加入培养基中,作为预处理。应用CCK-8比色法检测细胞存活率;双氯荧光素(DCFH-DA)染色荧光显微镜照相检测细胞内ROS水平;罗丹明123(Rh123)染色荧光显微镜照相检测线粒体膜电位(MMP);谷胱甘肽试剂盒检测GSSG/(GSSG+GSH)的比值。结果600μmol/LCoCl2明显地降低细胞存活率。在CoCl2处理H9c2心肌细胞前60min,应用500～2000μmol/LNAC能剂量依赖性地抑制CoCl2对心肌细胞的损伤作用,使细胞存活率显著升高。2000μmol/LNAC能明显地对抗CoCl2引起的氧化应激反应,使H9c2心肌细胞内GSSG/(GSSG+GSH)的比值及ROS水平明显降低,并明显地对抗CoCl2对MMP的抑制作用。结论NAC能显著地对抗化学性缺氧诱导的心肌细胞损伤,此心肌细胞保护作用与其降低GSSG/(GSSG+GSH)的比值及ROS水平,改善MMP等机制有关。
Objective To investigate whether reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) can protect H9c2 cardiomyocytes against chemical hypoxia-induced injury. Methods H9c2 cardiomyocytes were treated with cobalt chloride (CoCl 2), a chemical hypoxia mimics, to establish an experimental model of hypoxia-injured cardiomyocytes. NAC was added to the medium 60 min prior to CoCl2 treatment of H9c2 cardiomyocytes as a pretreatment. Cell viability was detected by CCK-8 colorimetric assay. Intracellular ROS was detected by fluorescence microscopy with DCFH-DA staining. Mitochondrial membrane potential (MMP) was detected by Rh123 staining. The ratio of GSSG / (GSSG + GSH) was determined by the enzyme-linked immunosorbent assay (ELISA). Results 600 μmol / L CoCl2 markedly decreased cell viability. In CoCl2 treatment of H9c2 myocardial cells 60min, the application of 500 ~ 2000μmol / LNAC dose-dependent inhibition of CoCl2 on myocardial cell injury, so that cell survival was significantly increased. 2000μmol / L LNAC could obviously antagonize the oxidative stress induced by CoCl2, significantly decreased the ratio of GSSG / (GSSG + GSH) and ROS in H9c2cells, and significantly inhibited the inhibitory effect of CoCl2 on MMPs. Conclusion NAC can significantly antagonize the hypoxia-induced injury of cardiomyocytes. The protective effect of cardiomyocytes is related to the decrease of the ratio of GSSG / (GSSG + GSH) and ROS, and the improvement of MMP.