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目的观察H2S对氯化钴(CoCl2)诱导的H9C2心肌细胞缺氧性损伤的影响,探讨其作用机制。方法用CoCl2处理H9C2心肌细胞,建立化学性缺氧诱导心肌细胞损伤的实验模型。NaHS(H2S的供体)在CoCl2处理H9C2心肌细胞前30 min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;PI染色流式细胞仪检测细胞凋亡率;Western blot法检测Cleaved Caspase-3的表达;GSSG试剂盒检测GSSG/(GSSG+GSH)的比值;双氯荧光素(DCFH-DA)染色荧光显微镜照相检测细胞内的活性氧(ROS);罗丹明123(Rh123)染色荧光显微镜照相检测线粒体膜电位(MMP)。结果在400~800μmol.L-1浓度范围内,CoCl2处理H9C2心肌细胞36 h,呈剂量依赖性地降低细胞存活率。在600μmol.L-1CoCl2处理H9C2心肌细胞前30 min,应用400μmol.L-1NaHS可明显地抑制CoCl2对细胞的损伤作用,使细胞存活率明显升高,细胞凋亡率及CleavedCaspase-3表达降低;并使H9C2心肌细胞内GSSG/(GSH+GSSG)比值及ROS水平明显降低,同时明显地改善MMP。结论H2S能明显地对抗化学性缺氧诱导的心肌细胞损伤,此保护作用与其降低GSSG/(GSH+GSSG)比值和ROS水平,改善MMP,抑制Caspase-3活化等机制有关。
Objective To observe the effect of H2S on cobalt chloride (CoCl2) -induced hypoxic injury of H9C2 cardiomyocytes and to explore its mechanism. Methods H9C2 cardiomyocytes were treated with cobalt chloride (CoCl2) to establish a model of hypoxia-induced cardiomyocyte injury. NaHS (donor of H2S) was added to the medium 30 minutes prior to CoCl2 treatment of H9C2 cardiomyocytes as a pretreatment. The cell viability was detected by CCK-8 colorimetric assay; the apoptosis rate was detected by PI staining; the expression of Cleaved Caspase-3 was detected by Western blot; the ratio of GSSG / (GSSG + GSH) The reactive oxygen species (ROS) in the cells were detected by DCFH-DA staining and rhodamine123 (Rh123) staining. The mitochondrial membrane potential (MMP) was detected by fluorescence microscope. Results In the concentration range of 400 ~ 800μmol.L-1, CoCl2 treatment of H9C2 cardiomyocytes 36 h, a dose-dependent manner to reduce cell viability. In the treatment of H9C2 cardiomyocytes with 600μmol.L-1CoCl2 for 30min, 400μmol.L-1NaHS significantly inhibited the injury of CoCl2, significantly increased the cell viability, and decreased the apoptosis rate and the expression of CleavedCaspase-3. And significantly decreased the ratio of GSSG / (GSH + GSSG) and ROS in H9C2 myocardial cells, and at the same time, significantly improved MMPs. Conclusions H2S can obviously antagonize the injury of cardiomyocytes induced by chemical hypoxia. The protective effect of H2S is related to its mechanism of decreasing the ratio of GSSG / (GSH + GSSG) and ROS, improving MMP and inhibiting the activation of Caspase-3.