硫化氢对抗化学性缺氧引起的心肌细胞损伤及其机制

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。

Hydrogen sulfide protects H9C2 cells against chemical hypoxia-induced injury and the underlying mechanisms

Aim To investigate the effect of H2S on cobalt chloride(CoCl2)-induced H9C2 cells injury and explore the possible mechanisms.Methods H9C2 cells were treated with CoCl2 at different concentrations as a chemical hypoxia-induced injury model.NaHS(a H2S donor) was added into cell medium for 30 min prior to treatment with CoCl2.Cell viability was measured by using cell counter kit(CCK-8).Apoptotic rate was evaluated by propidium iodide(PI) staining and flow cytometry(FCM).The expression of Cleaved caspase-3 was detected by Western blot.The ratio of GSSG/(GSSG+GSH) was calculated according to GSSG kit.Intracellular reactive oxygen species(ROS) was observed by 2′,7′-dichlorfluorescein-diacetate(DCFH-DA) staining and photofluorography.Mitochondrial membrane potential(MMP) was measured by Rhodamine123(Rh123) staining and photofluorography.Results H9C2 cell viability was dose-dependently decreased by CoCl2 at the concentrations from 400 to 800 μmol·L-1 for 36 h.Preconditioning of 400 μmol·L-1 NaHS protected H9C2 cells against CoCl2-induced injury,leading to a significant increase in cell survival rate,a decrease in apoptotic percent of H9C2 cells and Cleaved caspse-3 expression.Meanwhile,NaHS attenuated the ratio of GSSG to(GSSG+GSH) and ROS,and ameliorated MMP in H9C2 cells.Conclusion H2S significantly protects H9C2 cells against apoptotic injury induced by chemical hypoxia,which is associated with decreasing the ratio of GSSG to(GSSG+GSH),intracellular ROS and caspase-3 activation as well as ameliorating MMP.