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目的探讨活性氧清除剂N-乙酰半胱氨酸能否保护H9c2心肌细胞对抗化学性低氧引起的内质网应激。方法应用化学性低氧模拟物氯化钴处理H9c2心肌细胞,建立化学性低氧损伤心肌细胞的实验模型。在氯化钴处理H9c2心肌细胞前60 min把N-乙酰半胱氨酸加入培养基中,作为预处理。应用CCK-8比色法检测细胞存活率;Hoechst33258染色荧光显微镜照相术检测凋亡心肌细胞的形态学改变;双氯荧光素染色荧光显微镜照相检测细胞内活性氧水平;免疫印迹法检测内质网应激蛋白葡萄糖调节蛋白78的表达。结果在100～2 000μmol/L浓度范围内,氯化钴处理H9c2心肌细胞24 h,呈剂量依赖性地抑制细胞存活率。在12～48 h时间范围内,800μmol/L氯化钴处理H9c2心肌细胞呈时间依赖性地抑制细胞存活率。在氯化钴处理H9c2心肌细胞前60 min,应用2 000μmol/L的N-乙酰半胱氨酸不仅能抑制氯化钴对活性氧生成的促进作用,也能明显的抑制氯化钴诱导的细胞凋亡。不同浓度的氯化钴处理H9c2心肌细胞24 h,可使葡萄糖调节蛋白78表达增多,其中800μmol/L的氯化钴处理H9c2心肌细胞24 h时,葡萄糖调节蛋白78表达最多,800μmol/L的氯化钴处理H9c2心肌细胞不同时间,9 h时葡萄糖调节蛋白78表达最多。在氯化钴处理H9c2心肌细胞前60 min,应用2 000μmol/L的N-乙酰半胱氨酸能明显的抑制氯化钴诱导的葡萄糖调节蛋白78的表达。结论 N-乙酰半胱氨酸能显著地对抗化学性低氧诱导的心肌细胞损伤,此心肌细胞保护作用可能与其对抗化学性低氧引起的内质网应激有关。
Objective To investigate whether reactive oxygen species scavenger N-acetylcysteine protects H9c2 cardiomyocytes against endoplasmic reticulum stress induced by chemical hypoxia. Methods H9c2 cardiomyocytes were treated with cobalt chloride, a chemical hypoxia mimics, to establish an experimental model of hypoxic injury of cardiomyocytes. N-Acetylcysteine was added to the medium 60 min before cobalt chloride treatment of H9c2 cardiomyocytes as a pretreatment. The cell viability was detected by CCK-8 colorimetric assay. Morphological changes of apoptotic cardiomyocytes were detected by Hoechst33258 staining fluorescence microscope. The levels of reactive oxygen species (ROS) in cells were detected by fluorescence microscopy with fluorescence microscope. Endoplasmic reticulum Stress protein glucose regulation protein 78 expression. Results H9c2 cardiomyocytes were treated with cobalt chloride for 24 h in a concentration range of 100-2 000 μmol / L, and the cell viability was inhibited in a dose-dependent manner. Treating H9c2 cardiomyocytes with 800μmol / L cobalt chloride inhibited the cell survival rate in a time-dependent manner in a time period of 12-48 h. At 60 min before cobalt chloride treatment of H9c2 cardiocytes, application of 2 000 μmol / L N-acetylcysteine not only inhibited the promotion of reactive oxygen species by cobalt chloride, but also significantly inhibited the cobalt chloride-induced cells Apoptosis. Treatment of H9c2 cardiomyocytes with different concentrations of cobalt chloride for 24 h increased the expression of glucose regulated protein 78, in which 800 μmol / L cobalt chloride treated H9c2 cardiomyocytes for 24 h, with the highest expression of glucose regulated protein 78 and 800 μmol / L of chlorine Cobalt treatment of H9c2 cardiomyocytes at different times, 9 h, the highest expression of glucose regulatory protein 78. At 60 min before cobalt chloride treatment of H9c2 cardiocytes, application of 2 000 μmol / L N-acetylcysteine significantly inhibited cobalt chloride-induced glucose regulation protein 78 expression. Conclusion N-acetylcysteine can significantly antagonize the hypoxic-induced injury of cardiomyocytes, which may be related to its resistance to endoplasmic reticulum stress induced by chemical hypoxia.