清开灵注射液对缺氧/缺糖再给氧损伤时神经细胞线粒体膜电位的保护作用

Protect effects of Qingkailing injection on mitochondrion membrane potential during injury induced by hypoxia-hypoglycemia and reoxygenation in cultured rat hippocampal neurons

目的:以缺氧/缺糖再给氧为模型,观察清开灵注射液对神经细胞线粒体膜电位的保护作用。方法:四唑盐比色实验(MTT)检测细胞线粒体活性,激光共聚焦扫描显微镜检测细胞内游离钙离子含量([Ca2+]i),流式细胞术检测细胞凋亡百分率和线粒体膜电位。结果:缺氧-缺糖5 h再给氧3 h时,细胞内[Ca2+]i和细胞凋亡率明显增加,并随再给氧时间的延长而明显增高,线粒体膜电位和活性明显降低,并随再给氧时间的延长而进一步下降,清开灵注射液能显著降低细胞内[Ca2+]i浓度,抑制细胞凋亡的发生,提高线粒体膜电位和活性,与缺氧-缺糖再给氧组相比均有显著性差异(P<0.05,<0.01)。结论:清开灵注射液可抑制缺氧-缺糖再给氧损伤所致的线粒体膜电位的降低,从而具有稳定线粒体膜电位的作用,抑制细胞凋亡的发生,而对海马神经细胞起到保护作用,这种作用可能与其能抑制神经细胞内钙超载有关。

Objective： To investigate the protect effects of Qingkailing injection on mitochondrion membrane potential（MMP） during injury induced by hypoxia-hypoglycemia and reoxygenation in cultured rat hippocampal neurons. Method： Mitochondrion activity was measured by methyl thiazolyl tetrazolium（MTT）test. MMP and apoptosis were measured by flow cytometry, intracellular free calcium concentration （[Ca^2＋ ]i）was measured with confocal laser scanning microscopy. Result： Hypoxia-hypoglycemia cultures for 5 hour and reoxygenation for 3 hour induced intracellular[ Ca^2＋ ]i and apoptosis rate significantly increased. The effects were increased with the extending time of reoxygenation. MMP and mitochondrion activity declined significantly after 3 hour reoxygenation. The effects were declined with the extending time of reoxygenation. Qingkailing injection might have significantly decrease intracellular [ Ca^2 ＋ ]i and apoptosis rate, increase MMP and mitochondrion activity. Conclusion： Qingkailing Injection might have significantly inhibit the decline in MMP induced by hypoxia-hypoglycemia and reoxygenation, and had effects of stable it and anti-neuronal apoptosis.The effects might be related to inhibit overload of intracellular free calcium.