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目的 观察一氧化氮 (NO)和谷氨酸在内皮素 (ET) 1诱导培养神经元凋亡中的作用。方法 神经元培养取自新生SD大鼠大脑皮质。培养 5天后分 4组 :对照组、ET 1组 (2 0nM)、ET 1 +L NAME(N 硝基左旋精氨酸甲酯 ,NO合酶抑制剂 ,1 0 0mM)组和ET 1 +APV组 (N 甲基 D 天冬氨酸型受体拮抗剂 ,1 0 0 μM)。培养 2 4h后 ,收集细胞用流式细胞仪定量检测凋亡率。上清液中NO水平通过硝酸还原酶法检测亚硝酸盐浓度反映 ,谷氨酸浓度测定用高压液相法。结果 2 0nMET 1处理后 2 4h,培养神经元凋亡率较对照组显著增高 (P <0 0 0 1 )。L NAME和APV分别明显阻断ET 1诱导神经元凋亡的作用 ,与ET 1组比较 ,凋亡率降幅分别为 40 % (P<0 0 5)和 80 % (P <0 0 0 1 )。ET 1作用 2 4h后。神经元培养液中NO和谷氨酸浓度较对照组显著增高 (P <0 0 0 1 ) ,L NAME完全抑制了ET 1引起的培养液中NO的升高。结论 NO和谷氨酸参与了ET 1诱导培养大鼠大脑神经元凋亡过程 ,其中谷氨酸更为重要
Objective To observe the effect of nitric oxide (NO) and glutamic acid on neuronal apoptosis induced by endothelin (ET) 1. Methods Neuronal cultures were obtained from the cerebral cortex of neonatal SD rats. After 5 days of culture, the mice were divided into 4 groups: control group, ET 1 group (20 nM), ET 1 + L NAME group (N nitro L-arginine methyl ester, NO synthase inhibitor, 100 mM) Group (N-methyl D aspartate receptor antagonist, 100 μM). After culturing for 24 h, the cells were harvested and quantified by flow cytometry. The level of NO in the supernatant was measured by nitrite reductase assay, and the concentration of glutamate was measured by high pressure liquid chromatography. Results The apoptosis rate of cultured neurons was significantly higher than that of the control group (P <0.01 01) after 24 h treatment with 2nMET 1. L NAME and APV significantly blocked the ET 1 -induced neuronal apoptosis. Compared with the ET 1 group, the apoptotic rates were decreased by 40% (P <0 05) and 80% (P 0 01 0), respectively . ET 1 role after 2 4h. The concentrations of NO and glutamate in neuron culture medium were significantly higher than those in control group (P <0.01). L NAME completely inhibited the increase of NO in culture medium induced by ET 1. Conclusion NO and glutamate are involved in the neuronal apoptosis in rat brain induced by ET 1, of which glutamate is more important