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目的研究海人酸(KA)致大鼠海马谷氨酸(Glu)、ATP、细胞内Ca2+水平、神经元凋亡细胞数、线粒体膜电位、线粒体Na+-K+-ATP酶动态变化,探讨致大鼠神经元损伤的机制。方法雄性SD大鼠40只随机分成KA致组和对照组。KA致组大鼠海马杏仁核内注射KA(2μg.kg-1)后分为6 h组、1 d组、3 d组、7 d组,每组8只;对照组(n=8):海马杏仁核注射与致剂等容量的9 g.L-1盐水。采用高效液相色谱测定各组大鼠海马组织CA3区中Glu和ATP水平;流式细胞仪及原位末端标记法测定相应区域神经元凋亡情况;JC-1荧光染色流式细胞仪检测其线粒体膜电位;Fluo-3荧光染色流式细胞仪检测胞质内Ca2+水平;ATP酶检测试剂盒测定其线粒体Na+-K+-ATP酶活性。结果 1.KA致组大鼠海马组织中Glu水平自KA注射后3 d开始增加,7 d达到高峰。2.KA注射6 h海马神经元内Ca2+水平开始升高,线粒体膜电位下降,凋亡细胞数增高,至7 d最为显著。3.KA注射后1 d,海马线粒体Na+-K+-ATP酶活性显著下降,7 d最低。4.海马ATP、线粒体膜电位、Na+-K+-ATP酶活性与神经元凋亡均呈负相关(Pa<0.01),海马Glu水平、胞质内Ca2+水平与神经元凋亡均呈正相关(Pa<0.01)。结论线粒体凋亡通路参与KA致大鼠海马神经元损伤。
Objective To investigate the dynamic changes of glutamate (Glu), ATP, intracellular Ca2 + level, neuronal apoptotic cells, mitochondrial membrane potential and mitochondrial Na + -K + -ATPase in hippocampus of kainate (KA) Mechanism of neuronal damage in rats. Methods Forty male SD rats were randomly divided into KA group and control group. Rats in KA group were randomly divided into 6 h, 1 d, 3 d, and 7 d groups, with KA (2 μg.kg-1) : Hippocampal amygdala injection of 9 gL-1 saline at the same volume as ketamine. The levels of Glu and ATP in hippocampus CA3 of rats in each group were determined by high performance liquid chromatography. The apoptosis of neurons in the corresponding region was detected by flow cytometry and in situ end labeling. The expression of Glu and ATP was detected by flow cytometry Mitochondrial membrane potential; Fluo-3 fluorescence staining flow cytometry detection of intracellular Ca2 + levels; ATPase kit assay mitochondrial Na + -K + -ATPase activity. The results showed that the level of Glu in hippocampus of KA rats increased from 3 days after KA injection and reached the peak on the 7th day. The level of Ca2 + in hippocampal neurons began to increase at 6 h after injection of KA, the mitochondrial membrane potential decreased and the number of apoptotic cells increased, reaching the most significant level at 7 d. At 1 d after 3 KA injection, the activity of mitochondrial Na + -K + -ATPase in the hippocampus significantly decreased and reached its lowest at 7 d. The activities of ATP, mitochondrial membrane potential and Na + -K + -ATPase in hippocampus were negatively correlated with neuronal apoptosis (P <0.01), hippocampus Glu levels and intracellular Ca2 + levels were positively correlated with neuronal apoptosis (Pa <0.01). Conclusion Mitochondrial apoptotic pathway is involved in neuronal damage of hippocampus in KA rats.