P2X7受体在大鼠海马脑片和神经元突触体氧糖缺失时谷氨酸和γ-氨基丁酸释放中的作用

Role of P2X7 receptors in release of glutamate and γ-aminobutyric acid during oxygen-glucose deprivation in rat hippocampus and neuronal synaptosome

目的 评价P2X7受体在大鼠海马脑片和神经元突触体氧糖缺失（OGD）时谷氨酸（Glu）和γ-氨基丁酸（GABA）释放中的作用.方法 健康成年雄性SD大鼠,体重150～200 g,制备海马脑片和神经元突触体,放入95%O2-5%CO2预充饱和人工脑脊液（aCSF）中35 ℃下孵育30 min后,取大鼠海马脑片96张,随机分为3组（n=32）;取大鼠海马神经元突触体72份,随机分为3组（n=24）.对照组（C组）于95%O2-5%CO2预充饱和的aCSF中孵育60 min;OGD组于95%N2-5%CO2预充饱和的乏糖aCSF中35 ℃下孵育60 min;亮蓝G组（BBG组）于预先加入P2X7受体拮抗剂亮蓝G 1 μmol/L、95%O2-5%CO2预充饱和的aCSF中孵育20 min,随后移至2 ml含亮蓝G l μmol/L、95%N2-5%CO2预充饱和的乏糖aCSF中孵育60 min.于OGD即刻、20、40、60 min（T1-4）时,分别随机取海马脑片和神经元突触体各6份,测定Glu和GABA的释放量.各时点分别取海马脑片2张,光镜下观察病理学结果.结果 与C组比较,OGD组和BBG组T2-4时海马脑片Glu和GABA释放量增加（P＜0.05）;与OGD组比较,BBG组T3-4时海马脑片Glu释放量减少,T4时GABA释放量减少（P＜0.05）,病理学损伤减轻.与C组比较,OGD组和BBG组T2-4时海马神经元突触体Glu和GABA的释放量增加（P＜0.05）;与OGD组比较,BBG组T2-4时海马神经元突触体GABA释放量差异无统计学意义（P＞0.05）,Glu释放量增加（P＜0.05）.结论 P2X7受体介导了大鼠海马OGD时Glu和GABA的释放,其中胶质细胞上的P2X7起主导作用.

Objective To evaluate the role of P2X7 receptors in release of glutamate （Glu） and γ-aminobutyric acid （GABA） during oxygen-glucose deprivation （OGD） in rat hippocampus and neuronal synaptosome.Methods Healthy male SD rats weighing 150-200 g were decapitated. Their hippocampi were isolated and cut into slices 400 μm thick or made into neuronal synaptosomes. The hippocampal slices and neuronal synaptosomes were incubated in artificial cerebro-spinal fluid （aCSF） at 35℃ for 30 min and divided into 3 groups （ n = 32 or 24 each）： control group （group C）; group OGD and group OGD ＋ BBG （brilliant blue G, a specific P2X7 receptor antagonist）. OGD was induced by incubating the slices and synaptosomes in glucose-free aCSF aerated with 95% N2-5% CO2. In group OGD ＋ BBG the slices and synaptosomes were incubated in O2-glucose deprived aCSF containing BBG 1 μmol/L 2 ml. Release of Glu and GABA from hippocampal slices and synaptosomes was determined by HPLC at 0, 20, 40, 60 min of OGD （T1-4）. Hippocampal slices were examined with microscope.Results （ 1 ） The release of Glu and GABA from hippocampal slices and synaptosomes were significantly increased after OGD （ P 〈 0.05）. （2） Glu released from hippocampal slices was significantly decreased at T3-4 and Glu released from synaptosomes increased at T2-4 in group OGD ＋ BBG as compared with group OGD （ P 〈 0.05）. （3）GABA released from hippocampal slices was significantly decreased at T4 in group OGD ＋ BBG as compared with group OGD （ P 〈 0.05）. There was no significant difference in GABA released from synaptosomes between group OGD and OGD ＋ BBG （P 〉 0.05）. （4） Microscopic examination showed that OGD induced significant histopathological damage to hippocampal slices which was attenuated by BBG treatment. Conclusion P2X7 receptors mediates the release of Glu and GABA during OGD in rat hippocampus and the P2X7 receptors in glial cells plays a leading role.