NMDA受体在匹罗卡品癫痫小鼠海马星形胶质细胞活化中的作用及相关机制研究
首发时间:2016-10-18
摘要:目的:本研究旨在探讨在匹罗卡品(pilocarpine)诱导的小鼠癫痫持续状态模型(Status Epilepticus, SE)中,NMDA受体在海马星形胶质细胞增生中的作用及其可能的分子机制。方法:将32只6周龄雄性C57/BL6小鼠随机分为对照组(Ctrl)、癫痫持续状态组(SE)、癫痫持续状态后注射NMDA受体拮抗剂MK-801组(SE+MK-801)和单纯注射MK-801组(MK-801),每组8只。腹腔注射300mg/kg 匹罗卡品建立癫痫持续状态模型。利用免疫组织化学方法检测各组小鼠海马脑区星形胶质细胞的形态差异,同时利用western blot方法检测海马脑区GFAP蛋白的表达和转录因子cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)的磷酸化水平。结果:癫痫持续状态组小鼠海马星形胶质细胞出现显著活化现象,免疫组化结果显示SE组小鼠海马GFAP免疫反应强度(immunostaining intensity)显著高于对照组(p<0.01),而在癫痫持续状态后给予MK-801阻断NMDA受体则显著抑制了癫痫持续状态诱导的海马星形胶质细胞活化。与免疫组化结果一致,Western Blot 结果显示SE组小鼠海马GFAP蛋白表达水平显著高于对照组(p<0.05),而在癫痫持续状态后给予MK-801阻断NMDA受体则显著抑制了癫痫持续状态诱导的海马GFAP蛋白表达水平的升高。同时我们的研究结果发现,SE组小鼠海马CREB蛋白磷酸化水平显著高于对照组,而阻断NMDA受体则显著抑制了癫痫持续状态诱导的海马CREB蛋白磷酸化。结论:匹罗卡品癫痫小鼠持续状态7天后,海马星形胶质细胞被显著活化,而这一活化过程依赖于NMDA受体的激活。同时,海马星形胶质细胞活化的过程伴随CREB的磷酸化,提示海马CREB磷酸化参与了NMDA受体介导的匹罗卡品癫痫小鼠星形胶质细胞活化过程。
关键词: NMDA受体 匹罗卡品 癫痫 海马 星形胶质细胞活化
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Effect of NMDA receptor on hippocampal astrocyte activation in pilocarpine-induced epileptic mice
Abstract:In this paper, we determined to study the effect of NMDA receptor on hippocampal astrocyte activation in pilocarpine-induced epileptic mice and the possible mechanism involved. Methods Thirty two C57/BL6 mice were randomly divided into control group, Status Epilepticu (SE) group, NMDA receptor antagonist MK-801 treated SE group and MK-801 alone treated group, with 8 mice in each group. SE epilepsy model was established by intraperitoneal injection of 300mg/kg pilocarpine. Immunohistochemistry was employed to determine hippocampal astrocyte activation in different group of mice. Western blot was used to detect hippocampal GFAP protein content and transcription factor CREB phosphorylation level. Results Hippocampal astrocytes are largely activated in SE epileptic mice. Immunohistochemistry results showed that GFAP immunostaining intensities in the hippocampus of SE epileptic mice were significantly increased as compared to those of control mice. NMDA receptor antagonist MK-801 treatment after SE, however, suppressed SE-induced hippocampal astrocyte activation. Consistent with the immunohistochemistry results, our western blot showed that GFAP protein level in SE epileptic mice is significantly higher than that of control mice. MK-801 treatment after SE, however, suppressed SE-induced increase of GFAP protein level. Meanwhile, our western blot result showed that phosphorylation level of cAMP response element binding protein (CREB) is significantly increased in SE epileptic mice as compared to that of control mice. Blocking NMDA receptor by MK-801 however, suppressed the SE-induced increase of CREB phosphorylation. Conclusion Pilocarpine-induced SE activates hippocampal astrocytes, which are dependent on the activation of NMDA receptors. Meanwhile, hippocampal astrocyte activation in pilocarpine-induced SE epileptic mice is accompanied with CREB phosphorylation, suggesting CREB phosphorylation is involved in NMDA receptor-mediated hippocampal astrocytes activation in pilocarpine induced epileptic mice.
Keywords: NMDA receptors Pilocarpine Epilepsy hippocampus astrocyte activation
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NMDA受体在匹罗卡品癫痫小鼠海马星形胶质细胞活化中的作用及相关机制研究
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