预缺血通过NMDA受体抑制海马CA1区线粒体内细胞色素c向胞质释放的研究

Ischemia preconditioning decrease Cyt c release from mitochondria to cytosol through NMDA receptor in hippocampal CA1 region

目的探讨脑缺血/再灌注、预缺血及预缺血前给予NMDA受体特异性抑制剂MK801对大鼠海马CA1区神经元线粒体内细胞色素c(Cyt c)向胞质释放的影响。方法建立SD大鼠四动脉结扎全脑缺血及预缺血模型,给药组大鼠在预缺血前60 min给予腹腔注射MK801 3 mg/kg。将SD大鼠随机分为假手术组、脑缺血/再灌注组、脑缺血/再灌注加生理盐水组、预缺血前给予MK801组、预缺血前给予生理盐水组和预缺血组。检测线粒体的标志蛋白COXⅣ在分离后的线粒体和对应胞质组分中的分布来证明实验中线粒体和胞质是否得到成功分离,利用免疫印迹法分析不同处理组大鼠海马CA1区神经元细胞线粒体组分以及对应胞质组分中的Cyt c和COXⅣ水平。结果线粒体和胞质成功分离,COXⅣ只存在于各组的线粒体组分中,各组的胞质组分中均未检测到COXⅣ的免疫活性。脑缺血/再灌注组线粒体内Cyt c向胞质的释放与假手术组比较显著增加,差异有统计学意义(P<0.05)。预缺血组与脑缺血/再灌注组相比,线粒体内Cyt c向胞质的释放显著降低,差异有统计学意义(P<0.05)。预缺血前给予MK801组与预缺血组相比,线粒体中Cyt c向胞质释放显著增加,差异有统计学意义(P<0.05)。结论预缺血通过NMDA受体的介导抑制脑缺血/再灌注诱导增加线粒体内Cyt c向胞质的释放,为临床治疗脑缺血/再灌注性损伤提供了理论依据。

Objective To explore the influence of NMDA receptor specific inhibiter MK801 on the Cyt c release from mitochondria to cytosol in hippocampal CA1 of rats in cerebral ischemia/reperfusion,ischemia preconditioning and preischemia preconditioning.Methods The whole cerebral ischemia and ischemia preconditioning model were established by 4-vessel occlusion in adult male Sprague-Dawley （SD） rats,the rats in the administration group before ischemic preconditioning for 60 minutes was given intraperitoneal injection of MK801 3 mg/kg.SD rats were randomly divided into the sham operation group,the cerebral ischemia/reperfusion group,the cerebral ischemia/reperfusion plus normal saline group,the MK801 treatment before ischemia preconditioning group,the normal saline treatment before ischemia preconditioning group and the ischemia preconditioning group.Whether the mitochondria and cytosol were successfully separated in the experiment was demonstrated by detecting the distribution of the mitochondrial marker protein COX IV in the isolated mitochondria and corresponding cytosolic fractions.The mitochondrial components of the CA1 neurons in the hippocampus of different treatment groups and the level of Cyt c and COX Ⅳ in the corresponding cytosolic fractions was analyzed by using immunoblot method.Results Mitochondria and cytosol were separated successfully,COX Ⅳ was only found in the mitochondrial fractions of each group,and the COX Ⅳ immune activity were not detected in the cytoplasmic fractions of each group.Compared with the sham operation group,the release of Cyt c from mitochondria to cytosol in the cerebral ischemia/reperfusion group was significantly increased,with significant difference （P〈0.05）.Compared with cerebral ischemia/reperfusion group,the release of from mitochondria to cytosol in the ischemia preconditioning group was significantly decreased,with significant difference （P〈0.05）.Compared with the ischemia preconditioning group,the release of Cyt c from mitochondria to cytosol in the MK801 treatment before ischemia preconditioning group was significantly increased,with significant difference （P〈0.05）.Conclusion Ischemia preconditioning can obviously depress the release of Cyt c from mitochondria to cytosol induced by cerebral ischemia/reperfusion through NMDA recepter,which provides a theoretical basis for clinical treatment of cerebral ischemia/reperfusion injury.