Bcl-2、Bad在大鼠癫痫持续状态中的表达及rHuEpo干预的分子机制  

Effect of recombinant human erythropoietin on the expression of Bcl-2、Bad in hippocampus of status epileptic rats

作　　者：于江华[1] 史志勤[2] 史诺菲 苏旭东[1] 周毅[1] 王珊[1] 贾丽景[1] 赵博[2] 朱梦楚[2] 冯晓红[2] 尹阔场 王维平[1] YU Jianghua;SHI Zhiqin;SHI Nuofei(Department of Neurology,The Second Hospital of Hebei Medical University,Shijiazhuang,050000,Chin)

机构地区：[1]河北医科大学第二医院,河北石家庄050000 [2]河北医科大学,河北石家庄050000

出　　处：《中风与神经疾病杂志》2018年第5期388-394,共7页Journal of Apoplexy and Nervous Diseases

基　　金：国家自然科学基金面上项目(81671292);河北省卫计委重点课题(20160658)

摘　　要：目的观察PTZ致痫的SD大鼠SE时海马神经元损伤的情况,给予rHuEpo干预后的变化以及应用PI3K抑制剂LY294002后神经元凋亡以及Bcl-2、Bad的变化情况,对rHuEpo的作用机制进行更深一步的探讨。方法采用PTZ致痫大鼠SE模型,随机将大鼠分为正常对照组、模型组、rHuEpo干预组、LY294002干预组、LY294002对照组,用TUNEL法检测海马神经元凋亡状况;免疫组化SP法观察p-Akt、Bcl-2、Bad阳性细胞的表达;每组大鼠海马Bcl-2 mRNA、Bad mRNA的表达采用RT-PCR方法检测,每组大鼠海马p-Akt、Bcl-2、Bad蛋白的表达采用Western blot方法检测。结果 rHuEpo干预后可以上调p-Akt、Bcl-2 mRNA和Bcl-2蛋白的表达、减少Bad mRNA和Bad蛋白的表达,对神经元具有一定的保护作用;与rHuEpo干预组相比,PI3K抑制剂LY294002干预后,pAkt、Bcl-2 mRNA和蛋白的表达明显减少,海马Bad mRNA和蛋白的表达较rHuEpo干预组明显增加,rHuEpo的神经保护作用被明显减弱,差异具有统计学意义。结论从正反两个方面提示rHuEpo的抗凋亡作用可能通过对PI3K/Akt信号通路发挥影响,从而对凋亡线粒体途径中Bcl-2、Bad蛋白的表达程度产生调节,最终实现对抗癫痫持续状态凋亡损伤的神经保护作用。Objective To observe the hippocampal neuron damage of status epilepticus （SE） SD rats kindled by Pentylenetetrazol（PTZ）,and the changes of neurons apoptosis and the expression changes of Bad、Bcl-2 post the application of recombinant human erythropoietin（rHuEpo） and phosphatidyl inositol 3-kinase（PI3K） inhibitor LY294002,then discuss the possible mechanisms of rHuEpo furtherly. Methods The SE rats kindled by the PTZ were randomly divided into normal control group （normal saline,NS）,model group （PTZ＋NS）,rHuEpo treated group （PTZ＋rHuEpo）,LY294002 treated group（PTZ＋LY294002＋rHuEpo）,LY294002 control group （rHuEpo＋PTZ＋DMSO）. The apoptosis of hippocampal neurons were detected by TUNEL method;The positive expression neurons of phosphorylation protein kinase B（p-Akt）、Bcl-2 and Bad were detected by immunohistochemistry SP method;The expression of Bcl-2 mRNA、Bad mRNA in hippocampal neurons of each group of rats were detected through reverse transcription polymerase chain reaction （RT-PCR） method;The expression of p-Akt and Bcl-2、Bad protein in hippocampal neurons of each group of rats were detected through Western blot method. Results rHuEpo can promote the expression of p-Akt、 Bcl-2 protein and Bcl-2 mRNA while down-regulate the expression of Bad protein and Bad mRNA and play a neuroprotective role. The expression of Bad protein and Bad mRNA in hippocampus increased significantly while the p-Akt、Bcl-2、Bcl-2 mRNA decreased significantly compared with that in rHuEpo treated group post the application of PI3K inhibitor LY294002,which decreased the protective effects of rHuEpo and the difference was statistical significant. Conclusion From two aspects of positive and negative hints rHuEpo antiapoptotic effect may through the impact of PI3K/Akt signal pathway,thus mediate the expression level of Bcl-2 and Bad protein in the mitochondrial pathway of apoptosis,finally achieve the neuroprotective effect against injury of status epilepticus apoptosis.

关 键 词：重组人红细胞生成素 癫痫持续状态 磷脂酰肌醇3激酶/蛋白激酶B B淋巴细胞瘤2 Bcl-xl/Bcl-2相关死亡启动因子 

分 类 号：R742.1[医药卫生—神经病学与精神病学]