重组人红细胞生成素对癫痫持续状态大鼠海马p-Akt和caspase-9的影响及作用机制  

Effect of recombinant human erythropoietin on hippocampal p-Akt and caspase-9 expressions in rats with status epilepticus and the mechanism

作　　者：王维平[1] 史志勤[1] 于江华[2] 郭力[1] 王乐[3] 韩东亮[3] 袁栋才[3] 臧颖卓[1] 

机构地区：[1]河北医科大学第二医院神经内科,河北石家庄050000 [2]北京天坛医院神经介入科,北京100050 [3]衡水市哈励逊国际和平医院,河北衡水053000

出　　处：《南方医科大学学报》2010年第1期64-69,共6页Journal of Southern Medical University

基　　金：河北省自然科学基金(C2007000852);河北省科技攻关项目(07276101D-21)

摘　　要：目的观察重组人红细胞生成素(rHuEPO)对戊四氮(PTZ)点燃的癫痫持续状态(status epilepticus,SE)的SD大鼠海马神经元磷酸化蛋白激酶B(p-PKB/p-Akt)和半胱氨酸天冬氨酸蛋白酶9(Caspase-9)表达的影响,应用磷脂酰肌醇3激酶(PI3K)抑制剂LY294002进一步探讨rHuEPO作用的可能机制。方法采用PTZ点燃大鼠SE模型,将175只大鼠随机分为正常对照组(给予生理盐水腹腔注射)、PTZ组(腹腔注射PTZ点燃SE发作后30min腹腔注射生理盐水)、rHuEPO组(SE发作后30min腹腔注射rHuEPO5000U/kg)、LY294002组(SE发作后10min脑室注射5μlLY294002,SE发作后30min腹腔注射rHuEPO5000U/kg)、二甲基亚砜(DMSO)组(SE发作后10min脑室注射5μlDMSO,SE发作后30min腹腔注射rHuEPO5000U/kg),并给予相应处理。检测大鼠行为学和脑电图的改变,免疫组织化学法观察p-Akt、caspase-9的表达,RT-PCR方法检测各组大鼠海马caspase-9mRNA的表达;Westernblotting方法检测各组大鼠海马Akt、p-Akt蛋白的表达。结果与PTZ组比较,rHuEPO组p-Akt免疫反应阳性细胞和p-Akt蛋白表达水平均增高,caspase-9免疫反应阳性细胞及caspase-9mRNA表达水平均降低(P<0.05);与rHuEPO组比较,LY294002组p-Akt免疫反应阳性细胞和p-Akt蛋白表达水平均降低,caspase-9免疫反应阳性细胞及caspase-9mRNA表达水平均增高(P<0.05)。结论在SE大鼠模型中rHuEPO激活PI3K/Akt信号转导途径调整线粒体凋亡途径相关调控因子caspase-9,借以发挥抗凋亡的神经保护作用。Objective To observe the effect of recombinant human erythropoietin （rhuEPO） on p-Akt and caspase-9 expressions in the hippocampus of rats with status epilepticus （SE） and explore the neuroprotective mechanism of rhuEPO. Methods Adult male SD rats were randomized into control, PTZ, rHuEPO, LY294002 group, and DMSO groups and treated with normal saline （NS）, PTZ, PTZ＋rHuEPO, PTZ＋LY294002＋rHuEPO, and PTZ＋DMSO＋rHuEPO, respectively. The behavioral and electroencephalogram （EEG） changes of the rats were recorded, and the expressions of p-Akt and caspase-9 were detected using immunohistochemistry. The hippocampal expression of caspase-9 mRNA was detected using RT-PCR, and the expressions of Akt and p-Akt proteins were determined with Western blotting. Results The p-Akt-positive cell and p-Akt protein expression increased significantly while the caspase-9-positive cell and caspase-9 mRNA expression decreased in rHuEPO group as compared with those in PTZ group （P0.05）. LY294002 treatment prior to rHuEPO injection significantly abolished the effects of rHuEPO on caspase-9 and p-Akt immunohistochemical positivity and caspase-9 mRNA and p-Akt protein expressions （P0.05）. Conclusion Administration of rHuEPO activates the PI3K/Akt signaling pathway in SE rats and increases the expression of p-Akt protein to regulate the expression of caspase-9, a regulatory factor of the mitochondrial-dependent apoptotic pathway, and therefore provides anti-apoptotic and neuroprotective effects.

关 键 词：癫痫持续状态 重组人红细胞生成素 磷脂酰肌醇3激酶/蛋白激酶B 半胱氨酸天冬氨酸蛋白酶9 

分 类 号：R742.1[医药卫生—神经病学与精神病学]