摘要
目的观察重组人红细胞生成素(r Hu EPO)对戊四氮(PTZ)点燃的癫痫持续状态(SE)的SD大鼠海马神经元的影响,应用磷脂酰肌醇3激酶(PI3K)抑制剂LY294002,观察门冬氨酸特异半胱氨酸蛋白酶-9(Caspase-9)的变化情况,为r Hu EPO在癫痫诊疗中提供实验室依据。方法采用PTZ点燃大鼠SE模型,将大鼠随机分为正常对照组(生理盐水,NS)、PTZ组(PTZ+NS)、r Hu EPO组(PTZ+r Hu EPO)、LY294002组(PTZ+LY294002+r Hu EPO)、二甲基亚砜(DMSO)对照组(PTZ+DMSO+r Hu EPO),检测各组大鼠行为学和脑电图(EEG)的改变;TUNEL法检测海马神经细胞的凋亡情况;免疫组织化学法观察磷酸化蛋白激酶B(p-PKB/p-Akt)、Caspase-9的表达;反转录多聚酶链反应(RT-PCR)方法检测各组大鼠海马Caspase-9m RNA的表达,Western blot方法检测各组大鼠海马Akt、p-Akt、Caspase-9蛋白的表达。结果r Hu EPO可以下调Caspase-9的表达,发挥神经保护作用;加入PI3K抑制剂LY294002,海马Caspase-9蛋白、Caspase-9m RNA的表达较r Hu EPO组增加,减弱了r Hu EPO的保护作用(P<0.05)。结论 r Hu EPO在癫痫持续状态所致损伤中具有神经保护作用,应用PI3K/Akt抑制剂进一步佐证了该作用可能是通过了PI3K/Akt信号通路,通过对线粒体凋亡途径的相关调控因子Caspase-9的表达进行调控,发挥抗凋亡、促存活的神经保护作用。
Objective To observe the effects of recombinant human erythropoietin(r Hu EPO) in hippocampal neurons of status epilepticus(SE) SD rats kindled by Pentylenetetrazol(PTZ), and the changes of aspartate specific cysteine protease-9(Caspase-9) post the application of phosphatidyl inositol 3-kinase(PI3 K) inhibitor LY294002, then to provide laboratory basis for r Hu EPO in epilepsy diagnosis and treatment. MethodThe SE rats kindled by the PTZ were randomly divided into normal control group(normal saline, NS), PTZ group(PTZ+NS), r Hu EPO group(PTZ+r Hu EPO), LY294002 group(PTZ+LY294002+r Hu EPO), LY294002 solvent dimethyl sulfoxide(DMSO) control group(r Hu EPO+PTZ+DMSO). The behavior changes and electroencephalogram(EEG) recording of rats were detected; The apoptosis of hippocampal neurons were detected by TUNEL method; The expression phosphorylation protein kinase B(p-PKB/, p-Akt) and Caspase-9 were detected by immunohistochemistry method; the expression of Caspase-9 m RNA in hippocampal neurons of rats were detected through reverse transcription polymerase chain reaction(RT-PCR) method; The expression of Akt, p-Akt and Caspase-9 protein in hippocampal neurons of rats were detected through Western blot method. Results r Hu EPO can down-regulate the expression of Caspase-9 and play a neuroprotective role. The expression of Caspase-9 protein and Caspase-9 m RNA in hippocampus increased compared with that in r Hu EPO group post the application of PI3 K inhibitor LY294002, which decreased the protective effects of r Hu EPO and the difference was statistical significant. Conclusion The PI3 K/Akt signaling pathway is one of the pathways through that r Hu EPO play neuroprotective effects and testified from the two aspects of positive and negative. r Hu EPO regulates the expression of mitochondrial apoptotic pathway related factor Caspase-9 to inhibit apoptosis and promote neuronal survival.
作者
于江华
史志勤
史诺菲
苏旭东
周毅
李彬
王珊
贾丽景
赵博
朱梦楚
冯晓红
耿丽淳
Yu Jianghua;Shi Zhiqin;Shi Nuofei;Su Xudong;Zhou yi;Li Bin;Wang Shan;Jia Lijing;Zhao Bo;Zhu Mengchu;Feng Xiaohong;Geng Lichun(Department of Neurology,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,Chin)
出处
《脑与神经疾病杂志》
2018年第6期363-368,共6页
Journal of Brain and Nervous Diseases
基金
河北省卫计委重点课题(20160658)