Caspase-9在戊四氮所致癫痫持续状态大鼠神经元中的表达及重组人红细胞生成素的影响

Expression of Caspase-9 in neurons of status epileptic rats kindled by Pentylenetetrazol and the effect of recombinant human erythropoietin

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.