重组人促红细胞生成素对戊四氮致痫大鼠海马Bax表达的影响

Effect of recombinant human erythropoietin on the expression of Bax in hippocampus of status epileptic rats kindled by Pentylenetetrazol

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Bax的变化情况,探讨r Hu EPO为临床处理SE提供实验依据。方法采用PTZ点燃大鼠SE模型,将大鼠随机分为正常对照组(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)、Bax的表达;反转录多聚酶链反应(RT-PCR)方法检测各组大鼠海马Baxm RNA的表达,Western blot方法检测各组大鼠海马Akt、p-Akt、Bax蛋白的表达。结果 r Hu EPO可以增加p-Akt蛋白的表达、下调Bax蛋白的表达,发挥神经保护作用;加入PI3K抑制剂LY294002,p-Akt的表达较r Hu EPO组减少,海马Bax蛋白、Baxm 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),差异具有统计学意义。结论 r Hu EPO的抗凋亡从应用PI3K/Akt激活剂和抑制剂两个方面证实作用可能是通过了PI3K/Akt信号通路,通过对线粒体凋亡途径的相关调控因子Bax的表达进行调控,发挥神经保护作用。

Objective To observe the effects of recombinant human erythropoietin（r Hu EPO） in hippocampal neurons of status epilepticus（SE） SD rats kildled by Pentylenetetrazol（PTZ）, and the expression changes of Bax post the application of phosphatidyl inositol 3-kinase（PI3 K） inhibitor LY294002, then discuss the possible mechanisms of r Hu EPO. Method The 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 of phosphorylation protein kinase B（p-PKB/p-Akt） and Bax were detected by immunohistochemistry method; the expression of Baxm RNA in hippocampal neurons of rats were detected through reverse transcription polymerase chain reaction（RT-PCR） method; The expression of Akt, p-Akt and Bax protein in hippocampal neurons of rats were detected through Western blot method. Results r Hu EPO can promote the he expression of p-Akt protein while down-regulate the expression of Bax protein and play a neuroprotective role. The expression of Bax protein and Baxm RNA in hippocampus increased while the p-Akt decreased 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 Bax to inhibit apoptosis and promote neuronal survival.