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目的观察重组人红细胞生成素(recombinant human EPO,rHuEPO)对戊四氮(Pentylenetetrazol,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X-连锁凋亡抑制蛋白(X-linked inhibtor of apoptosis protein,XIAP)表达的影响,应用磷脂酰肌醇3激酶(phosphatidyl inositol3kinase,PI3K)抑制剂LY294002进一步探讨rHuEPO作用的可能机制。方法采用PTZ制作大鼠SE模型,将点燃后的大鼠随机分为PTZ组(PTZ+NS)、rHuEPO组(PTZ+rHuEPO)、LY294002组(PTZ+LY294002+rHuEPO)、LY294002溶剂二甲基亚砜(DMSO)对照组(PTZ+DMSO+rHuEPO),正常对照组腹腔注射生理盐水(n=35)。观察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p-Akt、XIAP的表达;RT-PCR法检测各组大鼠海马XIAPmRNA的表达;Western blot法检测各组大鼠海马Akt、p-Akt蛋白的表达。结果正常对照组仅见少量凋亡细胞,p-Akt和XIAP阳性细胞、p-Akt蛋白、XIAP mRNA均有少量表达;PTZ组与rHuEPO组、LY294002组、LY294002溶剂DMSO对照组比较,p-Akt和XIAP阳性细胞数、p-Akt蛋白表达及XIAP mRNA表达均减少(P<0.05),凋亡细胞数增加(P<0.05);rHuEPO组、LY294002溶剂DMSO对照组与LY294002组比较,p-Akt和XIAP阳性细胞数、p-Akt蛋白表达及XIAP mRNA表达均增加(P<0.05),凋亡细胞数减少(P<0.05)。结论rHuEPO在SE模型中活化了PI3K/Akt,提高p-Akt蛋白的表达,进而对线粒体凋亡途径的相关调控因子XIAP的表达进行了调控,从而介导线粒体凋亡途经,发挥抗凋亡、促存活的神经保护作用。
Objective To observe the effect of recombinant human EPO (rHuEPO) on hippocampal neuronal phosphorylation of protein kinase B (p-PKB) in status epilepticus (SE) / p-Akt) and X-linked inhibitor of apoptosis protein (XIAP), and to further investigate the possible effect of rHuEPO by using phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002 mechanism. Methods PTZ rats were randomly divided into three groups: PTZ group (PTZ + NS), rHuEPO group (PTZ + rHuEPO), LY294002 group (PTZ + LY294002 + rHuEPO), LY294002 solvent dimethyl (DMSO) control group (PTZ + DMSO + rHuEPO), normal control group were injected with saline (n = 35). The changes of behavior and EEG were observed. The apoptosis of hippocampus neurons was detected by TUNEL. The expressions of p-Akt and XIAP were detected by immunohistochemistry. The expression of XIAP mRNA in hippocampus was detected by RT-PCR. Western blot was used to detect the expression of Akt, p-Akt protein in hippocampus of rats in each group. Results There were only a few apoptotic cells in the normal control group. The expressions of p-Akt and XIAP positive cells, p-Akt protein and XIAP mRNA were all low in PTZ group. Compared with rHuEPO group, LY294002 group and LY294002 solvent DMSO control group, The number of XIAP positive cells, p-Akt protein and XIAP mRNA expression decreased (P <0.05), and the number of apoptotic cells increased (P <0.05). Compared with LY294002 group, the expression of p-Akt and LY294002 in rHuEPO group, The number of XIAP positive cells, p-Akt protein and XIAP mRNA expression increased (P <0.05), and the number of apoptotic cells decreased (P <0.05). Conclusions rHuEPO activates PI3K / Akt in SE model and enhances the expression of p-Akt protein, which in turn regulates the expression of XIAP, a regulator of mitochondrial apoptosis pathway, thereby mediating mitochondrial apoptosis and exerting antiapoptotic, Promote survival of neuroprotective effect.