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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)的成年雄性(SD)大鼠海马神经元磷酸化蛋白激酶B(p-PKB/p-Akt)、Bcl-2相关X蛋白(Bax)和X-连锁凋亡抑制蛋白(XIAP)表达的影响,并进一步探讨r Hu EPO作用的可能机制。方法于2010年3月,由河北省实验动物中心提供的健康清洁级成年SD大鼠。采用PTZ点燃大鼠SE模型,将大鼠随机分为B组(PTZ+0.9%氯化钠溶液)、C组(PTZ+r Hu EPO)、D组(PTZ+LY294002+r Hu EPO)、E组(PTZ+二甲基亚砜+r Hu EPO),同时另选取不进行SE制模的SD大鼠作为A组(0.9%氯化钠溶液),检测大鼠行为学和脑电图的改变;用原位末端转移酶标记技术(TUNEL)检测海马神经元的凋亡情况;免疫组织化学法观察p-Akt、Bax、XIAP的阳性细胞数;反转录多聚酶链反应(RT-PCR)方法检测各组大鼠海马Bax信使RNA(BaxmRNA)的表达;Western blot方法检测各组大鼠海马蛋白激酶B(Akt)、p-Akt、XIAP蛋白的表达。结果与B组比较,C组p-Akt、XIAP阳性细胞数和p-Akt、XIAP蛋白表达水平均增多,Bax阳性细胞数及BaxmRNA表达水平均减少,差异有统计学意义(P<0.05);与C组比较,D组p-Akt、XIAP阳性细胞数和p-Akt、XIAP蛋白表达水平均减少,Bax阳性细胞数及BaxmRNA表达水平均增多,差异有统计学意义(P<0.05)。结论 r Hu EPO可以提高p-Akt、XIAP阳性细胞数及蛋白表达水平,降低Bax阳性细胞数及BaxmRNA表达水平,减少海马神经元的凋亡,发挥神经保护作用;加入磷脂酰肌醇3激酶(PI3K)抑制剂LY294002后,海马p-Akt、XIAP阳性细胞数及蛋白表达减少、Bax阳性细胞数及BaxmRNA表达增加,海马神经元的凋亡增加,减弱了r Hu EPO的保护作用。因此,PI3K/Akt信号通路是r Hu EPO发挥神经保护作用的通路之一,其作用机制可能是r Hu EPO活化PI3K/Akt后,提高p-Akt蛋白及线粒体凋亡途径相关调控因子XIAP的表达,下调了Bax的表达,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 erythropoietin (r Hu EPO) on hippocampal neuronal phosphorylation of protein kinase B (p-PKB / p-STAT) induced by PTZ-induced status epilepticus (SE) Akt, Bcl-2 and XIAP, and to explore the possible mechanism of r-Hu EPO. Methods In March 2010, healthy adult SD rats were provided by Experimental Animal Center of Hebei Province. The rats were randomly divided into two groups: PTZ + 0.9% sodium chloride solution, PTZ + r Hu EPO, PTZ + LY294002 + r Hu EPO, E (PTZ + dimethylsulfoxide + r Hu EPO). At the same time, SD rats without SE model were selected as group A (0.9% sodium chloride solution) to detect changes of behavior and electroencephalogram in rats. The apoptosis of hippocampal neurons was detected by TUNEL. The numbers of positive cells of p-Akt, Bax and XIAP were detected by immunohistochemical method. The number of positive cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) The expression of Bax mRNA in hippocampus of rats in each group was detected by Western blot. The expression of protein kinase A (Akt), p-Akt and XIAP in hippocampus were detected by Western blot. Results Compared with group B, the expression of p - Akt and XIAP positive cells, p - Akt, XIAP protein and Bax positive cells and Bax mRNA in group C were significantly decreased (P <0.05). Compared with group C, the expression of p-Akt, XIAP, p-Akt and XIAP in group D were decreased, the number of Bax positive cells and the expression of Bax mRNA were increased in group D, with statistical significance (P <0.05). Conclusion r Hu EPO can increase the number of p-Akt, XIAP positive cells and protein expression, reduce the number of Bax positive cells and Bax mRNA expression, reduce the apoptosis of hippocampal neurons, play a neuroprotective role; add phosphatidylinositol 3 kinase PI3K inhibitor LY294002, hippocampal p-Akt, XIAP-positive cells and protein expression decreased, Bax-positive cells and Bax mRNA expression increased, hippocampal neuronal apoptosis increased, weakening the protection of r Hu EPO. Therefore, PI3K / Akt signaling pathway is one of the pathways by which Hupp EPO play a neuroprotective role. Its mechanism may be that after activation of PI3K / Akt by rhu EPO, the expression of p-Akt protein and XIAP, a regulator of mitochondrial apoptosis pathway, , Down-regulated the expression of Bax, which mediated the mitochondrial apoptotic pathway and exerted anti-apoptotic and pro-survival neuroprotective effects.