重组人红细胞生成素对癫痫持续状态大鼠海马线粒体凋亡途径相关调控因子的影响及作用机制

Effects of r HuE PO on the Regulatory Factors Related With the Apoptosis Pathway of Hippocampal Mitochondria in Rats With Status Epilepticus and the Possible Mechanism

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（ rHuEPO） on the expression levels of p- PKB / p- Akt,Bax and XIAP in hippocampal neurons of male mature SD rats with pentylenetetrazol（ PTZ） induced status epilepticus（ SE）,and to further investigate the possible mechanism. Methods The included male mature rats of clean grade were provided by Laboratory Animal Center of Hebei Province in March 2010. By using the PTZ kindling epileptic rat SE model,the SD rats were divided into four groups： group B（ PTZ ＋ 0. 9% sodium chloride solution）,group C（ PTZ ＋ rHuEPO）,group D（ PTZ ＋ LY294002 ＋ rHuEPO） and group E（ PTZ ＋ dimethyl sulfoxide ＋ rHuEPO）. Meanwhile,we assigned SD rats without SE model building into group A（ 0. 9% sodium chloride solution）. The behavioral and EEG changes were detected; TUNEL was used to check the apoptosis status of hippocampal neurons; immunohistochemistry was used to observe the number of cells with positive p- Akt, Bax and XIAP; the RT- PCR method was used to detect the expression level of BaxmRNA in hippocampus; Western blot method was employed to detect the protein expression levels of Akt,p- Akt and XIAP. Results Compared with group B,group C was higher（ P〈0. 05） in the number of cells with positive p- Akt and XIAP,the protein expression levels of p- Akt and XIAP and was lower in the number of cells with positive Bax and the protein expression level of BaxmRNA. Compared with group C,group D was lower in the number of cells with positive p- Akt and XIAP and the protein expression levels of p- Akt a and XIAP and was higher（ P〈0. 05） in the number of positive cells with positive Bax and the expression level of BaxmRNA. Conclusion rHuEPO can increase the number of cells with positive p- Akt and XIAP and the protein expression of p- Akt and XIAP. It can also decrease the number of cells with positive Bax and the expression level of BaxmRNA,reduce the apoptosis of hippocampal neurons,and has a neuroprotective effect. When PI3 K inhibitor LY294002 is added,the number of cells with positive p- Akt and XIAP and the protein expression of p- Akt and XIAP decrease,the number of cells with positive Bax and the expression of BaxmRNA increase,the apoptosis of hippocampal neurons increases,and the neuroprotective effect of rHuEPO is weakened. Therefore,the signal path of PI3 K / Akt may be one of the neuroprotective ways of rHuEPO. The possible mechanism may be that rHuEPO activating the PI3 K / Akt increases the expression levels of p- Akt and XIAP,decreases the expression level of Bax,thus mediating the path of mitochondrial apoptosis and playing a neuroprotective role of anti- apoptosis and survival promotion.