rhEPO预处理对胎鼠宫内缺血缺氧性脑损伤的保护作用及Caspase-3表达的影响

Protection Effect of Recombiant Human Erythropoietin Preconditioning Against Intrauterine Hypoxic-ischemic Brain Injury and its Influence on Expression of Caspase-3 Protein in Brain Tissue

目的探讨重组人促红细胞生成素(rhEPO)对胎鼠宫内缺血缺氧性脑损伤后神经细胞凋亡及Caspase-3蛋白表达的影响。方法将44只孕19dSD大鼠分为rhEPO治疗组(Treat组)、生理盐水缺血对照组(I/R组)和假手术对照组(Sham组)。Treat组于缺血缺氧前30min经尾静脉注入rhEPO(5000U/kg),I/R组于缺血缺氧前30min注入1mL生理盐水。各组分别于缺血再灌注后30min、3h、6h、24h和48h取胎鼠脑组织。假手术对照组经尾静脉注入1mL生理盐水后只进行开关腹手术,于术后24h取胎鼠脑组织。采用免疫组化方法检测活化的Caspase-3蛋白的表达,并通过末端脱氧核苷酸转移酶缺口标记(Tunel)法观察脑神经元凋亡情况。结果 I/R组与Treat组缺血再灌注24h和48h海马区均观察到凋亡的脑神经细胞存在,且凋亡神经元的数量随再灌注时间的延长而增加。Treat组与I/R组比较,Tunel阳性细胞数减少,在再灌注48h时,差异有统计学意义(P<0.01)。I/R组Caspase-3蛋白表达阳性强度随再灌注时间的延长逐渐增加,在再灌注48h时最高。与I/R组相比较,Treat组Caspase-3蛋白表达减弱,Caspase-3表达的面积积分光密度值减小,除24h外其余各时间点比较差异均有统计学意义(P<0.01)。结论 rhEPO预处理可抑制胎鼠宫内缺血缺氧后脑组织中Caspase-3蛋白的表达以及神经细胞的凋亡,对缺血缺氧性脑损伤具有一定的保护作用。

Objecitve To investigate the effects of recombine human erythropoietin （rhEPO） on neural cells apoptosis and the expression of Caspase-3 protein in brain tissue of fetal rats after intrauterine hypoxic-ischemic brain injury. Methods Forty-four Sprague-Dawley rats on 19 days of pregnancy were divided into rhEPO treated group, ischemia-reperfusion group and sham-operated group. Intrauterine hypoxic-ischemic injury of fetal rats was induced by bilateral occlusion of the utero-ovarian artery for 20 min. rhEPO （5000 U/kg） was injected into rats through caudal vein in rhEPO treated group while saline was injected into rats in hypoxic-ischemic group 30 min before hypoxic-ischemic injury. The brain samples in rhEPO treated group and hypoxic-ischemic group were obtained at 30 min,3 h,6 h,24 h and 48 h respectively after artery clamping. There was no hypoxic-ischemic injury in sham-operated group, so the brain samples were obtained at 24 hours after sham operation. Neuroapoptosis in brain tissue was measured by TdT mediated dUTP-biotin nick end labeling （Tunel） staining. The expression of Caspase-3 protein was observed by immunohistochemistry. Results The number of apoptosis cells in fetal rat hippocampus after intrauterine hypoxic-ischemic increased progressively with reperfusion. Compared with the I/R group, the number of apoptosis cells decreased in rhEPO treated group （P〈0.01）. The expression of Caspase-3 increased rapidly after 3 hours from the reperfusion in the I/R group. Compared with the I/R group, there was less expression of Caspase-3 in rhEPO treated group （P〈0.01）. Conclusion rhEPO showed the effects to inhibit the apoptosis of fetal neural cells and the expression of Caspase-3 protein due to intrauterine hypoxic-ischemic brain injury.