重组人促红细胞生成素经PI3K/Akt通路对心室颤动兔心肺复苏后的脑保护作用

Cerebral protection of recombinant human erythropoietin via PI3K/Akt pathway in ventricular fibrillation rabbits after cardiopulmonary resuscitation

目的探讨不同剂量及不同给药方式的重组人促红细胞生成素(rh-EPO)对心室颤动致心搏骤停(VFCA)兔心肺复苏(CPR)后的脑保护作用及其机制.方法将24只新西兰雄性白兔分为8组:模型组(A组)、假手术组(B组)、500 U/kg rh-EPO组(C组)、磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002组(D组)、500 U/kg rh-EPO+LY294002组(E组)、250 U/kg rh-EPO组(F组)、1000 U/kg rh-EPO组(G组)和5次500U/kg rh-EPO组(H组),每组各3只.采用电刺激致颤法制造兔VFCA-CPR模型.自主循环恢复(ROSC)后2、24、72 h进行各组动物神经功能缺损评分,并于ROSC后72 h取兔脑组织测定脑组织含水量.酶联免疫吸附剂测定(ELISA)检测脑组织S100钙结合蛋白β(S100β)、神经元特异性烯醇化酶(NSE)、水通道蛋白4(AQP4)、rh-EPO浓度.Western-blotting法检测脑组织PI3K、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)蛋白表达水平.同时采用2,3,5-三苯基氯化四氮唑(TTC)染色观察脑组织坏死程度.结果各组动物脑组织含水量(F=10.550,P<0.001),神经功能缺损评分(F=3.141,P=0.030),S100β(F=70.380,P<0.001)、NSE(F=30.680,P<0.001)、AQP4(F=19.970,P<0.001)、rh-EPO(F=27.340,P<0.001)浓度,PI3K(F=6.124,P=0.002)、AKT(F=4.567,P=0.009)、p-AKT(F=3.663,P=0.020)蛋白表达水平的比较,差异均有统计学意义.组间进一步两两比较显示,与A组比较,C组动物脑组织含水量、S100β及AQP4浓度均显著降低,rh-EPO浓度、PI3K及p-AKT蛋白表达水平均显著升高(P均<0.05).而C组与F组、G组和H组间脑组织含水量及S100β、AQP4浓度比较,差异均无统计学意义(P均>0.05).仅ROSC后2h,B组神经功能缺损评分显著低于A组(P<0.05).TTC染色显示,A组动物大脑皮质和脑干及其周围可见弥散性分布的白色坏死区域,而C组显示为少许的坏死区域.结论rh-EPO可减轻脑组织含水量,减少S100β、AQP4浓度,增加PI3K、p-AKT蛋白表达水平,从而对VFCA-CPR兔有脑保护作用,PI3K/Akt通路可能参与其中.

Objective To explore the cerebral protection and the mechanism of recombinant human erythropoietin(rh-EPO)at different doses and administration methods in ventricular fibrillation cardiac arrest(VFCA)rabbits after cardiopulmonary resuscitation(CPR).Methods The New Zealand white male rabbits were randomly divided into eight groups,namely the VFCA-CPR group(Group A),sham operation group(Group B),500 U/kg rh-EPO group(Group C),phosphatidylinositol-3-kinase(PI3K)inhibitor LY294002 group(Group D),500 U/kg rh-EPO and LY294002 group(Group E),250 U/kg rh-EPO group(Group F),1000 U/kg rh-EPO group(Group G),and 5-time 500 U/kg rh-EPO group(Group H),with three rabbits in each group.The electrical stimulation of ventricular fibrillation was used to create the rabbit VFCA-CPR model.The score of neurological deficit was detected at 2,24,and 72 h after restoration of spontaneous circulation(ROSC).The content of water in brain was measured at 72 h after ROSC.The levels of S100 beta calcium binding protein(S100β),neuron-specific enolase(NSE),aquaporin protein-4(AQP4),and erythropoietin(EPO)were detected by enzyme linked immunosorbent assay(ELISA).The protein expression levels of PI3K,protein kinase B(Akt),and phosphor-AKT(p-Akt)in brain were detected by western-blotting.Meanwhile,the degree of brain necrosis was observed by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Results The content of water(F=10.550,P<0.001),the score of neurological deficit(F=3.141,P=0.030),the levels of S100β(F=70.380,P<0.001),NSE(F=30.680,P<0.001),AQP4(F=19.970,P<0.001)and rh-EPO(F=27.340,P<0.001),and the protein expression levels of PI3K(F=6.124,P=0.002),AKT(F=4.567,P=0.009)and p-AKT(F=3.663,P=0.020)in brain all showed significant differences in the eight groups.Further pairwise comparison showed that the content of water,and the levels of S100βand AQP4 in brain were markedly decreased,while the level of rh-EPO,and the protein expression levels of PI3K and p-AKT were obviously increased in the Group C as compared with the Group A(all P<0.05).There were no significant differences in the content of water,and the levels of S100βand AQP4 between the Group C and Group F,and between the Group G and Group H(all P>0.05).The score of neurological deficit in the Group B was significantly lower than that in the Group A only at 2 h after ROSC(P<0.05).TTC staining showed that rabbits in the Group A showed diffuse white necrotic areas in and around the cerebral cortex and brainstem,while rabbits in the Group C showed few necrotic areas.Conclusion rh-EPO can reduce the content of water,decrease the levels of S100βand AQP4,and increase the protein expression levels of PI3K and p-AKT in brain,thereby having a brain protective effect on VFCA-CPR rabbits through the PI3K/Akt pathway.