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

作　　者：雷远丽 陈寿权[1] 宋文兴 陈沁 殷佳娜 李惠萍[3] 程俊彦[1] 党欣然 许华清 李章平[4] Lei Yuanli;Chen Shouquan;Song Wenxing;Chen Qin;Yin Jiana;Li Huiping;Cheng Junyan;Dang Xinran;Xu Huaqing;Li Zhangping(Department of Emergency Medicine,the First Afiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China;Department of Gastroenterology,the First Afiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China;Department of Internal Medicine,Xiaoshan First People's Hospital,Hangzhou 311200,China;Department of Emergency Medicine,the Quzhou Afiliated Hospital of Wenzhou Medical University(Quzhou People's Hospital),Quzhou 324000,China)

机构地区：[1]温州医科大学附属第一医院急诊科,浙江温州325000 [2]萧山市第一人民医院内科,杭州311200 [3]温州医科大学附属第一医院消化内科,浙江温州325000 [4]温州医科大学附属衢州医院(衢州市人民医院)急诊科,浙江衢州324000

出　　处：《中华危重症医学杂志（电子版）》2023年第6期453-460,共8页Chinese Journal of Critical Care Medicine:Electronic Edition

基　　金：温州市基础性科研项目(Y20210096);温州市基础性科研项目(Y20220085)。

摘　　要：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

关 键 词：促红细胞生成素 心室纤颤 心搏骤停 脑保护 磷脂酰肌醇3-激酶 兔 

分 类 号：R459.7[医药卫生—急诊医学]