体外膜肺氧合对心肺复苏大鼠海马凋亡蛋白及内质网应激的影响

Effects of ECMO on apoptosis-related proteins in hippocampus and endoplasmic reticulum stress in cardiopulmonary resuscitation rats

目的探讨体外膜肺氧合(ECMO)对心肺复苏(CPR)大鼠海马凋亡蛋白及内质网应激的影响。方法50只健康SD雄性大鼠随机分为正常组、假手术组、模型组、ECMO组和二甲双胍组,每组10只。二甲双胍组以200 mg/(kg·d)的剂量连续灌胃给予二甲双胍14 d,采用经食道交流电刺激60 s的方法建立心脏骤停(CA)模型,6 min后开始行常规CPR抢救;ECMO组经右颈外静脉及尾动脉插管建立ECMO,随后制备CA模型,ECMO辅助下行CPR;模型组CA模型制备完成后不进行治疗;假手术组只进行简单麻醉、分离动静脉及插管,不进行电刺激及CPR;正常组不进行任何处理。比较各组大鼠造模完成后及恢复自主循环后24 h神经功能评分和苏木素-伊红(HE)染色观察各组大鼠海马组织病理学形态;免疫荧光染色观察大鼠海马组织超氧化物歧化酶1(SOD1)、SOD2表达;TUNEL检测海马组织细胞凋亡情况;免疫组化及Western blot分析海马组织凋亡、内质网应激相关蛋白表达。结果造模完成后模型组、ECMO组和二甲双胍组神经功能评分均明显低于正常组和假手术组(P<0.05),提示造模成功。与正常组比较,模型组大鼠恢复自主循环后24 h神经功能评分和海马组织SOD1、SOD2、Bcl-2表达明显降低,海马组织细胞凋亡率、Bax、Caspase-3、GRP78、XBP1表达明显升高(P<0.05),海马组织病理损伤程度明显加重;与模型组比较,ECMO组和二甲双胍组大鼠恢复自主循环后24 h神经功能评分和海马组织SOD1、SOD2、Bcl-2表达明显升高,海马组织细胞凋亡率、Bax、Caspase-3、GRP78、XBP1表达明显下降(P<0.05),海马组织病理损伤程度明显减轻。结论ECMO辅助下CPR能够明显改善CA大鼠的神经功能,其机制可能与增加海马内源性抗氧化能力、调节内质网应激及抑制海马组织细胞凋亡有关。

Objective To investigate the effects of extracorporeal membrane oxygenation(ECMO)on apoptosis-related proteins in hippocampus and endoplasmic reticulum stress in cardiopulmonary resuscitation(CPR)rats.Methods 50 healthy male SD rats were randomly divided into normal group,sham operation group,model group,ECMO group and metformin group,with 10 rats in each group.In metformin group,200 mg/(kg·d)metformin was given for 14-day continuous gavage,and the models of cardiac arrest(CA)were established by means of transesophageal AC stimulation for 60 s.Routine CPR started 6 min later.In the ECMO group,ECMO was intubated through the right external jugular vein and caudate artery to establish ECMO,then CA models were prepared,and CPR was performed assisted by ECMO.Model group did not receive treatment after CA model preparation.The sham operation group only received simple anesthesia,arteriovenous separation and intubation,without electrical stimulation and CPR.The normal group did not receive any treatment.The neurological function scores of each group were compared after the completion of modeling and 24 h after the return of spontaneous circulation.The histopathological morphology of hippocampus was observed by hematoxylin-eosin(HE)staining.The expression of superoxide dismutase 1(SOD1)and SOD2 in rat hippocampus was observed by immunofluorescence staining.TUNEL was used to detect the apoptosis of hippocampal cells.Hippocampal apoptosis and the expression of endoplasmic reticulum stress related protein were analyzed by immunohistochemistry and Western blot.Results After modeling,the neurological function scores were significantly lower in the model group,ECMO group and metformin group than in the normal group and the sham operation group(P<0.05),indicating that the modeling was successful.Compared with normal group,the neurological function score and the expression of SOD1,SOD2 and Bcl-2 in hippocampus of model group were significantly decreased 24 h after the return of spontaneous circulation,the apoptosis rate of hippocampus,the expression of Bax,Caspase-3,GRP78 and XBP1 were significantly increased(P<0.05),and the pathological injury degree of hippocampus was more serious.Compared with model group,the neurological function score and the expression of SOD1,SOD2 and Bcl-2 in hippocampal tissue of ECMO group and metformin group were significantly increased 24 h after the return of spontaneous circulation,while the apoptosis rate of hippocampal tissue,the expression of Bax,Caspase-3,GRP78 and XBP1 were significantly decreased(P<0.05),and the degree of histopathological injury was significantly light.Conclusions ECMO-assisted CPR can significantly improve the neurological function of CA rats,which may be related to increasing the endogenous antioxidant capacity of hippocampus,regulating endoplasmic reticulum stress and inhibiting hippocampal apoptosis.