细胞外信号调节激酶抑制剂对心肺复苏后大鼠脑皮质中钙蛋白酶的影响  

Effect of extracellular signal-regulated kinase inhibitor on calpain in rat cerebral cortex after cardiopulmonary resuscitation

作　　者：戴春光[1] 王文艳[1] 杨叶桂 钟荣[1] Dai Chunguang;Wang Wenyan;Yang Yegui;Zhong Rong(Department of Critical Care Medicine,Affiliated Hospital of Guilin Medical University,Guilin 541001,Guangxi Zhuang Autonomous Region,China;Department of Critical Care Medicine,Second Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]桂林医学院附属医院重症医学科,广西壮族自治区桂林541001 [2]广西医科大学第二附属医院重症医学科,南宁530021

出　　处：《中华危重病急救医学》2022年第11期1200-1205,共6页Chinese Critical Care Medicine

基　　金：广西自然科学基金(2018GXNSFBA281045);广西卫生健康委科研基金(Z20190977);广西医疗卫生重点(培育)学科(2019-19)。

摘　　要：目的探讨细胞外信号调节激酶(ERK)抑制剂PD98059对脑钙蛋白酶(calpain)相关蛋白的影响,并了解calpain在脑缺血/再灌注损伤(CIRI)中的病理生理变化。方法将42只大鼠按随机数字表法分为假手术(Sham)组(n=6)、模型组(n=12)、二甲基亚砜(DMSO)对照组(n=12)和PD98059组(n=12)。采用经食道起搏电刺激法诱发心室纤颤制备心搏骤停-心肺复苏(CA-CPR)模型诱导大鼠CIRI;Sham组仅进行麻醉、气管插管及动静脉置管等基本操作,不进行CA和CPR。DMSO对照组和PD98059组在自主循环恢复(ROSC)30 min时分别经股静脉注射DMSO或PD980590.30 mg/kg;Sham组和模型组给予等量生理盐水。记录各组大鼠CPR持续时间,以及ROSC 24 h存活率和神经功能缺损评分(NDS);分别采用苏木素-伊红(HE)染色和尼氏染色后观察脑皮质病理学改变;采用蛋白质免疫印迹试验(Western blotting)检测磷酸化ERK(p-ERK)、ERK、钙蛋白酶抑制蛋白(calpastatin)、calpain-1和calpain-2的蛋白表达;采用双重免疫荧光法检测p-ERK与calpain-2共表达情况。结果各制模组大鼠CPR持续时间及各组大鼠24 h存活率差异均无统计学意义。模型组大鼠脑皮质细胞核明显变形固缩、细胞空泡和组织排列紊乱,尼氏小体较Sham组显著减少,NDS评分明显降低,且ERK磷酸化水平明显增高,calpain-2蛋白表达明显上调;DMSO对照组各项指标与模型组差异无统计学意义。而给予PD98059干预后,大鼠脑皮质病理损伤明显改善,尼氏小体明显增加,NDS评分较模型组明显升高〔分:75.0(72.0,78.0)比70.0(65.0,72.0),P<0.05〕,ERK磷酸化水平和calpain-2蛋白表达水平较模型组明显降低〔p-ERK(p-ERK/ERK):0.65±0.12比0.92±0.05,calpain-2蛋白(calpain-2/GAPDH):0.73±0.10比1.07±0.14,均P<0.05〕;各组脑皮质calpastatin和calpain-1蛋白表达水平差异均无统计学意义。双重免疫荧光染色显示,p-ERK与calpain-2在细胞质和细胞核中存在共表达,模型组p-ERK与calpain-2共表Objective To explore the effect of extracellular signal-regulated kinase(ERK)inhibitor PD98059 on calpain-related proteins in the brain,and to understand the pathophysiological changes of calpain in cerebral ischemia/reperfusion injury(CIRI).Methods Forty-two rats were divided into sham operation(Sham)group(n=6),model group(n=12),dimethyl sulfoxide(DMSO)control group(n=12),and PD98059 group(n=12)by random number table.The rat model of CIRI induced by cardiac arrest-cardiopulmonary resuscitation(CA-CPR)was reproduced by transesophageal electrical stimulation to induce ventricular fibrillation.In the Sham group,only the basic operations such as anesthesia,tracheal intubation,and arteriovenous catheterization were performed without CA-CPR.The rats in the DMSO control group and PD98059 group were injected with DMSO or PD980590.30 mg/kg via femoral vein,respectively,30 minutes after the restoration of spontaneous circulation(ROSC),and rats in the Sham group and model group were given the same amount of normal saline.The duration of CPR,24-hour survival rate and neurological deficit score(NDS)after ROSC were recorded.Hematoxylin-eosin(HE)staining and Nissl staining were used to observe the pathological changes of the cerebral cortex.The expressions of phosphorylated ERK(p-ERK),ERK,calpastatin,calpain-1,and calpain-2 were detected by Western blotting.The co-expression of p-ERK and calpain-2 was detected by double immunofluorescence.Results There were no significant differences in the duration of CPR and 24-hour survival rate among all groups.In the model group,the nuclei of the cerebral cortex were obviously deformed and pyknotic,cells vacuoles and tissues were arranged disorderly,Nissl corpuscles were significantly reduced,NDS scores were also significantly reduced,level of ERK phosphorylation was increased,and calpain-2 protein was significantly up-regulated compared with the Sham group.There was no significant difference in the above parameters between the DMSO control group and the model group.After intervention wit

关 键 词：心肺复苏 脑缺血/再灌注损伤 钙蛋白酶 细胞外信号调节激酶 PD98059 

分 类 号：R459.7[医药卫生—急诊医学]