治疗性低温通过激活Ⅲ型PI3K通路增加自噬减轻大鼠心肺复苏后的脑损伤  

Therapeutic hypothermia reduced brain damage on rats after cardiopulmonary resuscitation by activating Ⅲ-type PI3K pathway to increase autophagy

作　　者：肖盐[1] 赵旭明[1] 朱建良[1] 邹勤华 刘励军[1] Xiao Yan;Zhao Xuming;Zhu Jianliang;Zou Qinhua;Liu Lijun(Department of Emergency,Second Affiliated Hospital of Soochow University,Suzhou 215008,Jiangsu,China)

机构地区：[1]苏州大学附属第二医院急诊科,江苏苏州215008

出　　处：《中华危重病急救医学》2019年第1期55-60,共6页Chinese Critical Care Medicine

基　　金：江苏省苏州市医药卫生科技项目(lczx201404).

摘　　要：目的探讨Ⅲ型磷脂酰肌醇3激酶(PI3K)通路调节自噬在低温治疗心肺复苏(CPR)后脑保护机制中的作用。方法取健康雄性SD大鼠作为研究对象,制备窒息致心搏骤停后CPR模型,将自主循环恢复(ROSC)的60只大鼠按随机数字表法分为常温组、低温组及PI3K抑制剂3-甲基腺嘌呤(3-MA)预处理组,每组再将大鼠分为ROSC 24h和48h两个亚组,每个亚组10只。常温组控制肛温在(37.0±0.2)℃;低温组在ROSC后立即给予降温处理,目标温度32~34℃。3-MA预处理组于窒息前20min经脑室内注射10mmol/L3-MA5μL;其他组给予等量生理盐水。另取10只大鼠作为假手术(Sham)组,仅麻醉后置管。各组分别于ROSC24h和48h处死大鼠取脑组织,采用干湿重法测定脑组织含水量(BWC),采用蛋白质免疫印迹试验(Western Blot)测定脑组织自噬相关蛋白Beclin-1和微管相关蛋白1轻链3(LC3)、凋亡相关蛋白Bcl-2和天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)、Ⅲ型PI3K相关蛋白Vps34和Atg14的表达,透射电镜下观察脑组织超微结构改变。各组于ROSC48h进行神经功能缺损程度评分(NDS)。结果与Sham组比较,常温组大鼠ROSC24h脑皮质即明显水肿,开始出现凋亡和自噬现象,脑组织自噬、凋亡、Ⅲ型PI3K相关蛋白表达均明显升高,并呈一定时间依赖性,ROSC48h神经功能明显损害;低温干预后,大鼠脑水肿明显减轻,未见明显凋亡现象,且自噬现象有所增加,ROSC48h脑组织自噬相关蛋白Vps34、Atg14和Ⅲ型PI3K相关蛋白Beclin-1、LC3表达较常温组进一步升高(Vps34/GAPDH:0.25±0.03比0.15±0.04,Atg14/GAPDH:0.12±0.03比0.05±0.04,Beclin-1/GAPDH:0.060±0.002比0.018±0.002,LC3-Ⅱ/GAPDH:0.160±0.010比0.050±0.010,均P<0.05),凋亡相关蛋白Bcl-2、caspase-3表达较常温组明显下降(Bcl-2/GAPDH:0.05±0.03比0.20±0.04,caspase-3/GAPDH:0.050±0.002比0.140±0.015,均P<0.05),神经功能明显改善〔NDS(分):157±85比343±198,P<0.05〕。给予3-MA预处理可抑制低温干Objective To investigate the effect of Ⅲ-type phosphatidylinositide 3 kinase (PI3K) pathway adjusting autophagy on brain damage mechanism after cardiopulmonary resuscitation (CPR) and hypothermia treatment. Methods The asphyxia induce cardiac arrest-CPR model was reproduced on healthy male Sprague-Dawley (SD) rats. Sixty rats after restoration of spontaneous circulation (ROSC) were randomly divided into normothermia group, therapeutic hypothermia group and PI3K inhibitor 3-methyl adenine (3-MA) pretreatment group, differentiated by 24 hours and 48 hours after ROSC. Each group had 10 rats at each time point. The anal temperature in the normothermia group was maintained at (37.0±0.2)℃, and the rats in the hypothermia group were given cooling treatment immediately after ROSC, and the target rectal temperature was 32-34℃. In the 3-MA pretreatment group, 10 mmol/L 3-MA 5 μL was injected into the ventricle 20 minutes before asphyxia, and other groups were given the same amount of normal saline. Ten rats without CPR were included in Sham group only received anesthesia and catheterization. The rats were sacrificed at 24 hours and 48 hours after ROSC respectively, and the brain tissues were harvested, the brain water content (BWC) was measured by dry-wet weight method. Western Blot was used to determine the autophagy related proteins Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3), apoptosis related proteins Bcl-2 and caspase-3, and the Ⅲ- type PI3K pathway proteins Vps34 and Atg14. Ultrastructural changes in brain tissue were observed with transmission electron microscope. Neurological deficit scores (NDS) was obtained in each group at 48 hours after ROSC. Results Compared with Sham group, the cortex at 24 hours after ROSC in normothermic group showed obvious edema, apoptosis and autophagy began to appear under transmission electron microscope, and the expressions of autophagy, apoptosis and Ⅲ- type PI3K-related proteins in brain tissue were significantly increased in a time-dependent manner

关 键 词：心肺脑复苏 低温性治疗 自噬 凋亡 Ⅲ型PI3K通路 

分 类 号：R459.7[医药卫生—急诊医学]