药物诱导亚低温对缺血性脑卒中的神经保护作用及DRP-1调控线粒体功能在其中的潜在分子机制  

Neuroprotective effect of drug-induced hypothermia in ischemic stroke and the potential mechanism of DRP-1-mediated mitochondrial function

作　　者：江倩 王红蕊 朱玥荃 李响 耿晓坤[1,2] 李凤武 Jiang Qian;Wang Hongrui;Zhu Yuequan;Li Xiang;Geng Xiaokun;LiFengwu(China-America Institute of Neuroscience,Beijing Luhe Hospital,Capital Medical University,Beijing 101100,China;Department of Neurology,Beijing Luhe Hospital,Capital Medical University,Beijing 101100,China)

机构地区：[1]首都医科大学附属北京潞河医院神经研究所,101100 [2]首都医科大学附属北京潞河医院神经内科,101100

出　　处：《中华脑血管病杂志(电子版)》2024年第6期586-594,共9页Chinese Journal of Cerebrovascular Diseases(Electronic Edition)

基　　金：国家自然科学基金青年项目(82101436);首都医科大学2022年度科技计划、社科计划一般项目(KM202210025002);北京市通州区区财政经费(2024)。

摘　　要：目的探讨药物诱导亚低温对缺血性脑卒中急性期损伤的神经保护作用及线粒体裂变动力相关蛋白1(DRP-1)调控线粒体功能在其中的潜在分子机制。方法45只雄性SD大鼠随机分为3组,假手术组(sham组)、大脑中动脉栓塞模型(MCAO)组和MCAO模型+腹腔注射氯丙嗪联合异丙嗪(C+P)组,每组15只。构建SD大鼠2 h MCAO模型,再灌注后即刻腹腔注射8 mg/kg C+P,并在再灌注后2 h补充1/3 C+P药量。在建立MCAO模型前测量大鼠基础体温,并在腹腔注射C+P药物即刻、给药后5 min、10 min、20 min、30 min、1 h、2 h、3 h、6 h、12 h和24 h分别测量大鼠体温。再灌注48 h后应用三苯基氯化四氮唑(TTC)染色评价脑梗死体积;应用Ludmila Belayev 12分和Longa 5分评分评价短期神经功能缺损程度;再灌注后24 h应用TUNEL染色检测缺血半暗带细胞凋亡情况;并且应用ELISA检测乳酸脱氢酶(LDH)、死亡细胞、ATP、活性氧(ROS)及线粒体呼吸链复合体(COX I-Ⅳ)的水平,评价细胞损伤情况和线粒体功能;此外,再灌注后24 h应用实时荧光定量聚合酶链式反应和蛋白质印记法检测DRP-1和线粒体裂变1蛋白(Fis-1)的表达,评价线粒体裂变水平。多组间资料比较采用单因素方差分析,采用LSD-t检验或Dunnett&apos;s T3法进行组间两两比较。结果再灌注即刻给予C+P能够诱导大鼠亚低温状态,5 min内大鼠体温迅速下降,2 h后体温降至最低[(33.5±0.3)℃],12 h后体温恢复至正常水平。与MCAO模型组比较,C+P组大鼠脑梗死体积降低[(29.73±2.32)%vs(48.46±0.48)%],短期神经功能缺损程度降低[Longa 5分评分:(2.0±0.1)分vs(4.0±0.1)分;Ludmila Belayev 12分评分:(5.0±0.3)分vs(8.0±0.2)分],差异均具有统计学意义(5分评分t=2.917,P=0.008;12分评分t=2.475,P=0.029)。同时,与MCAO模型组相比,C+P组LDH、ATP、ROS及线粒体呼吸链复合体(COX I-IV)的水平降低,细胞凋亡和死亡细胞减少,差异均具有统计学意义(P均<0.05);此外,�Objective To investigate the neuroprotective effect of drug-induced hypothermia in acute phase of ischemic stroke,as well as to elucidate the underlying mechanism involving DRP-1-mediated mitochondrial function.Methods A total of 45 adult male Sprague-Dawley rats were randomly assigned to three groups:sham operation(Sham),middle cerebral artery occlusion(MCAO),and MCAO with chlorpromazine and promethazine treatment(C+P)(n=15 per group).Rats underwent 2 h of MCAO followed by 24 h or 48 h reperfusion.C+P was administered at the onset of reperfusion at a dose of 8 mg/kg,with one-third of the original dose injected 2 hours later to potentiate the drug&apos;s effects.Body temperature was monitored at pre-MCAO,initial drug,and 5 min,10 min,20 min,30 min,1 h,2 h,3 h,6 h,12 h,24 h after administration.Infarct volumes and neurological deficits were assessed using 2,3,5-triphenyltetrazolium chloride(TTC)staining and scoring systems at 48 hours.TUNEL staining was used to detected the apoptotic cell death at 24 h.The lactate dehydrogenase(LDH),cell death,ATP,reactive oxygen species(ROS)concentration,and mitochondrial respiratory chain complex(COXⅠ-Ⅳ)were evaluated by ELISA at 24 h.The mRNA and protein expressions of DRP-1 and Fis-1 were quantified by qPCR and western blotting at 24 h.LSD-t test or Dunnett&apos;s T3 method were used for inter-group comparisons.Results C+P administration(8 mg/kg)significantly reduced body temperature within 5 minutes post-MCAO in rats,reaching the lowest temperature[(33.5±0.3)℃]after 2 hours,and maintaining a significantly reduced temperature for up to 12 hours before returning to normal.Compared to the MCAO group,C+P significantly reduced infarct volumes[(29.73±2.32)%vs(48.46±0.48)%]and neurological deficits[5 score:(2.0±0.1)vs(4.0±0.1);12 score:(5.0±0.3)vs(8.0±0.2);(5 score:t=2.917,P=0.008;12 score:t=2.475,P=0.029)].C+P also significantly mitigated mitochondrial dysfunction(LDH,ATP,ROS and COX I-IV)and cell death apoptosis(P<0.05).mRNA and protein expression of DRP-1 and Fis-1 w

关 键 词：缺血性脑卒中 亚低温 线粒体裂变 缺血/再灌注损伤 细胞凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]