选择性脑亚低温对大鼠脑缺血再灌注时Drp1 SUMO2/3化修饰的影响  

Effect of selective cerebral mild hypothermia on SUMO2/3 modification of Drp1 in a rat model of cerebral ischemia-reperfusion

作　　者：刘浩飞 刘阳[1] 张香香 牛瑞娇 王明山[1] 时飞[1] 袁阳 张高峰 董瑞 Liu Haofei;Liu Yang;Zhang Xiangxiang;Niu Rujiao;Wang Mingshan;Shi Fei;Yuan Yang;Zhang Gaofeng;Dong Rui(Department of Anesthesiology,Qingdao Municipal Hospital Affiliated to Qingdao University,Qingdao 266071,China)

机构地区：[1]青岛大学附属青岛市市立医院麻醉科,青岛266071

出　　处：《中华麻醉学杂志》2023年第5期591-596,共6页Chinese Journal of Anesthesiology

基　　金：山东省自然科学基金(ZR2021MH365);山东省医学会临床科研专项资金(YXH2021ZX011)。

摘　　要：目的:评价选择性脑亚低温对大鼠脑缺血再灌注时动力相关蛋白1(Drp1)小泛素样修饰蛋白2/3(SUMO2/3)化修饰的影响。方法:清洁级健康雄性SD大鼠60只,6~8周龄,体质量240~260 g,采用随机数字表法分为4组(n=15):假手术组(S组)、脑缺血再灌注组(I/R组)、选择性脑亚低温组(HT组)和常温组(NT组)。4组大鼠在监测脑温及直肠温度下进行操作,S组仅暴露颈部动脉;其余3组采用阻塞大脑中动脉2 h恢复灌注的方法制备大鼠局灶性脑缺血再灌注损伤模型。HT组和NT组拔除线栓即刻于左侧颈内动脉以80 ml·kg^(-1)·h^(-1)的速率分别灌注4和37℃生理盐水15 min。于再灌注24 h时行改良神经功能缺损严重程度评分(mNSS)。随后对大鼠实施深麻醉断头取脑,TTC染色观察并计算脑梗死体积百分比;取脑皮质缺血半暗带组织,透射电镜下观察线粒体超微结构改变,免疫共沉淀法检测Drp1 SUMO2/3化修饰水平,Western blot法检测总Drp1(T-Drp1)和总细胞色素c(T-Cytc)表达,分离线粒体和胞浆后检测线粒体外膜Drp1(M-Drp1)和胞浆Cytc(C-Cytc)表达。结果:与S组比较,其余3组mNSS和脑梗死体积百分比升高,M-Drp1、T-Drp1、C-Cytc和T-Cytc表达上调,Drp1 SUMO2/3化修饰水平升高(P<0.05),线粒体碎片化加重,嵴断裂和空泡化明显;与I/R组比较,HT组mNSS和脑梗死体积百分比降低,M-Drp1、T-Drp1、C-Cytc和T-Cytc表达下调,Drp1 SUMO2/3化修饰水平升高(P<0.05),线粒体碎片化减轻、嵴断裂和空泡化减弱。NT组各指标与I/R组比较差异无统计学意义(P>0.05)。结论:选择性脑亚低温减轻大鼠脑缺血再灌注损伤的机制与进一步增加Drp1 SUMO2/3化修饰水平,降低Drp1与线粒体外膜结合,减少线粒体过度分裂有关。Objective To evaluate the effect of selective cerebral mild hypothermia on small ubiquitin-like modifier 2/3(SUMO2/3)modification of dynamin-related protein 1(Drp1)in a rat model of cerebral ischemia-reperfusion(I/R).MethodsSixty clean-grade healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 240-260 g,were divided into 4 groups(n=15 each)using a random number table method:sham operation group(S group),cerebral I/R group(I/R group),selective cerebral mild hypothermia group(HT group)and normal temperature group(NT group).The operation was performed under the monitoring of cerebral temperature and rectal temperature.Only the cervical blood vessels were exposed in S group,while focal cerebral I/R was induced by 2 h middle cerebral artery occlusion(MCAO)followed by 24 h reperfusion in anesthetized animals in the other three groups.In HT group and NT group,4 and 37℃normal saline was perfused through the left internal carotid artery at a rate of 80 ml·kg^(-1)·h^(-1) for 15 min,respectively.Modified neurological severity score(mNSS)was assessed at 24 h of reperfusion.Then the rats were sacrificed under deep anesthesia,brains were removed,brain tissues were obtained for determination of the percentage of cerebral infarct size(by TTC staining),and the ischemic penumbra tissues in the cerebral cortex were removed for examination of the ultra-structural changes of mitochondria(with a transmission electron microscope)and for determination of the SUMO2/3 modification of Drp1(by CO-IP),expression of total Drp1(T-Drp1)and total cytochrome c(T-Cytc)(by Western blot),and expression of mitochondrial outer membrane Drp1(M-Drp1)and cytoplasmic Cytc(C-Cytc)(by Western blot)after isolation of mitochondria and cytoplasm.ResultsCompared with S group,the mNSS and percentage of cerebral infarct size were significantly increased,the expression of M-Drp1,T-Drp1,C-Cytc and T-Cytc was up-regulated,and SUMO2/3 modification of Drp1 in ischemic penumbra area was increased(P<0.05),the fragmentation of mitochondria was aggravated,and cris

关 键 词：低温 人工 再灌注损伤 脑 小泛素相关修饰蛋白质类 

分 类 号：R743.3[医药卫生—神经病学与精神病学]