外源性硫化氢减轻大鼠局灶性脑缺血再灌注时神经元凋亡的机制:PINK1/Parkin通路介导的线粒体自噬  

Mechanism of exogenous hydrogen sulfide-induced reduction of apoptosis in neurons during focal cerebral ischemia-reperfusion in rats: PINK1/Parkin pathway-mediated mitochondrial autophagy

作　　者：冯磊[1] 叶政辉[1] 华伟[1] 王海云[1] Feng Lei;Ye Zhenghui;Hua Wei;Wang Haiyun(Department of Anesthesiology,The Third Central Hospital of Tianjin Tianjin Key Laboratory of Extracorporeal Life Support for Critical Diseases Tianjin Artificial Cell Engineering Technology Research Center Tianjin Institute of Hepatobiliary Disease,Tianjin 300170,China)

机构地区：[1]天津市第三中心医院麻醉科,天津市重症疾病体外生命支持重点实验室天津市人工细胞工程技术研究中心,天津市肝胆疾病研究所,300170

出　　处：《中华麻醉学杂志》2021年第3期358-362,共5页Chinese Journal of Anesthesiology

摘　　要：目的探讨外源性硫化氢(H2S)减轻大鼠局灶性脑缺血再灌注时神经元凋亡与PINK1/Parkin通路介导的线粒体自噬的关系。方法健康雄性SD大鼠216只,6~8周龄,体重250~270 g,采用随机数字表法分为4组(n=54):对照组(C组)、脑缺血再灌注组(I/R组)、H2S组、H_(2)S+3-甲基腺嘌呤(H_(2)S+3-MA组)。采用大脑中动脉栓塞法制备局灶性脑缺血再灌注损伤模型。H_(2)S+3-MA组于再灌注前15 min时腹腔注射3-MA 10 mg/kg,余组腹腔注射等容量生理盐水。H2S组和H_(2)S+3-MA组于再灌注即刻腹腔注射外源性H2S供体0.25%NaSH 10 mg/kg,余组腹腔注射等容量生理盐水。于再灌注1、3和7 d时行神经功能评分和corner实验(计算左侧转身百分比);取脑组织,确定脑梗死体积百分比、Bax、Bcl-2和caspase-3阳性细胞率、神经元凋亡率,Western blot法检测线粒体微管相关蛋白1轻链3(LC3)、PINK1和Parkin的表达,计算LC3-Ⅱ与LC3-Ⅰ表达的比值(LC3-Ⅱ/LC3-Ⅰ)。结果与C组比较,I/R组再灌注各时点神经功能评分降低,左侧转身百分比、脑梗死体积百分比、脑组织Bax、Bcl-2和caspase-3阳性细胞率、神经元凋亡率及LC3-Ⅱ/LC3-Ⅰ升高,PINK1和Parkin表达上调(P<0.05);与I/R组比较,H2S组再灌注各时点神经功能评分和Bcl-2阳性细胞率升高,左侧转身百分比、脑梗死体积百分比、脑组织Bax和caspase-3阳性细胞率、神经元凋亡率降低,PINK1和Parkin表达上调,LC3-Ⅱ/LC3-Ⅰ升高(P<0.05),H_(2)S+3-MA组上述指标差异无统计学意义(P>0.05);与H2S组比较,H_(2)S+3-MA组再灌注各时点神经功能评分和Bcl-2阳性细胞率降低,左侧转身百分比、脑梗死体积百分比、脑组织Bax和caspase-3阳性细胞率、神经元凋亡率升高,PINK1和Parkin表达下调,LC3-Ⅱ/LC3-Ⅰ降低(P<0.05)。结论外源性H2S抑制大鼠局灶性脑缺血再灌注时神经元凋亡的机制与增强PINK1/Parkin通路介导的线粒体自噬有关。Objective To investigate the relationship between the mechanism of exogenous hydrogen sulfide(H2S)-induced reduction of apoptosis in neurons during focal cerebral ischemia-reperfusion(I/R)and PINK1/Parkin pathway-mediated mitochondrial autophagy in rats.Methods Two hundred and sixteen healthy male Sprague-Dawley rats,aged 6-8 weeks,weighing 250-270 g,were divided into 4 groups(n=54 each)using a random number table method:control group(group C),I/R group,H2S group and H2S plus 3-methyladenine(3-MA)group(H_(2)S+3-MA group).Focal cerebral ischemia was induced by middle cerebral artery occlusion in anesthetized rats.In group H_(2)S+3-MA,3-MA 10 mg/kg was intraperitoneally injected at 15 min before the onset of reperfusion,while the equal volume of normal saline was given instead in the other groups.In H2S and H_(2)S+3-MA groups,0.25%NaSH(a donor of exogenous H2S)10 mg/kg was intraperitoneally injected at the onset of reperfusion,while the equal volume of normal saline was given instead in the other groups.At 1,3 and 7 days of reperfusion,neural function was scored,and corner test(the percentage of left turn was calculated)was performed.Brains were removed and brain tissues were obtained for determination of the cerebral infarct size,Bax,Bcl-2 and caspase-3 positive cells,cell apoptosis,and expression of mitophagy-related protein microtubule-associated protein 1 light chain 3(LC3),PINK1 and Parkin(by Western blot).The percentage of cerebral infarct size,rate of Bax,Bcl-2 and caspase-3 positive cells and apoptosis rate were calculated.The ratio of LC3-Ⅱexpression to LC3-Ⅰexpression(LC3-Ⅱ/LC3-Ⅰ)was also calculated.Results Compared with group C,the neural function score was significantly decreased,the percentage of left turn,percentage of cerebral infarct size,rate of Bax,Bcl-2 and caspase-3 positive cells,apoptosis rate of neurons,and LC3-Ⅱ/LC3-Ⅰwere increased,and the expression of PINK1 and Parkin was up-regulated at each time point of reperfusion in group I/R(P<0.05).Compared with group I/R,the neural func

关 键 词：硫化氢 再灌注损伤 脑 细胞凋亡 线粒体 自噬 PINK1 PARKIN 

分 类 号：R743.3[医药卫生—神经病学与精神病学]