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

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 function score and rate of Bcl-2 positive cells were significantly increased,the percentage of left turn,percentage of cerebral infarct size,rate of Bax and caspase-3 positive cells,and apoptosis rate of neurons were decreased,the expression of PINK1 and Parkin was up-regulated,and LC3-Ⅱ/LC3-Ⅰwere increased at each time point of reperfusion in group H2S(P<0.05),and no significant change was found in the parameters mentioned above in group H_(2)S+3-MA(P>0.05).Compared with group H2S,the neural function score and rate of Bcl-2 positive cells were significantly decreased,the percentage of left turn,percentage of cerebral infarct size,rate of Bax and caspase-3 positive cells,and apoptosis rate of neurons were increased,the expression of PINK1 and Parkin was down-regulated,and LC3-Ⅱ/LC3-Ⅰwas decreased at each time point of reperfusion in H_(2)S+3-MA group(P<0.05).Conclusion The mechanism by which exogenous H2S inhibits apoptosis in neurons during focal cerebral I/R is related to enhancing mitochondrial autophagy mediated by the PINK1/Parkin pathway in rats.