线粒体活性氧自由基抑制剂R(+)-普拉克索对脑缺血再灌注损伤大鼠JAK2-STAT3通路及炎性因子TNF-α的影响

doi:10.3969/j.issn.1006-7795.2021.02.012

摘要/Abstract

摘要： 目的研究线粒体活性氧自由基(reactive oxygen species,ROS)抑制剂R(+)-普拉克索[R(+)-PPX]对大脑中动脉梗塞(middle cerebral artery occlusion, MCAO)模型大鼠再灌注6 h后内源性酪氨酸激酶2(janus kinase 2,JAK2)-信号转换器和转录激活因子3(signal transducer and activator of transcription 3,STAT3)信号通路以及炎性因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的影响,探讨R(+)-PPX在再灌注早期保护脑缺血损伤的机制。方法将30只健康雄性SD大鼠依据数字表法随机分为假手术(Sham)组、MCAO组和R(+)-PPX组,每组10只。大鼠MCAO模型制作采用改良线栓法,并于右侧大脑中动脉缺血90 min后拔出线栓进行再灌注。术中监测大鼠肛温,使其维持在正常范围。于再灌注后6 h处死大鼠,迅速取脑,应用Western blotting法检测缺血侧脑组织p-JAK2蛋白水平,免疫荧光染色法检测脑组织冰冻切片缺血半暗带区p-STAT3、TNF-α的表达,采用超氧化物阴离子荧光探针(dihydroethidium,DHE)荧光染色法计数大鼠脑缺血半暗带区ROS阳性细胞数目,应用荧光双标法将ROS分别和p-STAT3、TNF-α在大鼠脑缺血半暗带区进行共定位,应用免疫荧光双标法对p-STAT3和TNF-α共定位。结果 1)与Sham组相比,MCAO组大鼠再灌注6 h后,缺血侧脑组织p-JAK2蛋白水平明显升高(P<0.05)。R(+)-PPX组大鼠缺血侧脑组织p-JAK2蛋白水平比MCAO组显著减少(P<0.05)。2)Sham组大鼠脑内未见p-STAT3阳性细胞。MCAO组大鼠再灌注6 h脑缺血半暗带区p-STAT3阳性细胞数比Sham组显著增加(P<0.05)。R(+)-PPX组大鼠缺血半暗带区p-STAT3阳性细胞数比MCAO组显著减少(P<0.05)。且ROS与p-STAT3在大鼠脑缺血半暗带区共定位。3)Sham组大鼠脑内未见TNF-α阳性细胞。MCAO组大鼠再灌注6 h脑缺血半暗带区TNF-α阳性细胞数比Sham组显著增加(P<0.05)。R(+)-PPX组大鼠缺血半暗带区TNF-α阳性细胞数比MCAO组显著减少(P<0.05)。且ROS与TNF-α在大鼠脑缺血半暗带区共定位。4)MCAO组大鼠再灌注6 h脑缺血半暗带区内,p-STAT3与TNF-α免疫荧光染色共定位。结论线粒体ROS抑制剂R(+)-PPX可能通过抑制脑缺血再灌注6 h大鼠缺血脑组织JAK2-STAT3通路激活,降低TNF-α的水平,从而在脑缺血再灌注损伤早期发挥神经保护作用。

关键词: 脑缺血再灌注, 线粒体活性氧自由基, R(+)-普拉克索, JAK2-STAT3通路, TNF-α

Abstract: Objective To study the effect of mitochondrial reactive oxygen species (ROS) inhibitor R(+)-pramipexole [R(+)-PPX] on the janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) signaling pathway and pro-inflammatory factor tumor necrosis factor-α (TNF-α) in middle cerebral artery occlusion (MCAO) rats after 6 h reperfusion, and further to investigate the protective mechanism of R(+)-PPX on cerebral ischemia injury at early stage of reperfusion.Methods Thirty healthy male Sprague-Dawley rats were divided into Sham group, MCAO group, and R(+)-PPX group (n=10), according to random number table. MCAO rat model was established with modified suture method, the rats underwent 90 min of right MCAO and then reperfusion by withdrawing the filament. The anus temperature of the rats was monitored during the operation. The rats were sacrificed after 6h reperfusion, and then the brain was collected quickly. The level of p-JAK2 protein in ischemic brain tissue was determined with Western blotting. The expressions of p-STAT3 and TNF-α in the ischemic penumbra of frozen sections of brain tissues were detected with immunofluorescence staining. The ROS positive cells in the ischemic penumbra was detected with dihydroethidium (DHE) staining. Colocalization of ROS with p-STAT3 or TNF-α was examined with double labeling immunofluorescence staining in the ischemic penumbra. Colocalization of p-STAT3 and TNF-α was analyzed with double labeling immunofluorescence staining. Results 1) Compared with Sham group, the level of p-JAK2 protein was upregulated obviously in ischemic brain tissue of MCAO rats after 6 h reperfusion (P<0.05). The level of p-JAK2 protein decreased significantly in the ischemic brain tissue of R(+)-PPX group compared with that of MCAO group (P<0.05). 2) No p-STAT3 positive cell was observed in Sham group. The p-STAT3 positive cells in the ischemic penumbra of MCAO group increased obviously after 6 h reperfusion compared with Sham group (P<0.05). The p-STAT3 positive cells decreased significantly in the ischemic penumbra of R(+)-PPX group compared with MCAO group (P<0.05). ROS was colocalized with p-STAT3 in the ischemic penumbra of rats. 3) No TNF-α positive cell was observed in Sham group. The TNF-α positive cells in the ischemic penumbra of MCAO group increased obviously after 6 h reperfusion compared with Sham group (P<0.05). The TNF-α positive cells decreased significantly in the ischemic penumbra of R(+)-PPX group compared with MCAO group (P<0.05). ROS was colocalized with TNF-α in the ischemic penumbra of rats. 4) p-STAT3 was colocalized with TNF-α with immunofluorescence staining in the ischemic penumbra of MCAO group after 6 h reperfusion. Conclusion The mitochondrial ROS inhibitor R(+)-PPX may play a neuroprotective role at early stage of cerebral ischemia/reperfusion injury via inhibiting the activation of the JAK2-STAT3 signaling pathway and reducing the level of TNF-α in ischemic brain tissue of MCAO rats after 6 h reperfusion.

Key words: cerebral ischemia/reperfusion, mitochondrial reactive oxygen species, R(+)-PPX, janus kinase 2-signal transducer and activator of transcription 3 signaling pathway, tumor necrosis factor-α

中图分类号:

R743.3

丁锚, 杨楠, 黄语悠, 师文娟, 闫峰, 赵咏梅, 刘克建. 线粒体活性氧自由基抑制剂R(+)-普拉克索对脑缺血再灌注损伤大鼠JAK2-STAT3通路及炎性因子TNF-α的影响[J]. 首都医科大学学报, 2021, 42(2): 232-238.

Ding Mao, Yang Nan, Huang Yuyou, Shi Wenjuan, Yan Feng, Zhao Yongmei, Liu Kejian. Effects of mitochondrial reactive oxygen species inhibitor R(+)-pramipexole on JAK2-STAT3 signaling pathway and pro-inflammatory factor TNF-α in rats with cerebral ischemia/reperfusion injury[J]. Journal of Capital Medical University, 2021, 42(2): 232-238.

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