细胞焦亡参与脑缺血再灌注损伤的初步研究  

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

作　　者：佘颜[1] 胡雨琴 张荆 蒿玉兴 蔡媛 张蝈蝈 王睿智 邓常清[1] 刘新春[2] SHE Yan;HU Yu-qin;ZHANG Jin;HAO Yu-xing;CAI Yuan;ZHANG Guo-guo;WANG Rui-zhi;DENG Chang-qing;LIU Xing-chun(Laboratory of Vascular Biology, Hunan University of Chinese Medicine, Changsha 410208, China;The First Affiliated Hospital of Hunan Traditional Chinese Medical College, Zhuzhou 412000, China)

机构地区：[1]湖南中医药大学血管生物学实验室,湖南长沙410208 [2]湖南中医药高等专科学校附属第一医院,湖南株洲412000

出　　处：《中国病理生理杂志》2019年第8期1379-1386,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81603415);湖南省自然科学基金资助项目(No.2017JJ3231);湖南省中医药管理局重点项目(No.201820);湖南省教育厅科研项目(No.15C1034);2016年度湖南省大学生研究性学习和创新性实验计划(No.959)

摘　　要：目的:探讨细胞焦亡在脑缺血再灌注后不同时点海马和皮质区的变化,从NLRP3炎症小体介导的经典焦亡途径探讨其机制,分析细胞焦亡在脑内不同部位损伤中的作用。方法:SD大鼠随机分成假手术组(sham组)与模型组(MCAO/R组),模型组又分为脑缺血再灌注6 h组(MCAO/R 6 h组)、12 h组(MCAO/R 12 h组)和24 h组(MCAO/R 24 h组)。右侧改良线栓法制备大鼠大脑中动脉缺血-再灌注模型。采用神经功能评分、2,3,5-氯化三苯基四氮唑(TTC)染色法和形态学观察评价神经细胞损伤程度;TUNEL和caspase-1免疫荧光双染检测细胞焦亡;Western blot法检测NLRP3、cleaved caspase-1、pro-caspase-1和白细胞介素1β(IL-1β)蛋白的表达。结果:脑缺血再灌注后不同时间均出现神经功能损伤,TTC染色显示随着再灌注时间的延长,脑梗死体积逐渐增大(P<0.05)。海马CA1区及皮质区出现组织结构疏松,间质水肿,神经细胞排列紊乱,胞核染色加深,细胞核碎裂,细胞数量减少等细胞死亡的典型形态学特征。免疫荧光双染检测表明,脑缺血再灌注后不同时点均出现细胞焦亡现象,海马CA1区和皮质区在再灌注12 h和24 h时最明显(P<0.05)。Western blot检测结果表明,NLRP3介导细胞经典焦亡通路中的NLRP3、cleaved caspase-1和pro-caspase-1、IL-1β蛋白表达在脑缺血再灌注后均不同程度上调,NLRP3蛋白在海马区再灌注12 h和24 h表达明显增高(P<0.05),皮质区再灌注6 h表达明显增高(P<0.05);pro-caspase-1蛋白在海马区再灌注各个时点表达均明显增高(P<0.05),皮质区再灌注24 h表达明显增高(P<0.05);cleaved caspase-1蛋白在海马区再灌注12 h表达明显增高(P<0.05),皮质区再灌注24 h表达明显增高(P<0.05);IL-1β蛋白表达在海马区再灌注24 h明显增高(P<0.05),皮质区再灌注6 h表达增高(P<0.05)。结论:细胞焦亡参与了脑缺血再灌注后神经细胞损伤,经典焦亡途径在海马和皮质区均发挥重要调控作用,以�AIM: To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS: SD rats were randomly divided into sham operation group(sham group) and model group(MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group(MCAO/R 6 h group), 12 h group(MCAO/R 12 h group)and 24 h group(MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion(MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride(TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β(IL-1β) was determined by Western blot. RESULTS: Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time(P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion(P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The pro

关 键 词：脑缺血再灌注损伤 细胞焦亡 NLRP3炎症小体 CASPASE-1 

分 类 号：R743.3[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]