大脑皮层MANF和NF-κB p65表达及其在大鼠脑缺血再灌注损伤中的作用  

Expression of MANF and NF-κB p65 in cerebral cortex cerebral ischemia-reperfusion injury in rats

作　　者：周道英 尹号 韩明明 杨成伟 李振宇 宋尧 黄祥 康芳 李娟[1] ZHOU Dao-ying;YIN Hao;HAN Ming-ming;YANG Cheng-wei;LI Zhen-yu;SONG Yao;HUANG Xiang;KANG Fang;LI Juan(Department of Anesthesiology,The First Affiliated Hospital of University of Science and Technology of China,Anhui Provincial Hospital,Hefei Anhui 230001;School of Graduate,Wannan Medical College,Wuhu Anhui 241002,China)

机构地区：[1]中国科学技术大学附属第一医院(安徽省立医院),安徽合肥230001 [2]皖南医学院研究生学院,安徽芜湖241002

出　　处：《蚌埠医学院学报》2023年第9期1179-1184,共6页Journal of Bengbu Medical College

基　　金：安徽省重点研究与开发计划(202104a07020023);安徽省自然科学基金(2108085MH323)。

摘　　要：目的:探讨大脑皮层中脑星形胶质细胞源性神经营养因子(MANF)和核转录因子(NF)-κB p65表达在大鼠脑缺血再灌注损伤(CIRI)中的作用。方法:选取成年雄性SD大鼠30只,随机分为假手术组(Sham组)和脑缺血再灌注组(I/R组),各15只。I/R组采用改良线栓法制备大鼠局灶性CIRI模型;Sham组不将线栓插入颈内动脉,其余处理方法同I/R组。大鼠缺血2 h再灌注24 h后采用神经功能缺损评分法观察行为学变化,头颅磁共振(MRI)和TTC染色观察大脑梗死灶面积,HE染色观察缺血区域大脑皮层的组织病理学变化和细胞损伤情况,Western blotting法检测MANF和NF-κB p65蛋白相对含量,免疫组织化学法检测MANF、NF-κB p65和CHOP蛋白表达情况,免疫荧光染色法观察MANF和NF-κB p65亚细胞定位,TUNEL法观察缺血区域大脑皮层细胞凋亡情况,ELISA法检测大鼠血清中肿瘤坏死因子-α、白细胞介素(IL)-1β和IL-6水平。结果:与Sham组比较,I/R组大鼠神经功能缺损评分增加(P<0.05),头颅MRI T2相呈现高信号的脑梗死区域,TTC染色中脑梗死灶区域呈现为白色,缺血侧大脑皮层HE染色可见神经细胞数量减少,部分细胞呈空泡样改变,大脑皮层明显水肿,表明模型建立成功。与Sham组比较,I/R组大鼠缺血侧大脑皮层MANF和NF-κB p65蛋白表达均增加(P<0.01和P<0.05),且共定位于细胞核,缺血侧大脑皮层细胞凋亡率明显增加(P<0.01),血清肿瘤坏死因子-α、IL-1β和IL-6表达水平均明显增加(P<0.01)。结论:MANF和NF-κB p65在CIRI中表达上调并发生核易位,MANF可能与NF-κB p65在细胞核内相互作用,抑制炎症反应,减轻CIRI,此外,MANF还可能通过抑制ERS,减轻神经元凋亡,从而缓解CIRI。Objective:To investigate the role of expression and changes of mesencephalic astrocyte-derived neurotrophic factor(MANF)and nuclear factor(NF)-κB p65 in cerebral cortex of rats with cerebral ischemia-reperfusion injury(CIRI).Methods:Thirty adult male SD rats were divided into Sham group and cerebral ischemia-reperfusion group(I/R group)according to random number table,15 rats in each group.The model of focal CIRI in rats was established by modified thread embolization methods.In the Sham group,the thread embolization was not inserted into the internal carotid artery,and the other treatments were the same as the I/R group.Behavioral changes were observed by neurological deficit score 24 h reperfusion after 2 h ischemia,cerebral infarction area was observed by cranial magnetic resonance(MRI)and TTC staining,and cerebral cortex histopathological changes and cell damage in ischemic area were observed by HE staining.The relative protein levels of MANF and NF-κB p65 were detected by Western blotting,and the protein expression levels of NF-κB p65,CHOP and MANF were detected by immunohistochemistry.The subcellular localization of MANF and NF-κB p65 were observed by immunofluorescence staining.The apoptosis of cerebral cortex cells in the ischemic area was observed by TUNEL method,and the levels of TNF-α,IL-1βand IL-6 in serum of rats were detected by ELISA.Results:Compared with the Sham group,the rats in the I/R group showed increased neurological deficit scores(P<0.05),the T2 phase of cranial MRI presented high signal cerebral infarction areas,the cerebral infarction foci areas appeared white in TTC staining,the HE staining of the cerebral cortex of the ischemic side showed a decrease in the number of neuronal cells,some of which showed vacuolated alterations,and the cerebral cortex was obviously edematous,which indicated that the model was established successfully.Moreover,compared with the Sham group,the contents of MANF and NF-κB P65 protein in ischemic cerebral cortex of rats in the I/R group were significant

关 键 词：脑缺血再灌注损伤 中脑星形胶质细胞源性神经营养因子 核转录因子-ΚB 炎症反应 细胞凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]