Robo4在脑缺血再灌注损伤大鼠小胶质细胞极化中的作用及其机制研究  

Role of Robo4 in the polarization of microglia after cerebral ischemia reperfusion injury in rats and its underlying mechanisms

作　　者：曹天然 刘青芳 潘美民 陈勇 Cao Tian-ran;Liu Qing-fang;Pan Mei-min;Chen Yong(Clinical Trial Research Center,The First Hospital of Changsha,Changsha,Hunan 410005,China;Department of Dermatology,The First Hospital of Changsha,Changsha,Hunan 410005,China;Neurology Center,The First Hospital of Changsha,Changsha,Hunan 410005,China)

机构地区：[1]长沙市第一医院临床试验研究中心,湖南长沙410005 [2]长沙市第一医院神经医学中心,湖南长沙410005 [3]长沙市第一医院皮肤科,湖南长沙410005

出　　处：《中国现代医学杂志》2023年第18期36-42,共7页China Journal of Modern Medicine

基　　金：湖南省自然科学基金-科卫联合项目(No:2022JJ70129)。

摘　　要：目的探讨环形交叉轴突导向受体同源物4(Robo4)蛋白对脑缺血再灌注损伤(CIRI)后小胶质细胞M1/M2极化的影响。方法选取60只SD大鼠,随机分为假手术(Sham)组、大脑中动脉短暂性闭塞/再灌注(tMCAO/R)组、tMCAO/R联合过表达Robo4慢病毒阴性载体(tMCAO/R+Lv-scramble)组、tMCAO/R联合过表达Robo4慢病毒载体(tMCAO/R+Lv-Robo4)组,每组15只。tMCAO/R+Lv-scramble组及tMCAO/R+Lv-Robo4组在复制tMCAO前7 d于脑室内注射慢病毒载体。Longa评分评估神经功能缺损,TTC法检测脑梗死面积,实时荧光定量聚合酶链反应及Western blotting检测大脑组织中Robo4的表达,Western blotting检测M1小胶质细胞标志物(iNOS和CD86)、M2小胶质细胞标志物(Arg-1和CD206)和Notch通路蛋白(Notch-1、Hes1和Hes5)表达。酶联免疫吸附试验检测TNF-α、IL-1β、IL-10和TGF-β水平。结果tMCAO/R组Longa评分较Sham组高(P<0.05),tMCAO/R+Lv-scramble组较tMCAO/R+Lv-Robo4组高(P<0.05)。tMACO/R组Robo4 mRNA和蛋白相对表达量较Sham组低(P<0.05),tMCAO/R+Lv-Robo4组较tMCAO/R+Lv-scramble组高(P<0.05)。tMACO/R组脑梗死面积较Sham组大(P<0.05),tMCAO/R+Lv-Robo4组较tMCAO/R+Lv-scramble组小(P<0.05)。tMACO/R组较Sham组高(P<0.05),tMCAO/R+Lv-Robo4组iNOS、CD86较tMCAO/R+Lv-scramble组低,Arg-1、CD206较tMCAO/R+Lv-scramble组高(P<0.05)。tMACO/R组较Sham组高(P<0.05),tMCAO/R+Lv-Robo4组TNF-α、IL-1β较tMCAO/R+Lvscramble组低,IL-10、TGF-β较tMCAO/R+Lv-scramble组高(P<0.05)。tMACO/R组Notch-1、Hes1和Hes5相对表达量较Sham组高,tMCAO/R+Lv-Robo4组较tMCAO/R+Lv-scramble组低(P<0.05)。结论Robo4可调控CIRI后小胶质细胞极化向M2表型转移,并调控Notch信号通路,缓解CIRI。Objective To explore the role of roundabout guidance receptor 4(Robo4)in M1/M2 polarization of microglia after cerebral ischemia-reperfusion injury(CIRI).Methods Sixty SD rats were selected and divided into Sham group,transient middle cerebral artery occlusion/reperfusion(tMCAO/R)group,tMCAO/R combined with Robo4 negative control lentiviral vectors(tMCAO/R+Lv-scramble)group,and tMCAO/R combined with Robo4 overexpression lentiviral vectors(tMCAO/R+Lv-Robo4)group via the random number table method,with 15 rats in each group. The tMCAO/R + Lv-scramble group and the tMCAO/R + Lv-Robo4 group were injected with 5 μL of Lv-scramble (4×108 TU/mL) or Lv-Robo4 (1×109 TU/mL) 7 days before the induction of tMCAO. After 24 hours of tMCAO/R induction, the Longa score was applied to evaluate neurological deficits of rats. Besides, the cerebral infarct size was measured via the 2, 3, 5-triphenyltetrazolium chloride (TTC) method after 5 days of tMCAO/R induction. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of Robo4 in brain tissues, and the expressions of M1 microglia markers (iNOS and CD86), M2 microglia markers (Arg-1 and CD206) and proteins associated with the Notch signaling pathway (Notch-1, Hes1 and Hes5) were detected by Western blotting. The levels of tumor necrosis factor α (TNF α), interleukin-1β (IL-1β), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected by ELISA. Results Compared with the Sham group, the tMCAO/R group had higher Longa score, larger cerebral infarction size, higher protein expressions of iNOS, CD86, Arg-1, CD206, Notch-1, Hes1 and Hes5 in the brain tissues, higher levels of TNF-α, IL 1β, IL-10 and TGF-β, and lower expressions of Robo4 (P < 0.05). Compared with the tMCAO/R + Lv-scramble group, the tMCAO/R + Lv-Robo4 group had lower Longa score, smaller cerebral infarct size, lower protein expressions of iNOS, CD86, Notch-1, Hes1 and Hes5 in the brain tissues, lower levels of TNF-α and IL-1β, highe

关 键 词：脑缺血再灌注损伤 环形交叉轴突导向受体同源物4 NOTCH信号通路 M1/M2极化 小胶质细胞 

分 类 号：R743.31[医药卫生—神经病学与精神病学]