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

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+Lv-scramble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。

Abstract:

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, higher expressions of Robo4, higher protein expressions of Arg-1 and CD206, and higher levels of IL-10 and TGF-β (P < 0.05).Conclusions Robo4 regulates the polarization of microglia after CIRI through the Notch signaling pathway, thereby promoting the recovery of neurological function.