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脑缺血再灌注小鼠M1极化的小胶质细胞分泌的可溶性Robo4对血脑屏障完整性的破坏作用

黄锦龙¹, 李宸¹, 杨亮亮¹, 高洋¹, 赵普远¹, 杨志刚^1,2,*

¹复旦大学附属中山医院，上海 200032；²国家放射与治疗临床医学研究中心，上海 200032

摘要

本文旨在研究缺血再灌注过程中小胶质细胞的极化对血脑屏障的调控作用和机制。构建脑缺血再灌注的小鼠模型后，小鼠外周血中IL-6、TNF-α表达明显升高，脑组织中IL-6和iNOS mRNA表达增加，iNOS的蛋白表达水平增加，小胶质细胞呈现出明显的M1极化趋势。在体外对脑小胶质细胞Bv-2进行糖氧剥夺后复氧复糖(oxygen-glucose deprivation/reoxygenation, OGD/R)处理，在细胞培养上清中检测到TNF-α、IL-6表达明显上升，细胞内IL-6、iNOS和CD86 mRNA表达增加，IL-6和iNOS蛋白表达增加。RNAseq检测结果显示，诱导M1极化的Bv-2细胞和OGD/R处理的Bv-2细胞中Robo4 (roundabout guidance receptor 4)的表达都显著增加。进一步研究发现M1极化的Bv-2细胞和OGD/R处理的Bv-2细胞外泌可溶性Robo4蛋白(soluble roundabout guidance receptor 4, sRobo4)也显著增加，并且Robo4重组蛋白、M1极化Bv-2细胞培养上清或OGD/R处理的Bv-2细胞培养上清都能够使内皮细胞的紧密结构破坏。此外，Robo4在急性脑大血管闭塞取栓术后再灌注的患者血清中也有高表达。上述结果显示，脑缺血再灌注过程中M1极化的小胶质细胞分泌Robo4增加可能是引起血脑屏障损伤的原因之一，Robo4有可能作为血脑屏障功能保护的治疗靶点之一。

关键词： 脑缺血再灌注; 血脑屏障; 脑小胶质细胞; M1极化; sRobo4

The destructive role of soluble Robo4 secreted by the M1-polarized-microglia during cerebral ischemia-reperfusion in blood-brain barrier integrity

HUANG Jin-Long¹, LI Chen¹, YANG Liang-Liang¹, GAO Yang¹, ZHAO Pu-Yuan¹, YANG Zhi-Gang^1,2,*

¹Zhongshan Hospital, Fudan University, Shanghai 200032, China；²National Clinical Research Center for Interventional Medicine, Shanghai 200032, China

Abstract

This project was aimed to investigate the role and the underlying mechanism of microglia polarization on blood-brain barrier (BBB) during cerebral ischemia-reperfusion. After construction of the mouse model of cerebral ischemia-reperfusion, upregulated IL-6 and TNF-α in peripheral blood and increased IL-6 and iNOS in ischemia tissues were confirmed. The supernatant expression of TNF-α and IL-6, as well as IL-6, iNOS and CD86 mRNA, was significantly increased in the of Bv-2 cells after oxygen-glucose deprivation/reoxygenation (OGD/R) model was constructed in vitro. For further understanding the expression pattern of RNAs, the next-generation RNA sequencing was performed and upregulation of Robo4 (roundabout guidance receptor 4) was found both in M1-polarized and OGD/R treated Bv-2 cells, which was also confirmed by RT-qPCR. Extracellular soluble Robo4 (sRobo4) protein also increased in the supernatant of M1-polarized and OGD/R treated Bv-2 cells. Treating bEND3 cells with the Robo4 recombinant protein, M1-polarized Bv-2 cell supernatant or OGD/R Bv-2 cell supernatant decreased trans-endothelial electrical resistance (TEER), suggesting the injury of BBB. In addition, Robo4 was also highly expressed in the serum of patients who experienced acute ischemia stroke and mechanical thrombectomy operation. All the results suggest that increased secretion of Robo4 by M1-polarized-microglia during cerebral ischemia-reperfusion is most likely one of the causes of BBB injury, and Robo4 may be one of the therapeutic targets for BBB functional protection. 

Key words: cerebral ischemia-reperfusion; blood-brain barrier; microglia; M1 polarization; sRobo4

收稿日期：　　录用日期：

通讯作者：杨志刚　　E-mail: yang.zhigang1@zs-hospital.sh.cn

DOI: 10.13294/j.aps.2022.0052

引用本文：

黄锦龙, 李宸, 杨亮亮, 高洋, 赵普远, 杨志刚. 脑缺血再灌注小鼠M1极化的小胶质细胞分泌的可溶性Robo4对血脑屏障完整性的破坏作用[J]. 生理学报 2022; 74 (4): 513-524.

HUANG Jin-Long, LI Chen, YANG Liang-Liang, GAO Yang, ZHAO Pu-Yuan, YANG Zhi-Gang. The destructive role of soluble Robo4 secreted by the M1-polarized-microglia during cerebral ischemia-reperfusion in blood-brain barrier integrity. Acta Physiol Sin 2022; 74 (4): 513-524 (in Chinese with English abstract).