南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (11): 1705-1711.doi: 10.12122/j.issn.1673-4254.2022.11.15

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黄芪多糖减轻大脑中动脉闭塞模型大鼠的血脑屏障损伤：基于抑制P2X7R通道

袁 巧，谢丽英，陈朝俊   

  1. 广州中医药大学，广东 广州 510800；广州中医药大学附属广州中西医结合医院脑病科，广东 广州 510800
  • 出版日期:2022-11-20 发布日期:2022-11-30

Astragalus polysaccharide protects against blood-brain barrier damage in MCAO rats by inhibiting P2X7R channel

YUAN Qiao, XIE Liying, CHEN Chaojun   

  1. Guangzhou University of Chinese Medicine, Guangzhou 510800, China; Department of Encephalopathy, Guangzhou Hospital of Integrated Traditional Chinese and Western Medicine, Guangzhou 510800, China
  • Online:2022-11-20 Published:2022-11-30

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摘要： 目的 研究黄芪多糖（APS）通过P2X7R通道对大脑中动脉闭塞（MCAO）模型大鼠血脑屏障的影响。方法 建立血脑屏障体外模型及OGD模型，通过试漏实验对体外血脑屏障的形成进行初步判断，LC-MS检测APS对血脑屏障体外模型通透性的影响。将18只SD大鼠随机分为3组：对照组、MCAO+生理盐水组、MCAO+APS组，6只/组。对照组大鼠腹腔注射生理盐水，连续3 d；建立MCAO模型，造模成功后，MCAO+生理盐水组大鼠腹腔注射生理盐水，连续3 d；MCAO+APS组大鼠腹腔注射APS（45 mg/kg），连续3 d。取大鼠脑组织、血清，进行依文思蓝（EB）含量测定，制作标准曲线得到吸光度值换算成EB含量以评价血脑屏障的通透性；运用ELISA检测各组大鼠脑组织中三磷酸腺苷（ATP）含量变化情况；运用Western blot检测各组大鼠脑组织基质金属蛋白酶-9（MMP-9）、P2X7R表达水平。结果 经过APS处理可修复ATP或OGD状态下血脑屏障的完整性。与对照组相比，MCAO+生理盐水组、MCAO+APS组大鼠脑组织EB含量增多（P<0.01）、血脑屏障通透性增大（P<0.01），与MCAO+生理盐水组相比，MCAO+APS组大鼠脑组织EB含量减少（P<0.05），血脑屏障通透性改善（P<0.05）。与对照组相比，MCAO模型大鼠脑组织中ATP含量减少（P<0.05），MMP-9、P2X7R表达水平升高（P<0.01）；与MCAO+生理盐水组相比，MCAO+APS组ATP含量有所恢复（P<0.01），MMP-9 、P2X7R表达水平降低（P<0.05）。结论 黄芪多糖通过稳定脑缺血缺氧状态下的内环境，下调MMP-9表达水平，改善血脑屏障的通透性，起到对MCAO模型大鼠脑组织保护作用；并且其作用机制可能与抑制P2X7R通道相关。

关键词: 黄芪多糖；血脑屏障；MMP-9；P2X7R通道

Abstract: Objective To investigate the protective effect of astragalus polysaccharide (APS) against blood-brain barrier in a rat model of middle cerebral artery occlusion (MCAO) and the role of P2X7R channel in the protective mechanism. Methods In rat microglial cell models of oxygen and glucose deprivation (OGD) or ATP treatment, the formation of blood-brain barrier in vitro was assessed using the leak test, and the effect of APS on the permeability of the blood-brain barrier was determined using LC-MS. In 12 SD rats, MCAO model was established followed by treatment with intraperitoneal injection of normal saline (n=6) or APS (45 mg/kg, n=6) for 3 consecutive days, with another 6 rats without MCAO receiving saline injections as the control group. The permeability of the blood-brain barrier of the rats was evaluated by determining Evans blue (EB) extravasation, and ATP content in the brain tissue was detected using ELISA; the expression levels of matrix metalloproteinase-9 (MMP-9) and P2X7R in the brain tissue were detected with Western blot. Results In the in vitro cell model of OGD or ATP treatment, APS treatment obviously promoted the repair of blood-brain barrier integrity. In the rat models, the EB content in the brain tissue and the blood-brain barrier permeability increased significantly in MCAO+saline group and MCAO+APS group as compared with those in the control group (P<0.01). Compared with saline treatment, APS treatment significantly decreased EB content in the brain tissue and improved the blood-brain barrier permeability in the MCAO rats (P<0.05). MCAO caused a significant reduction of ATP content and obviously increased the expression levels of MMP-9 and P2X7R in the brain tissue of the rats (P<0.01), and these changes were significantly alleviated after APS treatment (P<0.01 or 0.05). Conclusion APS can protect the brain tissue of MCAO rats by stabilizing the internal environment, down-regulating the expression of MMP-9 and improving the permeability of blood-brain barrier under cerebral ischemia and hypoxia, and its mechanism may involve the inhibition of P2X7R channel.

Key words: astragalus polysaccharides; blood-brain barrier; matrix metalloproteinase-9; P2X7R channel