黄芪甲苷通过促进小胶质细胞/巨噬细胞M2型极化抑制大鼠脑缺血后炎症反应

doi:10.3785/j.issn.1008-9292.2020.12.02

浙江大学学报（医学版）

2020, Vol. 49

Issue (6): 679-686 DOI: 10.3785/j.issn.1008-9292.2020.12.02

专题报道

黄芪甲苷通过促进小胶质细胞/巨噬细胞M2型极化抑制大鼠脑缺血后炎症反应

郑心甜¹(

),甘海燕¹,李琳²,胡小伟²,方燕²,储利胜^2,*(

)

1. 浙江中医药大学药学院, 浙江杭州 310053
2. 浙江中医药大学基础医学院, 浙江杭州 310053

Astragaloside Ⅳ inhibits inflammation after cerebral ischemia in rats through promoting microglia/macrophage M2 polarization

ZHENG Xintian¹(

),GAN Haiyan¹,LI Lin²,HU Xiaowei²,FANG Yan²,CHU Lisheng^2,*(

)

1. College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China
2. College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China

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摘要：

目的: 研究黄芪甲苷对大鼠脑缺血后小胶质细胞/巨噬细胞M1/M2极化及炎症反应的影响。方法: 将48只大鼠随机分为手术对照组、模型对照组和黄芪甲苷组。采用线栓法建立大鼠大脑中动脉阻塞模型。黄芪甲苷组造模后即刻腹腔注射黄芪甲苷（40 mg/kg），随后1次/d，连续给药3 d。各组术后第1、3天采用改良的神经损伤严重程度评分（mNSS）和角试验进行神经功能评价；术后第3天采用2，3，5-氯化三苯基四氮唑染料（TTC）染色检测脑梗死体积；实时逆转录PCR检测M1型小胶质细胞/巨噬细胞表面标志物CD86、诱导型一氧化氮合酶（iNOS）和促炎因子TNF-α、IL-1β、IL-6的mRNA表达，以及M2型小胶质细胞/巨噬细胞表面标志物CD206、精氨酸酶1（Arg-1）、类几丁质酶3样分子1/2（YM1/2）及抗炎因子IL-10、TGF-β的mRNA表达；免疫荧光双标记法检测脑缺血周边区CD16/32/Iba1和CD206/Iba1的表达。结果: 与模型对照组比较，黄芪甲苷组mNSS分值降低、右转次数减少（P < 0.05或P < 0.01），脑梗死体积减小（P < 0.01），M1型小胶质细胞/巨噬细胞标志物CD86、iNOS、TNF-α、IL-1β和IL-6的mRNA表达下调（均P < 0.01），M2型小胶质细胞/巨噬细胞标志物CD206、Arg-1、YM1/2、IL-10和TGF-β的mRNA表达上调（均P < 0.01），脑缺血区CD16/32⁺/Iba1⁺细胞数量减少（P < 0.05），CD206⁺/Iba1⁺细胞数量增加（P < 0.01）。结论: 黄芪甲苷对大鼠脑缺血损伤有保护作用，可能与促进小胶质细胞/巨噬细胞从M1型向M2型转化、抑制炎症反应有关。

关键词： 黄芪甲苷; 小胶质细胞/巨噬细胞; 极化; 炎症; 脑缺血; 大鼠, Sprague-Dawley

Abstract:

Objective: To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on microglia/macrophage M1/M2 polarization and inflammatory response after cerebral ischemia in rats. Methods: Forty eight male SD rats were randomly divided into sham operation control group, model control group and AS-Ⅳ group with 16 rats in each. Focal cerebral ischemia model was induced by occlusion of the right middle cerebral artery (MCAO) using the intraluminal filament. After ischemia induced, the rats in AS-Ⅳ group were intraperitoneally injected with 40 mg/kg AS-Ⅳ once a day for 3 days. The neurological functions were evaluated by the modified neurological severity score (mNSS) and the corner test on d1 and d3 after modelling. The infarct volume was measured by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining on d3 after ischemia. The expression of M1 microglia/macrophage markers CD86, inducible nitric oxide synthase (iNOS) and pro-inflammatory factors TNF-α, IL-1β, IL-6, M2 microglia/macrophages markers CD206, arginase-1 (Arg-1), chitinase-like protein (YM1/2) and anti-inflammatory factors interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) was detected by real-time RT-PCR. The expression of CD16/32/Iba1 and CD206/Iba1 was determined by double labeling immunefluorescence method in the peripheral area of cerebral ischemia. Results: Compared with model control group, AS-Ⅳ treatment improved neurological function recovery and reduced infarct volume after ischemia (P < 0.05 or P < 0.01). The qRT-PCR results showed that AS-Ⅳ treatment down-regulated the expression of CD86, iNOS, TNF-α, IL-1β, IL-6 mRNA (all P < 0.01), and up-regulated the expression of CD206, Arg-1, YM1/2, IL-10 and TGF-β mRNA (all P < 0.01). Furthermore, the results of immunefluorescence labeling showed that AS-Ⅳ treatment reduced the number of CD16/32⁺/Iba1⁺ cells (P < 0.05) and increased the number of CD206⁺/Iba1⁺ cells (P < 0.01) after cerebral ischemia. Conclusion: The findings suggest that AS-Ⅳ ameliorates brain injury after cerebral ischemia in rats, which may be related to inhibiting inflammation through promoting the polarization of the microglia/macrophage from M1 to M2 phenotype in the ischemic brain.

Key words: Astragaloside Ⅳ Microglia/macrophage Polarization Inflammation Cerebral ischemia Rats, Sprague-Dawley

收稿日期: 2020-08-14 出版日期: 2021-01-14

CLC:

R285.5

基金资助: 国家自然科学基金(81073075, 81873028)

通讯作者: 储利胜 E-mail: 2395059108@qq.com;cls2004@zcmu.edu.cn

作者简介: 郑心甜(1995-), 女, 硕士研究生, 从事中药促进脑缺血损伤修复的机制研究; E-mail:2395059108@qq.com; https://orcid.org/0000-0003-0418-3601

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引用本文:

郑心甜,甘海燕,李琳,胡小伟,方燕,储利胜. 黄芪甲苷通过促进小胶质细胞/巨噬细胞M2型极化抑制大鼠脑缺血后炎症反应[J]. 浙江大学学报（医学版）, 2020, 49(6): 679-686.

ZHENG Xintian,GAN Haiyan,LI Lin,HU Xiaowei,FANG Yan,CHU Lisheng. Astragaloside Ⅳ inhibits inflammation after cerebral ischemia in rats through promoting microglia/macrophage M2 polarization. J Zhejiang Univ (Med Sci), 2020, 49(6): 679-686.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.12.02 或 http://www.zjujournals.com/med/CN/Y2020/V49/I6/679

表 1 实时逆转录PCR引物序列

图 1 各组脑组织梗死体积比较

图 2 各组M1型小胶质细胞/巨噬细胞标志物mRNA表达比较(n=4)

图 3 各组M2型小胶质细胞/巨噬细胞标志物mRNA表达比较(n=4)

图 4 各组小胶质细胞/巨噬细胞CD16/32+/Iba1+检测结果

图 5 各组小胶质细胞/巨噬细胞CD206+/Iba1+检测结果

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