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J Zhejiang Univ (Med Sci)  2020, Vol. 49 Issue (6): 679-686    DOI: 10.3785/j.issn.1008-9292.2020.12.02
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 Lisheng2,*()
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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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 wordsAstragaloside Ⅳ      Microglia/macrophage      Polarization      Inflammation      Cerebral ischemia      Rats, Sprague-Dawley     
Received: 14 August 2020      Published: 14 January 2021
CLC:  R285.5  
Corresponding Authors: CHU Lisheng     E-mail:;
Cite this article:

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.

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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 
引物名称 引物序列(5′-3′)
iNOS:诱导型一氧化氮合酶; Arg-1:精氨酸酶1;YM1/2:类几丁质酶3样分子1/2;GAPDH:甘油醛-3-磷酸脱氢酶.
Tab 1 Primer sequences for real-time RT-PCR
Fig 1 Infarct volume in each group
Fig 2 mRNA expression of M1 microglia/macrophage markers in each group (n=4)
Fig 3 mRNA expression of M2 microglia/macrophage markers in each group (n=4)
Fig 4 Immunofluorescence results of CD16/32+/Iba1+ microglia/macrophage in each group
Fig 5 Immunofluorescence results of CD206+/Iba1+ microglia/macrophage in each group
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