The Effect of Dual Antiplatelet Therapy with Aspirin and Clopidogrel on Inflammatory Response in Bone Marrow and Brain after Cerebral Ischemia Injury in Mice

doi:10.3969/j.issn.1673-5765.2022.06.009

Abstract

Abstract:

Objective To explore the effect of dual antiplatelet therapy with aspirin and clopidogrel (ASA+CPG) for 21 days on the neutrophil inflammatory factors in bone marrow and microglia polarization after cerebral ischemia injury in mice.

Methods Male C57BL/6 mice were randomly divided into control group and dual antiplatelet (ASA+CPG) group, 10 mice per group. A model of transient cerebral ischemia was constructed by occluding the distal middle cerebral artery (ischemia for 60 minutes, then restore to reperfusion). A combination of aspirin(12 mg/kg, 0.3 mg per mouse) and clopidogrel (12 mg/kg, 0.3 mg per mouse) or drinking water (100 μL) were administered by gavage at the onset of reperfusion, once a day and for 21 days. After 21 days, the mice were killed and neutrophils were isolated from bone marrow. The expression of neutrophil related inflammatory factors of IL-1β, IL-6, IL-10, inducible nitric-oxide synthase (iNOS), TNFα, arginase1 (Arg1), transforming growth factor β (TGFβ), CD206, CD32, chitinase-like 3 (YM1/Chil3) were detected by real time PCR. The number, morphology and polarization of microglia cells were observed by labelled CD16, CD206 and Iba1 using brain tissue immunofluorescence staining.

Results Compared with the control group, the level of IL-1β (0.703±0.124 vs. 1.000±0.203, P=0.019) decreased and the level of YM1 (2.173±0.968 vs. 1.000±0.282, P=0.019) and CD206 (1.361±0.203 vs. 1.000±0.260, P=0.048) increased in myeloid neutrophils in ASA+CPG group at 21 days after cerebral ischemia. There was no statistical difference in the number of microglial cells in peri-infarct area between the two groups at 21 days after cerebral ischemia; while the number of CD16 positive microglia (M1 type) cells (119.4±37.43 vs. 220.2±63.07, P<0.001) decreased, and the number of CD206 positive microglia (M2 type) cells (198.2±40.16 vs. 122.8±29.88, P<0.001) increased in ASA+CPG group at 21 days after cerebral ischemia. The microglia cells transformed into ramified microglia cells in ASA+CPG group.

Conclusions Combination treatment with ASA+CPG may protect against cerebral ischemia through inhibiting myeloid neutrophils inflammation, improving ramified microglia cells and activating microglia cells transforming into M2 anti-inflammatory type microglia cells.

Key words: Cerebral ischemia; Aspirin; Clopidogrel; Neutrophil; Microglia

摘要：

目的探讨小鼠脑缺血损伤后阿司匹林和氯吡格雷联合应用21 d对骨髓中性粒细胞炎症因子及脑内小胶质细胞极化的影响。

方法雄性C57BL/6小鼠随机分为对照组、阿司匹林联合氯吡格雷组（双抗组）2组，每组10只。通过结扎大脑中动脉远端制作脑缺血再灌注模型（缺血60 min，解开结扎实现再灌注），再灌注即刻通过灌胃给予饮用水100 μL（对照组）和阿司匹林（剂量12 mg/kg，每只实际用量0.3 mg）与氯吡格雷（剂量12 mg/kg，每只实际用量0.3 mg）混悬液100 μL（双抗组），每日1次，连续21 d。21 d后处死小鼠，分离骨髓中性粒细胞，通过实时荧光定量PCR检测骨髓中性粒细胞相关炎症因子，包括IL-1β、IL-6、IL-10、诱导型一氧化氮合酶（inducible nitric-oxide synthase，iNOS）、TNFα、精氨酸酶1（arginase1，Arg1）、转化生长因子β（transforming growth factor β，TGFβ）、CD206、CD32、几丁质酶样蛋白（chitinase-like3，Chil3/YM1）的表达情况。脑组织行冰冻切片，免疫荧光染色标记离子化钙结合适配分子1（ionizedcalcium binding adapter molecule 1，Iba1）、CD16和CD206，观察小胶质细胞数量、形态变化与极化状态。

结果与对照组相比，治疗21 d后双抗组小鼠骨髓中性粒细胞IL-1β的相对表达下降（0.703±0.124vs. 1.000±0.203，P =0.019）；YM1（2.173±0.968 vs. 1.000±0.282，P =0.019）和CD206（1.361±0.203vs. 1.000±0.260，P =0.048）的相对表达上调。治疗21 d后双抗组与对照组梗死周边区的小胶质细胞数量差异无统计学意义，但双抗组CD16阳性（M1型）小胶质细胞数量降低（119.4±37.43个/平方毫米vs. 220.2±63.07个/平方毫米，P <0.001），CD206阳性（M2型）小胶质细胞数量增加（198.2±40.16个/平方毫米 vs. 122.8±29.88个/平方毫米，P <0.001），且双抗组小胶质细胞形态上呈多分支网状结构。

结论阿司匹林与氯吡格雷联合应用可能通过抑制骨髓中性粒细胞炎症反应，增加脑内小胶质细胞分支，并激活小胶质细胞向抗炎表型（M2型）转化，从而发挥神经保护作用。

关键词: 缺血性脑损伤; 阿司匹林; 氯吡格雷; 中性粒细胞; 小胶质细胞

DONG Wen, JIANG Mingyu, CHEN Wentao, LIU Wenqian, CHEN Qingfang, ZHAO Shunying, YE Weizhen, WEN Shaohong, LIU Xiangrong. The Effect of Dual Antiplatelet Therapy with Aspirin and Clopidogrel on Inflammatory Response in Bone Marrow and Brain after Cerebral Ischemia Injury in Mice[J]. Chinese Journal of Stroke, 2022, 17(06): 605-610.

董雯, 姜鸣钰, 陈文涛, 刘文倩, 陈青芳, 赵顺英, 叶维贞, 温少红, 刘向荣. 阿司匹林和氯吡格雷联合应用对小鼠脑缺血损伤后骨髓及脑内炎症反应的影响[J]. 中国卒中杂志, 2022, 17(06): 605-610.

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