阿司匹林和氯吡格雷联合应用 对小鼠脑缺血恢复期RAGE及 sRAGE表达的影响

doi:10.3969/j.issn.1673-5765.2022.08.006

摘要/Abstract

摘要： 目的探索阿司匹林和氯吡格雷联合应用对小鼠大脑中动脉远端缺血再灌注模型恢复期晚期糖基化终末产物受体（receptor for advanced glycation end products，RAGE）及可溶性晚期糖基化终末产物受体（soluble receptor for advanced glycation end products，sRAGE）表达的影响及其机制。
方法 60只C57BL/6J雄性小鼠随机分为假手术组、溶剂组、阿司匹林和氯吡格雷联合组（双抗组），每组20只。通过压迫大脑中动脉远端制作脑缺血再灌注模型（缺血60 min再灌注），再灌注即刻灌胃，溶剂组给予饮用水100 μL，双抗组给予阿司匹林（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，每日1次，连续21 d。缺血1、3、5、7、9、11、14、21 d对小鼠进行神经功能评价。缺血21 d取材，酶联免疫吸附法检测小鼠血清sRAGE表达水平；免疫印记检测脑组织RAGE表达水平；实时荧光定量PCR检测CD16、CD32、CD11b、诱导型一氧化氮合成酶（inducible nitric-oxide synthase，iNOS）、CD206、精氨酸酶1（arginase1，Arg1）、转化生长因子β（transforming growth factor β，TGF-β）、几丁质酶样蛋白（chitinase-like 3，Chil3/Ym1/2）等炎症因子及RAGE的mRNA表达情况；免疫荧光染色标记RAGE、神经元特异核蛋白（neuronal nuclei，NeuN）、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP），观察RAGE与神经元和星形胶质细胞共定位情况。
结果双抗组小鼠3 d时胡须碰触评分和神经功能总分、9 d时神经功能总分优于溶剂组，差异有统计学意义（P=0.0067、0.0140、0.0406）。缺血再灌注21 d时，双抗组小鼠血清sRAGE表达水平高于溶剂组（1.099±0.541 ng/mL vs. 0.319±0.341 ng/mL，P=0.0120）；脑组织iNOS（1.250±0.318 vs. 1.843±0.301，P=0.0164）及RAGE（2.105±0.300 vs. 2.732±0.249，P=0.0071）的mRNA相对表达较溶剂组下降。在小鼠脑梗死周边区RAGE与神经元具有共定位，与星形胶质细胞无共定位；双抗组RAGE+NeuN+细胞数量少于溶剂组（328.798±35.183个/平方毫米 vs. 814.437±165.758个/平方毫米，P=0.0012）。
结论阿司匹林和氯吡格雷联合应用可能通过下调RAGE、上调sRAGE的表达水平，减轻炎症反应，从而发挥脑保护作用。

文章导读： 阿司匹林和氯吡格雷联合应用可在小鼠脑缺血恢复期调控RAGE表达，减轻炎症，发挥脑保护作用。

关键词: 阿司匹林; 氯吡格雷; 缺血再灌注; 晚期糖基化终末产物受体; 可溶性晚期糖基化终末产物受体

Abstract:

Objective To explore the effects and mechanisms of aspirin combined with clopidogrel (ASA+CPG) on the expression of receptor for advanced glycation end products (RAGE) and soluble receptor for advanced glycation end products (sRAGE) in the convalescent stage of distal middle cerebral artery occlusion-reperfusion model in mice.
Methods 60 male C57BL/6J mice were randomly divided into sham operation group, vehicle group and ASA+CPG group, 20 mice per group. A model of transient cerebral ischemia was performed by occluding the distal middle cerebral artery (ischemia for 60 minutes, then restore to reperfusion). 100 μL suspension of aspirin (12 mg/kg, 0.3 mg per mouse), clopidogrel (12 mg/kg, 0.3 mg per mouse) and water were administered by gavage at the onset of reperfusion, once a day for 21 days. The neurological function of mice was evaluated on day 1, 3, 5, 7, 9, 11, 14 and 21 after ischemia. Samples were harvested 21 days after ischemia. The serum sRAGE expression level was detected by enzyme-linked immunosorbent assay (ELISA). Immunoblotting was used to evaluate RAGE expression in brain tissues. Real-time quantitative PCR was applied to detect the mRNA expression levels of CD16, CD32, CD11b, inducible nitric oxide synthase (iNOS), CD206, arginase 1 (Arg1), transforming growth factor β (TGF-β), chitinase-like 3 (Chil3/Ym1/2) and RAGE. RAGE, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) were labelled by immunofluence to observe the colocalization of RAGE with neurons and astrocytes.
Results The vibrissae touch score at day 3, neurological function total score at day 3 and day 9 in ASA+CPG group were better than those in vehicle group (P=0.0067, 0.0140, 0.0406). At 21 days after ischemia, the level of serum sRAGE in ASA+CPG group was higher than that in vehicle group (1.099±0.541 ng/mL vs. 0.319±0.341 ng/mL, P=0.0120). The mRNA expression levels of iNOS (1.250±0.318 vs. 1.843±0.301, P=0.0164) and RAGE (2.105±0.300 vs. 2.732±0.249, P=0.0071) in ASA+CPG group decreased compared with vehicle group. RAGE colocalized with neurons but not with astrocytes in peri-infarct area of ischemic mice. The number of RAGE+NeuN+ cells in ASA+CPG group was less than that in vehicle group (328.798±35.183 vs. 814.437±165.758 per square millimeter, P=0.0012).
Conclusions Combined treatment with aspirin and clopidogrel plays cerebral ischemia protective effect by down-regulate RAGE and up-regulate sRAGE expression to alleviate inflammatory response.

Key words: Aspirin; Clopidogrel; Ischemia-reperfusion; RAGE; sRAGE

温少红, 刘向荣, 赵顺英, 董雯, 陈青芳, 陈文涛, 李子孝, 王春娟. 阿司匹林和氯吡格雷联合应用对小鼠脑缺血恢复期RAGE及 sRAGE表达的影响[J]. 中国卒中杂志, 2022, 17(08): 821-828.

WEN Shaohong, LIU Xiangrong, ZHAO Shunying, DONG Wen, CHEN Qingfang, CHEN Wentao, LI Zixiao, WANG Chunjuan. Effects of Aspirin Combined with Clopidogrel on the Expression of RAGE and sRAGE during the Convalescence of Cerebral Ischemia in Mice[J]. Chinese Journal of Stroke, 2022, 17(08): 821-828.

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