阿司匹林和氯吡格雷对体外血小板黏附内皮细胞基质活性的影响及其机制研究

Effects of Aspirin and Clopidogrel on the Adhesion Activity of Platelet to Endothelial Cell Matrix in Vitro and Its Mechanism

目的体外研究阿司匹林和氯吡格雷对血小板黏附内皮细胞基质活性的影响及其机制,从理论上寻找两种药物联合效果优于单药的原因。方法于2013年在第三军医大学动物中心购买健康雌性SD大鼠42只,采用随机数字表法,将SD大鼠分为阿司匹林组(8只)、氯吡格雷组(8只)、阿司匹林联合氯吡格雷组(联合组,8只)、对照组(8只)以及用于原代细胞分离培养10只。分离培养原代大鼠血管内皮细胞,利用50μg/ml氧化修饰低密度脂蛋白(ox-LDL)建立受损血管内皮细胞模型并采用100 mg/kg阿司匹林、10 mg/kg氯吡格雷、100 mg/kg阿司匹林联合10 mg/kg氯吡格雷以及200μl蓖麻油制作内皮细胞基质板;血浆血小板分离制备前3 d,阿司匹林组喂养阿司匹林100mg·kg-1·d-1,氯吡格雷组喂养氯吡格雷10 mg·kg-1·d-1,联合组喂养阿司匹林100 mg·kg-1·d-1,氯吡格雷10mg·kg-1·d-1,对照组喂养蓖麻油200μl/d。采用ELISA法检测黏附内皮细胞基质上的血小板数量;采用蛋白质免疫印迹法检测阿司匹林和氯吡格雷对血管内皮细胞蛋白血栓调节蛋白(TM)、氧化型低密度脂蛋白受体1(LOX-1)、CD40表达的影响。结果经阿司匹林和氯吡格雷处理的血小板对内皮细胞黏附活性的影响:4组50、100、150、200μl血小板黏附内皮细胞基质活性比较,差异均有统计学意义(P<0.05),其中氯吡格雷组和联合组50、100、150、200μl血小板黏附内皮细胞基质活性均低于对照组和阿司匹林组,阿司匹林组150μl血小板黏附内皮细胞基质活性低于对照组(P<0.05)。血小板黏附经阿司匹林和氯吡格雷处理的内皮细胞基质活性的影响:4组50、100、150、200μl血小板黏附内皮细胞基质活性比较,差异均有统计学意义(P<0.05),其中联合组50、100、150、200μl血小板黏附内皮细胞基质活性分别低于对照组、阿司匹林组和氯吡格雷组(P<0.05)。蛋白质免疫印迹法显示,阿司匹林和氯吡格雷均能抑制由ox-LDL引起的TM表达的减少。ox-LDL能明显诱导血管内皮细胞LOX-1表达,阿司匹林能明显抑制由ox-LDL引起的LOX-1和IL-6的表达;而氯吡格雷则能明显抑制内皮细胞CD40的表达。结论阿司匹林和氯吡格雷均能从上调TM和抑制炎性因子两方面降低血小板对内皮细胞基质的黏附作用。但两者联合更能抑制由ox-LDL引起的炎性因子的表达,降低血小板对内皮细胞基质的黏附。

Objective To study the effects of aspirin and clopidogrel on the adhesion activity of platelet to endothelial cell matrix in vitro and its mechanism, and to find the reasons why combination use of two drugs is better than monotherapy. Methods A total of 42 healthy female SD rats were bought from the animal centre of Third Military Medical University. By using random number table method,SD rats were divided into aspirin group（8 rats）,clopidogrel group（8 rats）,aspirin and clopidogrel（combination）group（8 rats）,control group（8 rats）,the other 10 rats were used for primary isolation and culture of cells. The isolated and cultured primary rat vascular endothelial cells were treated with ox - LDL to establish damaged vascular endothelial cell model. 100 mg/ kg aspirin,10 mg/ kg clopidogrel,combination of 100 mg/ kg aspirin and 10 mg/ kg clopidogrel,and 200 μl castor oil were used respectively to prepare endothelial cell matrix plate. 3 days before the separation and preparation of platelet from plasma,rats in aspirin group were fed with 100 mg＆#183;kg - 1 ＆#183;d - 1 aspirin,rats in clopidogrel group were fed with 10 mg＆#183;kg - 1 ＆#183;d - 1 clopidogrel,rats in aspirin and clopidogrel group were fed with 100 mg＆#183;kg - 1 ＆#183;d - 1 aspirin and 10 mg＆#183;kg - 1 ＆#183;d - 1 clopidogrel,rats in control group were fed with castor oil 200 μl per day. ELISA assay was used to evaluate the amount of platelets which adhered to the endothelial matrix. Western blotting technology was used to evaluate the effects of aspirin and clopidogrel on expression of thrombomodulin（TM）,lectin like oxidized low density lipoprotein receptor- 1（LOX - 1）and CD40 in vascular endothelial cells. Results The adhesion activity of aspirin/ clopidogrel - treated platelet to endothelial cell matrix：there were significant differences in adhesion activity of aspirin/ clopidogrel - treated（50,100,150, 200 μl）platelet to endothelial cell matrix among 4 groups（P ﹤ 0. 05）,adhesion activity of aspirin/ clopidogrel - treated platelet to endothelial cell matrix in clopidogrel group and combination group was significantly lower than that in control group and aspirin group,respectively,adhesion activity of aspirin/ clopidogrel - treated（150 μl） platelet to endothelial cell matrix in aspirin group was significantly lower than that in control group（P ﹤ 0. 05）. The adhesion activity of platelet to aspirin/ clopidogrel -treated endothelial cell matrix：there were significant differences in adhesion activity of platelet to aspirin/ clopidogrel - treated （50,100,150,200 μl） endothelial cell matrix among 4 groups（ P ﹤ 0. 05 ）,adhesion activity of platelet to aspirin/clopidogrel - treated（50,100,150,200 μl）endothelial cell matrix in combination group was significantly lower than that in control group,aspirin group,clopidogrel group,respectively（P ﹤ 0. 05）. According to western blotting results,both aspirin and clopidogrel could inhibit ox - LDL - induced decreasing expression of TM. Ox - LDL could induce LOX - 1 expression in vascular endothelial cells,aspirin could inhibit ox - LDL - induced expression of LOX - 1 and IL - 6 obviously,clopidogrel could inhibit CD40 expression in endothelial cells obviously. Conclusion Both aspirin and clopidogrel can reduce adhesion activity of platelet to endothelial cell matrix through increasing TM level and inhibiting inflammatory factors. The combination use of the two drugs can inhibit ox - LDL - induced expression of inflammatory cytokines obviously,and reduce the adhension of platelet to endothelial cell matrix.