大鼠模型中糖尿病导致氯吡格雷治疗后血小板高反应性的机制研究

目的在大鼠模型中探讨糖尿病对氯吡格雷治疗后血小板高反应性(HTPR)的影响及其可能的影响机制。方法造模成功的雄性SD大鼠45只,随机分为空白组11只、氯吡格雷组11只、糖尿病组11只和糖尿病+氯吡格雷组(实验组)12只,普通饲料饲养8周,采用灌胃法给予氯吡格雷,糖尿病模型采用链脲佐菌素(STZ)一次性注射法建立。流式细胞术检测CD62P水平和细胞质内Ca2+水平,ELISA法检测同型半胱氨酸(Hcy)、高敏C反应蛋白(hs-CRP)、超氧化物歧化酶(SOD)、丙二醛(MDA)、血栓素A2(TXA2)、前列环素(PGI2)及NO等水平,RT-PCR和Westernblot检测细胞色素450(CYP450)、蛋白激酶C(PKC)及P2Y12受体基因和蛋白表达。结果糖尿病组和实验组血糖、Hcy、MDA、TXA2、hs-CRP水平、P2Y12基因和蛋白及PKC蛋白表达显著高于空白组和氯吡格雷组(P<0.01);PGI2、SOD及NO、ADP诱导的血小板聚集抑制率(ADP-IR)、CYP450蛋白表达显著低于空白组和氯吡格雷组(P<0.01)。实验组ADP-IR显著低于糖尿病组(45.64±13.31)%vs(80.14±4.30)%,P<0.01]。糖尿病组CD62P、细胞质内Ca2+水平明显高于其他组(P<0.01);且实验组明显高于空白组和氯吡格雷组(P<0.05,P<0.01);氯吡格雷组细胞质内Ca2+水平显著低于空白组(P<0.05)。各组PKC和CYP450基因表达比较,差异无统计学意义(P>0.05)。ADP-IR与SOD和TXA2水平呈负相关(P<0.05,P<0.01);CD62P水平与细胞质内Ca2+水平呈正相关(P<0.01)。ADP-IR与CD62P水平呈负相关(r=-0.3567,P=0.015)。结论糖尿病导致氯吡格雷HTPR的机制为血小板功能异常、血小板表面受体表达上调及氯吡格雷活化相关酶系表达减少。

Mechanism of DM-induced high platelet reactivity to clopidogrel treatment in a rat model

Objective To study the effect of DM on high platelet reactivity to clopidogrel treatment in a rat model and its mechanism.Methods Forty-five male SD rats were randomly divided into blank control group(n=11),clopidogrel treatment group(n=11),DM group(n=11),DM+clopidogrel treatment group(n=12).The animals were fed on a common forage for 8 weeks and treated with clopidogrel by gastric gavage.A DM model was established by injecting streptozotocin.The serum levels of CD62P and intracytoplasmic calcium were measured by flow cytometry and those of Hcy,hs-CRP,SOD,MDA,TXA2,PGI2 and NO were measured by ELISA.Expressions of CYP450,P2Y12 and PKC were detectedd by RT-PCR and Western blot respectively.Results The serum levels of blood glucose,Hcy,MDA,TXA2,hs-CRP and the expression levels of P2Y12 and PKC were significantly higher while the serum levels of HDL-C,PGI2,SOD,NO,the ADP-induced platelet aggregation inhibition rate and the CYP450 expression level were significantly lower in DM group and DM+clopidogrel treatment group than in blank control group and clopidogrel treatment group(P<0.01).The ADP-induced platelet aggregation inhibition rate was significantly lower in DM+clopidogrel treatment group than in DM group(45.64%±13.31%vs80.14%±4.30%,P<0.01).The serum CD62P and intracytoplasmic calcium levels were significantly higher in DM group than in blank control group,clopidogrel treatment group and DM+clopidogrel treatment group(P<0.01)and in DM+clopidogrel treatment group than in blank control group and clopidogrel treatment group(P<0.05,P<0.01).The serum intracytoplasmic calcium level was significantly lower in clopidogrel treatment group than in blank control group(P<0.05).The ADP-induced platelet aggregation inhibition rate was negatively related with the serum SOD and TXA2 levels(P<0.05,P<0.01)while the serum CD62P level was positively related with the serum intracytoplasmic calcium level(P<0.01).Linear logistic regression analysis showed that the ADP-induced platelet aggregation inhibition rate was negatively related the serum CD62P level(r=-0.3567,P=0.015).Conclusion Platelet dysfunction,upregulated expression of platelet surface receptor,and downregulated expression of clopidogrel-activated enzymes are the mechanisms of DM-induced high platelet reactivity to clopidogrel treatment.