普伐他汀和CRP对ADP诱导的血小板凝血酶受体PAR-1表达的调节

Modulation of PAR-1 expression by pravastatin and C reactive protein in vitro blood platelets

目的探讨普伐他汀对二磷酸腺苷(ADP)诱导的血小板PAR-1表达的影响及机制。方法体外分离富血小板血浆,分别给予C反应蛋白(CRP)、普伐他汀干预和ADP刺激进行体外研究。试验分组分别为:对照组,单纯ADP组,低浓度普代他汀+ADP组,高浓度普伐他汀组+ADP组,CRP组,普伐他汀+CRP联合组。采用流式细技术检测PAR-1和LOX-1平均荧光强度(MFI)。采用酶联免疫试验检测TXB2和F1+2水平。结果 5μmol/L ADP刺激能促使血小板PAR-1表达增加35%。50μg/mL CRP显著降低ADP诱导的血小板PAR-1的表达(P<0.01)。1μmol/L、10μmol/L普伐他汀均显著降低ADP诱导的血小板PAR-1的表达(P<0.01)。联合应用CRP和普伐他汀更能降低ADP诱导的血小板PAR-1表达,较单独使用CRP或普伐他汀降低更显著(P<0.05)。单纯ADP刺激后TXB_2较基础时明显增高(P<0.01),50μg/mL CRP、10μmol/L普伐他汀干预后ADP刺激的TXB_2分别下降为(112.68±24.48)pg/mL、(146.48±46.54)pg/mL,与单纯ADP刺激比较,差异均有统计学意义(P<0.01)。50μg/mL CRP显著增加ADP诱导的F1+2水平(P<0.01),10μmol/L普伐他汀对ADP诱导F1+2的生成无明显影响。普伐他汀呈浓度依赖性的方式降低ADP诱导的血小板LOX-1表达(1μmol/L和10μmol/L普伐他汀处理后MFI分别为:1.80±0.19和1.62±0.16),与单纯ADP刺激后LOX-1表达(MFI:3.16±0.23)比较,差异有统计学意义(P<0.01)。50μg/mL CRP对ADP刺激的血小板LOX-1表达无明显影响。结论 PAR-1在ADP诱导的血小板活化中起重要作用,普伐他汀和CRP通过不同机制明显降低ADP诱导的血小板PAR-1的表达,提示在炎症状态下他汀仍能起着重要的抗血栓作用。

Objective To study the modulation of protease-activated receptor-1（PAR-1） expression by pravastatin and C reactive protein（CRP）in vitro blood platelets.Methods Platelet-rich plasma（PRP）was isolated from peripheral blood,PRP were treated with CRP,pravastatin and ADP stimulation in vitro study.Experimental groups：blank control group,simple ADP stimulated group,low concentration of pravastatin＋ADP group,high concentration pravastatin group＋ADP group,CRP group,pravastatin＋CRP united group.PAR-1 and LOX-1 expression on platelets were detected by flow cytometry,the result were shown by mean fluorescence intensity（MFI）.TXB2 and F1＋2 levels were detected by enzyme-linked immunosorbent assay.Results The 5μmol/L ADP stimulation significantly increased PAR-1expression on platelets by 35%.The 50μg/mL CRP significantly reduced platelet PAR-1 expression induced by ADP（P〈0.01）.1,10μmol/L pravastatin significantly reduced platelet PAR-1expression induced by ADP（P〈0.01）.Platelet PAR-1 expression induced by ADP was further reduction by combination treatment of CRP,which were significantly reduced compared with treatment of CRP or pravastatin alone（P〈0.05）.Simple ADP stimulation significantly increased TXB2 level（P〈0.01）.50μg/mL CRP and 10μmol/L pravastatin respectively reduced TXB2 level treated by ADP to（112.68±24.48）pg/mL and（146.48±46.54）pg/mL.Both were reduced significantly compared with ADP stimulation alone（P〈0.01）.The 50μg/mL CRP significantly increased level of prothrombin fragment 1＋2 induced by ADP（P〈0.01）,10μmol/L pravastatin,in contrast,did not influence F1＋2 level.Pravastatin reduced platelet LOX-1 expression induced by ADP in a concentration dependent manner,MFI of LOX-1on platelets treated by 1μmol/L and 10μmol/L pravastatin were 1.80±0.19 and 1.62±0.16 respectively,both were reduced significantly compared with that treated by ADP alone（MFI：3.16±0.23）,P〈0.01.The 50μg/mL CRP had no significant effect on the expression of LOX-1 stimulated by ADP.Conclusion PAR-1 served as a critical mechanism to relay the platelet activation process induced by ADP.CRP and pravastatin reduced PAR-1 expression in platelet induced by ADP in different way.It is suggested that statins can still play an important role in the antithrombotic effect in the inflammatory state.