葡萄籽原花青素体外抗血小板聚集的机制研究

目的: 研究葡萄籽原花青素(PC)体外抗血小板聚集的可能机制。方法: 应用血小板聚集仪研究PC、二亚苯基碘鎓(DPI,非特异性NADPH氧化抑制剂)和夹竹桃麻素(apocynin,特异性NADPH氧化酶抑制剂)对胶原诱导的健康志愿者血小板最大聚集率的影响。用化学发光仪检测PC对血小板NADPH氧化酶活性、一氧化氮(NO)含量和超氧阴离子(O₂^-·)水平的影响。用流式细胞术观察PC对血小板活化标志物PAC-1和CD62P表达率的影响。结果: PC(100 μmol/L)、apocynin(10 μmol/L)和DPI(100 μmol/L )均可显著抑制胶原蛋白诱导的血小板最大聚集率(P<0.01)。胶原蛋白激活的血小板NO含量显著降低,O₂^-·含量显著增加,100 μmol/L PC可使二者明显恢复(P<0.05)。添加了100 μmol/L PC的样本中,血小板NADPH氧化酶活性受到明显的抑制(P<0.01),血小板PAC-1 和CD62P表达率也显著降低(P<0.05)。结论: 葡萄籽原花青素可能通过抑制NADPH氧化酶的活性,进而影响血小板NO和O₂^-·含量,在一定程度上阻断血小板活化过程,最终达到抗血小板聚集的作用。

In vitro anti-platelet aggregative effect of procyanidins isolated from grape seeds

AIM: To study the possible anti-platelet aggregative mechanisms of procyanidins(PC) isolated from grape seeds in vitro.METHODS: Platelet-rich plasma(PRP) and platelet-poor plasma(PPP) were prepared from the blood of healthy volunteers.PC,diphenylene iodonium(DPI,a nonspecific NADPH oxidase inhibitor) and apocynin(a specific NADPH oxidase inhibitor) were used to observe the effects on collagen-induced platelet maximum aggregation rate using platelet aggregometer.The influences of PC on platelet NADPH oxidase activity,NO content and superoxide anion() level were evaluated by chemiluminescence spectrometer.The role of PC in the expression of activated platelet markers(PAC-1 and CD62P) was observed by flow cytometry.RESULTS: PC(100 μmol/L),apocynin(10 μmol/L) and DPI(100 μmol/L) significantly inhibited collagen-induced maximum platelet aggregation rate(P<0.01).In collagen-activated platelets,NO content reduced and  level increased,both of which were recovered by PC at concentration of 100 μmol/L(P<0.05).PC also obviously inhibited NADPH oxidase activity(P<0.01),and significantly down-regulated PAC-1 and CD62P expression(P< 0.05) in platelets.CONCLUSION: Procyanidins isolated from grape seeds have the anti-platelet aggregation function through inhibiting NADPH oxidase activity,further influencing platelet NO and O-·2 levels.