四氢姜黄素经PI3K/Akt信号通路对人血小板活化和聚集的调控作用

目的：探讨姜黄素的主要肠道代谢物四氢姜黄素（tetrahydrocurcumin，THC）对血小板活化和聚集的影响及其可能的分子机制。方法：在体外实验中，用不同浓度的THC（0、0.5、1、10 μmol/L）提前与健康人纯化血小板共同孵育40 min，然后加入凝血酶激活血小板2 min，用流式细胞术测定血小板表面CD62P和CD63的表达量，用酶联免疫吸附法测定血小板释放血小板因子-4（platelet factor-4，PF4）和趋化因子配体-5（chemokine ligand 5，CCL5）水平，用血小板聚集仪检测血小板释放ATP水平和血小板最大聚集率，用Western blot蛋白免疫印迹法检测血小板磷酸肌醇-3-激酶（phosphoinositide 3-kinase，PI3K）和Akt蛋白的磷酸化水平。结果：与模型组（血小板悬液中加入0.05%二甲基亚砜）相比，THC能抑制凝血酶诱导的血小板表面CD62P和CD63的表达，抑制PF4、CCL5和ATP的释放，降低血小板最大聚集率，下调PI3K和Akt蛋白的磷酸化水平，且呈浓度依赖效应，其中10 μmol/L的浓度下作用效果显著（P＜0.01、P＜0.001）。PI3K的特异性激动剂740 Y-P可部分逆转THC对PF4和CCL5释放和血小板聚集的抑制作用（P＜0.05、P＜0.01）。结论：THC具有显著抑制血小板活化和聚集的作用，其机制可能是THC可下调PI3K/Akt介导的信号通路。

Regulatory Effects of Tetrahydrocurcumin on Human Platelet Activation and Aggregation through PI3K/Akt Signalling Pathway

Objective: The aim of the current study was to investigate the effect of tetrahydrocurcumin (THC), the major intestinal metabolite of curcumin, on human platelet activation and aggregation, as well as the possible mechanisms. Methods: Human gel-filtered platelets were pre-incubated with various concentrations of THC (0, 0.5, 1 and 10 μmol/L) for 40 minutes in vitro, followed by activation with thrombin for two minutes. Platelet surface expression of CD62P and CD63 were determined by flow cytometry. Enzyme linked immunosorbent assay (ELISA) was used to measure the levels of secretion of platelet factor-4 (PF4) and chemokine ligand 5 (CCL5). The levels of ATP released from platelets and platelet aggregation were detected in an aggregometer. Moreover, the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt were measured by Western blot. Results: Compared with the control group (with 0.05% of dimethyl sulfoxide (DMSO)), THC inhibited thrombin-induced platelet surface expression of CD62P and CD63 and the release of PF4, CCL5 and ATP, reduced the maximum platelet aggregation rate, and down-regulated the phosphorylation levels of PI3K and Akt in a concentration-dependent manner, more obvious effect being observed at 10 μmol/L (P < 0.01, P < 0.001). A specific agonist of PI3K, 740 Y-P, could partially reverse the inhibitory effect of THC on the release of PF4 and CCL5 and platelet aggregation (P < 0.05, P < 0.01). Conclusion: THC attenuates platelet activation and aggregation possibly through inhibiting PI3K/Akt signaling pathway.