人参二醇通过调节PI3K/Akt/GSK3β信号通路诱导血小板聚集参与止血过程

20-(S) Panoxadiol Effects PI3K/Akt/GSK3β Signaling Pathway to Induce Platelets Aggregation to Participate in Hemostatic Process

目的目的研究人参二醇(PD)的止血作用并探讨其作用机制。方法在体内研究中,采用小鼠断尾和肝划痕方法,观察PD对小鼠断尾出血时间和肝划痕出血时间的影响,应用血常规分析仪检测血常规;在体外研究中,采用血凝分析仪检测凝血四项;应用血小板聚集仪检测血小板聚集度;应用流式细胞仪检测人洗涤血小板中P-选择素(CD62P)和糖蛋白IIb/IIIa(PAC-1)表达;蛋白免疫印迹(Western blot)法检测人血小板的相关蛋白表达。结果与空白对照组比较,PD能明显降低小鼠断尾和肝划痕的出血时间;显著增加大鼠血浆中红细胞分布宽度标准差(RDW-SD)、红细胞分布宽度变异系数(RDW-CV)、血小板数量(PLT)、血小板压积(PCT)和大型血小板比率(P-LCR);PD能显著缩短大鼠血清的活化部分凝血活酶时间(APTT)和增加纤维蛋白原的含量(FIB);在血小板聚集的结果中,大鼠和人的血小板聚集率随着PD浓度的增加而显著增加,当PD浓度为140μM时,最大聚集率分别为51.40%和33.40%。PD激活血小板,促进CD62P的释放和增加糖蛋白Ⅱb/Ⅲa的表达。同时,PD能显著增加磷酸肌醇3激酶(Phosphoinositide3-kinase,PI3K)、蛋白酶B(Protein Kinase B,Akt)和糖原合酶激酶3β(glycogen synthase kinase 3 beta,GSK3β)磷酸化水平。结论PD具有止血作用,作用机制是通过激活内源性凝血途径和血小板的PI3K/Akt/GSK3β信号通路促进血小板聚集,形成血栓,参与止血过程。

Objective To investigate the hemostatic effect of panoxadiol(PD) and explore its mechanism.Methods In vivo experiments, the tail amputation model and liver scratch model experiments were used to observe the effect of PD on the bleeding time. Blood routine was investigated by blood analyzer. In vitro experiments, we used the coagulation analyzer detected coagulation parameters. The effect of PD on the rate of the washed platelet aggregation was observed by applying a platelet aggregometer. CD62P expression rate and PAC-1 binding rate were analyzed with a flow cytometer. Western blot analyzed the related proteins of human platelets.Results Compared with the control group, PD could significantly shorten the tail bleeding time and liver bleeding time of mice;PD significantly increased red blood cell distribution width-standard deviation(RDW-SD) and red blood cell distribution width-coefficient of variation(RDW-CV) in red blood cell parameters, and markedly increased platelet count(PLT), plateletcrit(PCT) and plateletlarger cell ratioin(P-LCR) in platelet parameters. In the high-and medium-dose group, PD significantly decreased APTT;different concentrations of PD could significantly increase the content of fibrinogen(FIB). With the increase of PD concentration, the rat/human platelet aggregation rate increased dramatically, when the dose of PD was augmented to 140 μM. The rat and human platelet aggregation rate was around 51.40% and 33.40%, respectively. PD could activate platelet to release the CD62P and increase the glycoprotein Ⅱb/Ⅲa(PAC-1) expression. The results of western blot showed that PD increased phosphorylation level of PI3K, Akt and GSK3β.Conclusion PD has a hemostatic effect. The mechanism of hemostatic effect of PD is related to activate the internal coagulation pathway and PI3K/Akt/GSK3β signaling pathway to induce platelets aggregation to form a blood clot to achieve hemostatic.