蔓荆子黄素通过磷脂酰肌醇-3激酶/蛋白激酶B信号通路抗血小板活化的研究

Study on antiplatelet activation of vitexicarpin via phosphatidylinositol-3 kinase protein kinase B signaling pathway

目的蔓荆子黄素对血小板活化的作用及机制探讨。方法采集健康志愿者肘静脉血并制备洗涤血小板,分为空白组(等量Tyrode's缓冲液),对照组(1‰二甲基亚砜),低、高剂量实验组(1,2μmol·L^(-1)蔓荆子黄素),阿司匹林组(1 mmol·L^(-1))或替罗非班组(0.5μg·mL^(-1)),各组均在37℃孵育10 min。用聚集仪检测不同激动药诱导的血小板聚集,用流式细胞仪检测血小板表面P-选择素表达和血小板激活复合物-1(platelet-activating compound-1,PAC-1)活化,用蛋白质印迹法检测血小板磷酸化磷脂酰肌醇-3激酶(phosphorylate-phosphatidylinositol-3 kinase,p-PI3K)/PI3K、磷酸化蛋白激酶B(phosphorylate-protein kinase B,p-Akt)/Akt、磷酸化糖原合成酶激酶-3(phosphorylate-glycogen synthase kinase-3β,p-GSK3β)/GSK3β蛋白的表达水平。结果低、高剂量实验组和对照组、阿司匹林组对胶原诱导的血小板聚集率分别为(62.67±2.03)%,(1.33±0.88)%,(69.33±0.67)%和(0.33±0.33)%,p-PI3K/PI3K蛋白表达量分别为0.93±0.49,0.48±0.19,1.14±0.02和0.50±0.21,p-Akt/Akt蛋白表达量分别为0.94±0.01,0.53±0.35,1.15±0.03和0.45±0.24,p-GSK3β/GSK3β蛋白表达量分别为0.98±0.03,0.50±0.02,1.19±0.06和0.42±0.03。低、高剂量实验组和对照组、替罗非班组在血小板表面PAC-1活化率分别为(65.83±1.61)%,(50.53±2.11)%,(80.90±0.49)%和(5.20±0.46)%,P-选择素表达率分别为(30.10±1.78)%,(23.80±0.95)%,(46.60±1.31)%和(19.67±0.87)%。低、高剂量实验组的上述指标与对照组比较,差异均有统计学意义(P<0.05,P<0.01)。结论蔓荆子黄素抑制血小板活化,可能是通过抑制PI3K/Akt信号通路实现的。

Objective To investigate the effect and mechanism of vitexicarpin on platelet actvation.Methods Blood was collected from the cubital vein of healthy volunteers and washed platelets were prepared.They were divided into blank group(equivalent Tyrode’s buffer),control group(1‰dimethyl sulfoxide),experimental-L,-H groups(1,2μmol·L^(-1)vitexicarpin)and aspirin group(1 mmol·L^(-1))or tirofiban group(0.5μg·mL^(-1)),each group was incubated at 37°C for 10 min.Detection of platelet aggregation induced by different agonists using aggregometer.Flow cytometry was used to detect the expression of P-selectin and activation of platelet-activating compound-1(PAC-1)on platelet surface.Detecting the platelet phosphorylate-phosphatidylinositol-3 kinase(p-PI3K)/PI3K,phosphorylate-protein kinase B(p-Akt)/Akt and phosphorylate-glycogen synthase kinase-3β(p-GSK3β)/GSK3βprotein expression by Western blotting.Results The platelet aggregation rates induced by collagen in experimental-L,-H groups,control group and aspirin group were(62.67±2.03)%,(1.33±0.88)%,(69.33±0.67)%and(0.33±0.33)%;the protein expression levels of p-PI3K/PI3K were 0.93±0.49,0.48±0.19,1.14±0.02 and 0.50±0.21;the protein expression levels of p-Akt/Akt were 0.94±0.01,0.53±0.35,1.15±0.03 and 0.45±0.24;p-GSK3β/GSK3βprotein expression levels were 0.98±0.03,0.50±0.02,1.19±0.06 and 0.42±0.03,respectively.The activation rates of PAC-1 on the platelet surface of experimental-L,-H groups,control group and tirofiban group were(65.83±1.61)%,(50.53±2.11)%,(80.90±0.49)%and(5.20±0.46)%;the expression rates of P-selectin were(30.10±1.78)%,(23.80±0.95)%,(46.60±1.31)%and(19.67±0.87)%.Experimental-L and experimental-H groups were compared with the control group and the results were significantly different in above results(P<0.05,P<0.01).Conclusion Vitexicarpin inhibited platelet activation was achieved through the inhibition of PI3K/Akt signaling pathway possibly.