鸦胆子油对血小板聚集的抑制作用及其机制的体外研究

目的 以人洗涤血小板为模型,体外观察鸦胆子油对血小板聚集的抑制作用,并探讨其可能机制.方法 将不同浓度鸦胆子油与人洗涤血小板作用,分为4组:空白组(加丙酮)、阴性对照组(加丙酮,0%),9.0%鸦胆子油组及22.5%鸦胆子油组.以血小板聚集分析仪检测ADP、AA、胶原和凝血酶诱导的血小板最大聚集率、以流式细胞仪检测活化血小板膜FIB-R和P-选择素表达水平,以SDS-PAGE分析血小板肌动蛋白聚合体的变化.结果 9.0%鸦胆子油组ADP、AA、胶原和凝血酶诱导的血小板聚集率分别为(57.7±4.0)%、(62.2±3.9)%、(66.9±5.0)%和(71.8±5.1)%,均低于阴性对照(0%)组的(75.3±4.1)%、(79.3±4.8)%、(80.6±5.4)%和(84.1±6.2)%,差异均有统计学意义(t值分别为7.312、8.785、9.123和9.466,P均<0.01);22.5%组分别为(39.2±3.5)%、(45.8±3.4)%、(51.2±3.9)%和(56.7±4.8)%,显著低于9.0%组,差异有统计学意义(t值分别为9.123、10.221、9.533和10.243,P均<0.01).22.5%鸦胆子油组对血小板聚集的抑制率分别为47.9%、42.2%、36.5%和32.6%,显著高于9.0%组的23.4%、21.6%、17.0%和14.6%,差异有统计学意义(χ~2值分别为13.08、9.77、9.70和8.93,P均<0.01).血小板聚集水平与鸦胆子油浓度呈负相关(r=-0.952,-0.961,-0.970和-0.975,P<0.01).9.0%鸦胆子油组ADP诱导的血小板FIB-R表达水平和P-选择素表达水平分别为(64.7±4.0)%、(3.91±0.21)%,均低于阴性对照组的(85.5±4.6)%和(5.05±0.27)%,差异有统计学意义(t值分别为11.35、7.81,P均<0.01);22.5%鸦胆子油组分别为(36.2±3.9)%和(2.34±0.15)%,低于9.0%组,差异有统计学意义(t值分别为14.35、14.80,P均<0.01);22.5%鸦胆子油组对FIB-R的抑制率为57.7%,高于9.0%组的24.3%,差异有统计学意义(χ~2=23.05,P<0.01).9.0%和22.5%鸦胆子油组与空白组在相对分子质量43 000为肌动蛋白区带的吸光度值之比分别为1.77±0.12和1.68±0.10,明显低于阴性对照组与空白组的比值2.22±0.15,差异有统计学意义(t=7.21、8.14,P值均<0.01);22.5%胆鸦子油组为1.68±0.10,与9.0%组间差异无统计学意义(t=2.77,P>0.05).结论 鸦胆子油通过抑制活化血小板膜纤维蛋白原受体的表达,并抑制肌动蛋白聚合以及释放反应,从而剂量依赖性地抑制血小板的聚集反应,可能有抗血栓的功效.

The in vitro inhibitory effects of brucea javanica oil on platelet aggregation and the related mechanism

Objective To observe the in vitro inhibitory effects of brucea javanica oil on human washed platelet and explore the possible mechanism.Methods Human washed platelets were mixed withdifferent concentration of brucea javanica oil which were divided into four groups[untreated control group,negative control group,9.0% of brucea javanica oil group,and 22.5%of brueea javanica oil group].The maximal ratio of platelet aggregation induced by adenosine liphosphate(ADP),arachidonic acid(AA),collagen,and thrombin,respectively,was measured with platelet aggregation analyzer.The expressions of fibrinogen receptor(FIB-R)and P-selectin(CD_(62p))on external membrane of activated platelet were determinated with flow cytometry.The F-actin of cytoskeletal structure in activated platelet was detected by SDS-PAGE.Results At 9.0% of brucea javanica oil,the maximal ratio of platelet aggregation[(57.7±4.0)%,(62.2±3.9)%,(66.9±5.0)%and(71.8±5.1)%]induced by ADP,AA,collagen,and thrombin,was significantly lower than that[(75.3±4.1)%,(79.3±4.8)%,(80.6±5.4)%,(84.1±6.2)%]at negative control(0% of brucea javanica oil)(P<0.01),but makedly higher than that[(39.2±3.5)%,(45.8±3.4)%,(51.2±3.9)%and(56.7±4.8%)]at 22.5%,respectively(P<0.01).The inhibitory rate of platelet aggregation(47.9%,42.2%,36.5%and 32.6%)at 22.5%of brucea javanica oil was notably higher than that(23.4%,21.6%,17.0%and 14.6%)at 9.0%,respectively(P<0.001).There was a negative correlation between brucea javanica oil concentration and the aggregation ratio of platelet stimulated by the four agonists,respectively(r=-0.952,-0.961,-0.970,-0.975,P<0.001).At 9.0% of brucea javanica oil,the expression levels of FIB-R[(64.7±4.0)%]and CD_(62p) [(3.91±0.21)%] of platelet activated by ADP were significantly lower than that[(85.5±4.6)%and (5.05±0.27)%]at negative control,but remarkably higher than that[(36.2±3.9)%and(2.34±0.15)%]at 22.5%,respectively(P<0.01).There was a much higher inhibitory rate of platelet aggregation(57.7%)at 22.5%than that(24.3%)at 9.0%(P<0.01).The ratios(1.68±0.10 and 1.77±0.12)of F-actin photodensity at 22.5%and 9.0%to that in blank control were significantly lower than that(2.22±0.15)at negative control(P<0.01)but there was no statistical difference between the ratios in the group of 9.0%and 22.5%brucea javanica(P>0.05).Conclusions brucea javanica oil has special inhibitory effect on activated platelet and thrombosis in a dose-dependent manner.The mechanism is to inhibit the expression of fibrinogen receptor on external membrane of activated platelet,which is also related to the inhibition of F-actin and secretion of platelet.