银杏内酯B抑制血小板活化研究

Inhibitional effect of ginkgolide B on platelet activation

目的探讨银杏内酯B在动物模型中的抗凝作用及对内皮型一氧化氮合酶(e NOS)表达的影响。方法选择大鼠、小鼠和家兔为本次研究动物,均随机分为银杏内酯B高、中、低剂量组(各组剂量为:大鼠高剂量组60 mg/kg、中剂量组30 mg/kg、低剂量组15 mg/kg;小鼠高剂量组120 mg/kg、中剂量组60 mg/kg、低剂量组30 mg/kg;家兔高剂量32.0 mg/kg、中剂量16 mg/kg、低剂量8 mg/kg)、溶剂对照组和阳性对照阿司匹林组,连续灌胃5 d后,分别测定大鼠血流旁路血栓形成抑制率、小鼠凝血时间和家兔载体血小板聚集率。Western blot检测大鼠血小板和内皮细胞中所含的e NOS蛋白和Ser1177位磷酸化表达及NO含量。结果银杏内酯B高、中剂量均有明显抑制大鼠颈总动脉-颈外静脉血流旁路血栓的形成(q分别=9.87、4.56;P均<0.05),银杏内酯B高、中、低剂量延长小鼠凝血时间(q分别=32.02、13.58、3.98,P均<0.05);银杏内酯B各组均对血小板活化因子(PAF)、二磷酸腺苷(ADP)、血小板花生四烯酸(AA)诱导的家兔血小板聚集率有明显的抑制作用(q分别=17.26、9.46、4.91、14.67、10.18、4.91、9.51、7.01、4.44,P均<0.05);经银杏内酯B高、中剂量给药处理后,血小板中的e NOS总蛋白含量和磷酸化水平均有明显增加(q分别=19.54、9.08、28.41、11.86,P均<0.05);银杏内酯B120 mg/kg处理5 d后大鼠的循环血中NO的含量高于溶剂对照组(q=11.24,P<0.05);银杏内酯B 120μg/ml和60μg/ml处理48 h后的培养基中NO的含量高于溶剂对照组(q分别=8.36、15.68,P均<0.05)。结论银杏内酯B在大鼠、小鼠和家兔的动物模型中均表现出良好的抑制凝血的作用,银杏内酯B处理内皮细胞能激活e NOS信号通路,表明其抗凝机制与激活血小板中e NOS、增加NO释放量有关。

Objective To investigate the anticoagulant effect of ginkgolide B （GB） in animal models and the effect on endothelial nitric oxide synthase （eNOS）. Methods Three species （mice, rats and rabbits） were randomly divided into three groups treated with high, medium and low dosage of GB. The high dosage was 60 mg/kg in mice, 120 mg/kg in rat and 32 mg/kg in rabbit, the medium dosage was 30 mg/kg in mice, 60 mg/kg in rat and 16 mg/kg in rabbit, the low dosage was 15 mg/kg in mice, 30 mg/kg in rat and 8 mg/kg in rabbit. At meanwhile, control group and aspirin group were set up. After five days of continuous oral administration, suppression rate of venous blood flow bypass thrombosis in mice, clotting time in rat and platelet aggregation in rabbit were measured. The eNOS content and the phosphorylation level with- in platelet and endothelial cells were measured with western blot. Results High and medium dosages of GB significantly inhibited the formation of carotid artery-external carotid venous blood flow bypass in mice（q=9.87, 4.56, P〈0.05）. High, medium and low dosages of GB significantly prolong the clotting time in rat （q=32.02, 13.58, 3.98, P〈0.05）. High, medium and low dosages of GB significantly inhibited the platelet aggregation induced by PAF, ADP, AA in rabbit （q= 17.26, 9.46, 4.91, 14.67, 10.18, 4.91, 9.51, 7.01, 9.51,P〈0.05）. After given High and medium dosages of GB, the GB high, the eNOS protein content and the phosphorylation level of platelet were significantly increased （q=19.54, 9.08, 28.41, 9.08,P〈0.05）. After given 120 mg/kg GB for 5 days, the NO in blood of mice was significantly higher than control group （q=11.24, P〈0.05）. After given 120 μg/ml and 60μg/ml GB treatment for 48 hours, the NO in endothelial cells of mice was significantly higher than control group（q=8.36, 15.68, P〈0.05）. Conclusion The of GB had good anticoagula-tion effect on animal models of mice, rat and rabbit. The eNOS signaling pathway can be activated by GB treatment which indicating that its anticoagulation isrelated to activating eNOS in platelet and increasing the release of NO.