Objective To study the effect of Hirudo (水蛭) extract on the release of von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) from cultured human umbilical vein endothelial cells (HUVECs) induced by thrombin, and preliminarily investigate the anti-coagulating mechanism of the extract. Methods The HUVECs were cultured in vitro. The well grown 2 - 3 generation HUVECs were divided into five groups: the control, thrombin challenge, and high,medium, and low concentration Hirudo extract groups. Different concentrations of Hirudo extracts (600, 300,150 mg/L) were given after administration of thrombin (10 kU/L) in the high, medium, and low concentration Hirudo extract groups. The control group was given equal amount of RPMI1640 culture medium. The HUVECs were cultured for 12 hours and the supernatant was then collected in each group. The concentrations of vWF, PAI-1, and t-PA were measured with enzyme linked immunosorbent assay (ELISA).Results Compared with the control group, the concentrations of vWF and PAI-1 (μg/L) were significantly increased [vWF: (11.01±0.54)％ vs. (7.05±0.49)％; PAI-1: 72.26±0.85 vs. 55.89±1.26, both P＜0.01] and the concentration of t-PA (μg/L) was markedly decreased in thrombin challenge group (15.15±1.41 vs. 34.29±1.22, P＜0.01). Compared with the thrombin challenge group, the concentrations of vWF and PAI-1 [vWF: (9.48±0.64)％, (8.57±0.50)％, (7.97±0.44)％; PAI-1: 58.30±0.90, 62.69±1.01,67. 47±1.28] were substantially decreased, and that of t-PA (32. 59±1.46, 26. 40±0. 82, 21. 06±1.13) was markedly increased (all P＜0. 01) in the high, medium, low concentration Hirudo extract groups. These results showed that the Hirudo extract could significantly inhibit the release of vWF and PAI-1, and enhance the release of t-PA from HUVECs induced by thrombin. Conclusion The Hirudo extract has a noticeable effect of anticoagulation. Its mechanism may be related to the regulation of the release of vWF from endothelial cells, of the balance of the fibrinolysis system and the protection of damaged vascular endothelial cells.%目的 观察水蛭提取液对凝血酶诱导人脐静脉内皮细胞(HUVECs)释放血管性血友病因子(vWF)、纤溶酶原激活物抑制剂-1(PAI-1)、组织型纤溶酶原激活物(t-PA)的影响,探讨水蛭抗凝的作用机制.方法 体外培养 HUVECs,将生长良好的2～3代细胞分为对照组、凝血酶刺激组及水蛭提取液高、中、低剂量组5组.将生药浓度600、300、150 mg/L的水蛭提取液加入到10 kU/L凝血酶刺激的HUVECs培养12 h,对照组加等量RPMI 1640培养液.用酶联免疫吸附法(ELISA)检测细胞培养上清液中vWF、PAI-1、t-PA的含量.结果 与对照组比较,凝血酶刺激组细胞培养上清液中vWF、PAI-1(μg/L)含量显著升高[vWF:(11.01±0.54)%比(7.05±0.49)%;PAI-1:72.26±0.85比55.89±1.26,均P<0.01],t-PA(μg/L)含量显著降低(15.15±1.41比34.29±1.22,P<0.01).与凝血酶刺激组比较,水蛭提取液高、中、低剂量组vWF、PAI-1含量[vWF:(9.48±0.64)%、 (8.57±0.50)%、 (7.97±0.44)%;PAI-1:58.30±0.90、 62.69±1.01、 67.47±1.28]显著降低,t-PA含量(32.59±1.46、 26.40±0.82、 21.06±1.13)显著升高(均P<0.01).说明水蛭提取液能显著减弱凝血酶诱导HUVECs释放vWF、PAI-1,增强凝血酶促进HUVECs表达t-PA.结论 水蛭具有明显的抗凝作用,其作用机制可能为参与调节内皮细胞释放vWF,调节纤溶平衡及保护受损的血管内皮细胞.
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