补阳还五汤有效组分对血管内皮细胞抗血栓功能及蛋白激酶C的影响

Effect of Buyang Huanwu Decoction and its active fraction on antithrombosis in cultured vascular endothelial cells and protein kinase C

目的探讨补阳还五汤及其有效组分生物碱、苷对凝血酶诱导的血管内皮细胞抗血栓功能改变及蛋白激酶C(PKC)活化的影响。方法以凝血酶(10 U/mL)作用于培养的人脐静脉内皮细胞(ECV304),同时加入药物,24 h后测定上清液中组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物-1(PAI-1)水平、细胞组织因子(TF)、组织因子途径抑制物(TFPI)、凝血酶调节蛋白(TM)mRNA表达及PKCα蛋白表达。结果凝血酶作用内皮细胞后,tPA释放增加(P<0.01),TF、TFPI mRNA表达增强(P<0.05),而PAI-1释放及TM mRNA表达无显著性变化(P>0.05);PKCα表达增强(P<0.05)。补阳还五汤可抑制凝血酶诱导的tPA释放增加(P<0.01),生物碱对tPA释放增加无显著影响(P>0.05);苷(1.25 mg/mL)可使凝血酶诱导的tPA分泌增加(P<0.05);原方、生物碱(2 mg/mL)和苷(5 mg/mL)均可抑制内皮细胞分泌PAI-1(P<0.01)。原方、生物碱(1,2 mg/mL)和苷均可抑制凝血酶诱导的TF mRNA表达增强;生物碱(0.5和1 mg/mL)可抑制TFPI mRNA表达(P<0.05);各药对TM mRNA表达无显著影响;原方、生物碱和苷均可抑制凝血酶诱导的内皮细胞PKCα表达的增强(P<0.01)。PKC激活剂佛波酯(PMA)刺激内皮细胞后,PKCα被激活(P<0.01);PKC抑制剂H7作用于PMA刺激的内皮细胞后,PKCα表达增强不明显,PAR-1受体抑制剂CATG作用于凝血酶刺激的ECV304细胞后,PKCα表达显著抑制(P<0.05)。结论凝血酶可诱导内皮细胞抗血栓性发生变化。补阳还五汤原方、生物碱和苷对凝血酶诱导的血管内皮细胞抗凝、纤溶功能的改变具有调节作用,使血管内皮细胞的抗凝、纤溶作用趋于正常,其作用可能主要是通过抑制凝血酶诱导的PKCα的激活而介导的。生物碱和苷类有效组分可能为该方抗血栓作用的药效物质基础。

Objective To investigate the antithrombosis effects of Buyang Huanwu Decoction （BHD） and its active fractions on cultured vascular endothelial cells （VEC） following the stimulation of thrombin and their mechanism. Methods The human umbilicus vein endothelial cells （ECV 304） were cultured in media containing 10 U/mL thrombin and different dosese of drugs. The levels of tPA and PAI-1 were detected by ELISA, the expression of TF, TFPI, and TM mRNA was measured by RT-PCR, and the expression of PKCα was examined by immunohistochemical staining 24 h later. Results Compared to the control without any treatment, the release of tPA was increased evidently （P〈0.01）, and the expression of E-selectin, CD31 and TF, TFPI mRNA was upregulated （P〈0.05）; While no significant change had been found in the release of PAI-1 and the expression of TM mRNA （P〉0. 05）. Compared to the data after the mere stimulus of thrombin, each dose of BHD increased the release of tPA （P〈0.01）; None of the doses of alkaloid had significant effect on the release of tPA （P〉0. 05）; And glycoside （1.25 mg/ mL） increased the release of tPA （P〈0.05）. Alkaloid （2 mg/mL）, glycoside （5 mg/mL）, and each dose of BHD reduced the release of PAI-1 of VEC （P〈0. 01）. The data of RT-PCR detection indicated that the expression of TF mRNA was reduced by the treatment of alkaloid （1 and 2 mg/mL）, all doses of BHD, and glycoside; Alkaloid （0. 5, and 1 mg/mL） downregulated the expression of TFPI mRNA （P〈0.05）; While no significant change had been found in the expression of TM mRNA after the treatment of all doses of drugs. The expression of PKC in each drug treatment group was greatly decreased when compared to that in the mere stimulus of thrombin （P〈0.01）. PKC activator （PMA） significantly enhanced the PKCα expression （P〈0.01）, H7 （PKC inhibitor） could inhibit PKCα activation sitimulated by PMA. The expression of PKCostimulated by thrombin was inhibited obviously after adding CATG （PAR-1 inhibitor） （P 〈 0.05）. Conclusion Thrombin could induce thrombotic changes in cultured VEC, including anticoagulation and fibrinolytic functions. Those changes can be adjusted to the control level by the treatment of BHD, alkaloid, and glycoside. BHD may ameliorate the thrombotic changes caused by thrombin through inhibiting the activation of PKC. The similar effects of alkaloid and glycoside against thrombosis indicate that these two constituents are two effective pharmacological components through which BHD plays its role of antithrombosis.