Effects Of Buyang Huanwu Decoction And Its Active Fractions On Antithrombosis And Its Mechanism In Cultured Vascular Endothelial Cells

Objective:To investigate the antithrombosis effects of Buyang Huanwu Decoction(BHD) and its active fractions and its mechanism on cultured vascular endothelial cells(VEC) following the stimulation of thrombin.Methods:The endothelial cells(ECV-304) were cultured in mediums containing 10u/ml thrombin and different doses of drugs.24 hours later, the vigour of VEC and LDH activity were measured by colorimetry,the level of tPA and PAI-1 were detected by ELISA,the expressions of E-selectin,CD31 and PKC_αwere examined by immunohistochemical staining,and the expressions of TF,TFPI and TM mRNA were measured by RT-PCR.Results:①Compared to the control without any treatment,after thrombin stimulus,the cell vigour of VEC was significantly decreased (P＜0.01);the LDH activity was increased obviously(P＜0.05),the release of tPA was increased evidently(P＜0.01),and the expressions of E-selectin, CD31 and TF,TFPI mRNA were upregulated(P＜0.05);while no significant change had been found in the release of PAI-1 and the expression of TM mRNA.②Compared to the data after the mere stimulus of thrombin,different doses of BHD,Alkaloid and Glycosid showed no significant effect on cell vigour and LDH activity induced by thrombin.Each dose of BHD decreased the release of tPA(P＜0.01);none of the doses of Alkaloid had significant effect on the release of tPA;and Glycoside(1.25mg/ml) increased the release of tPA(P＜0.05).Alkaloid Alkaloid(2mg/ml),Glycoside(5mg/ml) and each dose of BHD reduced the release of PAI-1 of VEC(P＜0.01).BHD(50,100mg/ml) downregulated the expression of E-selectin(P＜0.05);and BHD(25,50mg/ml),Alkaloid(1mg/ml) and Glycoside(5mg/ml) downregulated the expression of CD31.The data of RT-PCR detection indicated that The expression of TF mRNA was reduced by the treatment of Alkaloid(1,2mg/ml),all doses of BHD and Glycoside; Alkaloid(0.5,1mg/ml) downregulated the expression of TFPI mRNA; while no si(?)nificant change had been in the expression of TM mRNA after the treatment of all doses of drugs.③The expression of PKC in each drug treatment group was greatly decreased when compared to that in the mere stimulus of thrombin(P＜0.01).Conclusion:Thrombin can induce thrombotic changes in cultured VEC, including anticoagulation,fibrinolytic functions and the activation of platelet.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 play its role of antithrombosis.