Influence Of Puerarin On The Expression Of Tissue Factor Induced By Angiotensinâ…¡ And Its Mechanism In Vascular Endothelial Cells

Tissue factor(TF)is an initiator in physiological coagulation and pathological thrombus.It is a membrane glycoprotein with 47 kD.It is not only the receptor of FⅦ/FⅦa but also functions as a necessary cofactor of FⅦa activating FⅩand FⅨ。Considerable evidence has accumulated to indicate that there is a alternatively spliced(asTF)in vivo. asTF maybe have dual function on procoagulation and anticoagalation. But the exact biological and pathological impacts of asTF remains to be elucidated.Renin-angiotensin system(RAS)is the most important regulative system,and plays a significant role in maintaining homeostasis.Recently many reports indicated that angiotensinⅡhas not only the action of boosting blood pressure,but also affects the balances of coagulation-anticoagulation and fibrinolysis-antifibrinolysis.It is widely accepted that angiotensinⅡparticipates in the development of atherosclerosis and acute cerebrocardiac infarction.Puerarin is the major component extracted from the Chinese herb, Radix.Puerarin and is generally used as an antithrombotic in Chinese clinical practice.Puerarin is also known to exert cytoprotective and antioxidant effects on endothelial cell and antithrombotic effects in experimental animal models.However,whether it affects the TF gene expression induced by angiotensinⅡin endothelial cells and its exact mechanism remains unclear yet.AIM The purposes of this study were to observe the effects of puerarin on the expression of TF induced by angiotensinⅡin human umbilical vein endothelium derived cell line(HUVECs)and to elucidate the inhibitory mechanism of puerarin on the expression of TF induced by angiotensinⅡ.METHODS HUVECs were cultured in DMEM.TF activity was determined with one-stage clotting assay measuring total cellular procoagulant activity(PCA).PCA was recognized as TF activity by FⅦdeficient plasma and TF monoclonal antibody methods.TF antigen was determined with enzyme linked immunosorbent assay(ELISA).TF mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR).Immunohistochemical analysis was performed to evaluate the activation of NF-κB.RESULTS①A gradual increase in PCA(r=0.9528,P＜0.05)was observed in HUVECs stimulatived with increasing concentration of AngⅡ(10^-10-10^-6mol/l).②Puerarin(62.5～500 mg/l)alone could not inhibit the expression of TF in HUVECs(P＞0.05).③When pretreated with puerarin(62.5～500 mg/l),puerarin inhibited the expression of TF activity,TF antigen and mRNA induced by AngⅡin dose-dependent fashion(P＜0.05),and 500mg/l was the strongest concentration.500 mg/l puerarin decreased TF activity,TF antigen in HUVECs on a time dependent manner,reaching a maximum level after 12 h.④L-NAME alone,which is the inhibitor of NOS,had no marked effects on PCA and TF mRNA in HUVECs,but L-NAME significantly blocked the inhibitory effects of puerarin on TF PCA and mRNA expression induced by AngⅡ(P＜0.05).⑤Immunohistochemical analysis demonstrated that NF-κB translocation from cytoplasm to the nucleus was observed after treatment of endothelial cells with AngⅡ.puerarin inhibited AngⅡ-induced NF-κB translocation.CONCLUSIONS The present data suggest that①puerarin can inhibit the expression of TF induced by AngⅡin vascular endothelial cells,and this effect of puerarin could be exerted at mRNA level.②NO pathway participates in the inhibitory effects of puerarin on the expression of TF induced by AngⅡ.③NF-κB translocation plays an important role in AngⅡ-induced TF expression and puerarin can inhibit this NF-κB translocation.