Study On The Effect Of BYHWD And Its Active Fractions On The Expression Of Vasoactive Substances And Adhesion Molecules And The Signal Transduction Mechanism In VECs

Object:The first purpose of the study was to observe the effect of BYHW decoction and its active fractions on the expression of vasoactive substances and adhesion molecules induced by thrombin in human umbilical vein endothelium derived cell line ECV-304.The second was to research the possible action mechanism of BYHW decoction from cell signal transduction.Method:ECV-304 stimulated with thrombin were cultured in RPMI-1640 and then given different dose of BYHWD,alkaloid and glycoside.The activities and injury degree of ECV-304 were observed with MTT measuring the OD and LDH activity.The contents of NO and the activities of TNOS, iNOS and eNOS were measured in different groups.ET-1 and 6-keto-PGF_1α were measured with radioimmunoassay(RIA).PGI₂ synthase mRNA and ECE mRNA were examined by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR).Immunohistochemical analysis was performed to evaluate the expression of PECAM-1,ICAM-1,E-Selectin, PKCα,p38MAPK,ERK and NF-κB.Results:The activities of ECV-304 were significantly decreased stimulated by thrombin(P＜0.05).The cells' activities in BYHWD,alkaloid and glycoside groups were not increased comparing with the thrombin group(P＞0.05).Thrombin lowered the secretion of NO and the activities of eNOS of ECV-304(P＜0.05).Compared with thrombin group,the contents of NO in BYHWD groups were increased(P＜0.05).In alkaloid groups and BYHWD groups,the level of 6-keto-PGF_1α and the expression of PGI₂ synthase mRNA were significantly higher than those in thrombin groups.The level of ET-1 in thrombin group was higher than in the blank group(P＜0.05),while the difference between thrombin and drug groups had no statistic value(P＞0.05).No distinct difference of the expression of ECE mRNA existed among all the groups of ECV-304(P＞0.05).Thrombin improved significantly the expression of adhesion molecules,such as PECAM-1,ICAM-1 and E-selectin in ECV-304,while drugs lowered the level(P＜0.05).Thrombin increased the expression of protein kinase C(PKC) of ECV-304,and the drugs restrained the effect(P＜0.05).The expression of p38MAPK,ERK or NF-κB showed no remarkable difference among all groups of ECV-304(P＞0.05).Conclusion:BYHWD and its active fractions could improve VECs releasing the vasodilator material—NO and PGI₂,but couldn't significantly affect the secretion of vasoconstrictive substance—ET-1.BYHWD could reduce the increasing expression of adhesion molecules in VECs stimulated by thrombin, and alkaloid and glycoside were the possible functional components. BYHWD could prevent the activating of PKC system,and then restrain VECs' activation stimulated by thrombin.The experiment result also suggested that alkaloid and glycoside may be associated with it.The drugs including BYHWD and its main effective fractions had no influence on the expression of p38MAPK,ERK or NF-κB in VECs.It suggested that the function of BYHWD on VECs may be related to other cell signal transduction pathway.