[Effect and signal transduction mechanism of active components of Buyang Huanwu Decoction on vascular adhesion...

To explore the effect and possible cell signal transduction mechanism of active components (alkaloids and glucosides) of Buyang Huanwu Decoction (BHD) on the vascular adhesion molecule (VAM) expression induced by thrombin (Thr).

(1) Endothelial cells (ECV-304) were cultured in vitro and stimulated by Thr, then cultured in media containing BHD whole recipe (BHDw), its alkaloids (BHDa) or glycosides (BHDg), respectively. The expressions of E-selectin, CD31, CD54, and protein Kinase Calpha PKCa) were detected by immunocytochemical method. (2) Cultured ECV-304 cells were treated with Thr, Thr + 1-(5-isoquinolinesulfonyl)-2-mythylpiperazine dihydrochloride (H7), phorbol myristate acetate (PMA), PMA + H7, Thr + cathepsin-6 (CATG) as well as Thr plus BHDw, BHDa, and BHDg, respectively after then, changes of PKCalpha, phospho-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-extracellular signal regulated kinase (p-ERK) and nuclear factor-kappaB (NF-kappaB) expressions were observed.

(1) Expressions of PECAM-1, ICAM-1 and E-selectin in ECV-304 cells were significantly enhanced after thrombin stimulation (P < 0.05). When compared with the thrombin group, the three expressions were lower in the BHDw treated group (P < 0.05); while in groups treated with BHDa and BHDg, only expressions of PECAM-1 and ICAM-1 were lower (P < 0.05), but no obvious difference in E-selectin was shown. (2) Expression of endothelial PKCalpha increased after thrombin stimulation (P < 0.01), which could be enhanced by PMA (PKC activator, P < 0.01); but inhibited by PMA + H7 (PKC inhibitor), CATG (PAR-1 inhibitor, P < 0.05) as well as by BHDw, BHDa and BHDg (P < 0.05). (3) Expressions of p-p38 MAPK and p-ERK in ECV-304 cells showed no remarkable change after thrombin stimulation, or after reacted with PMA, H7, CATG, BHDw, BHDa and BHDg. (4) Similar to that of p-p38MAPK and p-ERK, NF-kappaB was unchanged in all the reactions.

BHD could down-regulate the increasing of VAM expression induced by thrombin in VECs; BHDa and BHDg might be the active components in the recipe. The effect of thrombin is mainly mediated through activation of PKC; while p-p38MAPK, ERK or NF-kappaB are not the chief signal transduction pathways. And BHD and its effective components may antagonize the thrombin activation on VECs through inhibiting the activation of PKCalpha.