OBJECTIVETo 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).

METHODS(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.

RESULTS(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.

CONCLUSIONSBHD 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.

Cell Adhesion Molecules ; metabolism ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; E-Selectin ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; NF-kappa B ; metabolism ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Protein Kinase C-alpha ; metabolism ; Signal Transduction ; Thrombin ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

The aim of this study was to investigate the effect of GW003 on the ability of granulocyte colony forming in vitro of bone marrow cells. The bone marrow samples was collected from normal rhesus, the patients with leukemia in stages of remission and chemotherapy respectively, and the nucleated cells were separated and cultured for 12 days after addition of different concentrations of GW003 or rhG-CSF, or G-CSF mutant. Then the amount of colony-forming unit-granulocyte-macrophage was counted. The results indicated that GW003 could enhance the ability of bone marrow nucleated cells of rhesus to forming CFU-GM in vitro, and its effect was much better than that of rhG-CSF or G-CSF mutant at the same concentration(®). The GW003 showed dose-response relationship to CFU-GM level (r = R(2) = 0.965, P = 0.003, in a certain concentration), the GW003 also could enhance CFU-GM formation of marrow nucleated cells in leukemic patients, especially for patients receiving chemotherapy. The GW003 could relieve the marrow suppression caused by chemotherapy significantly. It is concluded that the GW003 can significantly improve the ability of bone marrow cells to form granulocyte colony in vitro as well as effectively alleviate bone marrow suppression.
Adult ; Animals ; Bone Marrow Cells ; drug effects ; Cell Line, Tumor ; Colony-Forming Units Assay ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; drug effects ; Granulocytes ; drug effects ; Humans ; Macaca mulatta