thromboplastin and anisodamine

To explore the mechanism of anisodamine in treating infectious shock through studying effect of anisodamine on endotoxin lipopolysaccharide (LPS) induced expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in vascular endothelial cells (EC).. Human umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. In order to evaluate a possible contribution of the nuclear factor-kappa B (NF-kappa B) pathway on the transductive effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B binding oligonucleotides.. LPS could significantly strengthen the expression of HUVEC PAI-1 protein and TF activity and its mRNA, this effect of LPS could be markedly weakened after adding Anisodamine dose-dependently. Anisodamine could also completely block the LPS induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC.. The possible mechanism of anisodamine in treating infectious shock may be through antagonizing LPS induced HUVEC TF and PAI-1 expression, and the antagonism might be, at least partially, transduced by path of NF-kappa B.

Topics: Cells, Cultured; Culture Media, Conditioned; Drugs, Chinese Herbal; Endothelial Cells; Humans; NF-kappa B; Plasminogen Activator Inhibitor 1; RNA, Messenger; Solanaceous Alkaloids; Thromboplastin; Umbilical Veins

By study on the effect of anisodamine on lipopolysaccharide-induced expression of tissue factor (TF) in vascular endothelial cells (EC), the mechanism of anisodamine antithrombosis, as well as in the treatment of bacteraemic shock was investigated. Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method. TF activity was measured in the lysates of HUVEC by using a single step clotting assay. Specific mRNA expression was detected by Northern blotting. In order to evaluate a possible contribution of the nuclear factor (NF)-kappa B pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVECs and NF-kappa B-binding oligonucleotides. The results showed that treatment of HUVEC with LPS resulted in a significant increase in TF activity. Anisodamine dose-dependently inhibited LPS-induced upregulation of TF. These effects was also confirmed on the level of specific TF mRNA expression by Northern blotting. Furthermore, EMSA showed that anisodamine completely abolished LPS-induced NF-kappa B DNA binding activity in nuclear extracts from HUVECs treated with LPS together with anisodamine. The results suggest that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced TF expression in these cells. Its interference with the NF-kappa B pathway might--at least in part--contribute to this effect. The ability of anisodamine to counteract LPS effect on endothelial cells might be one underlying mechanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.

Topics: Cells, Cultured; Drugs, Chinese Herbal; Electrophoretic Mobility Shift Assay; Endothelium, Vascular; Humans; Lipopolysaccharides; NF-kappa B; RNA, Messenger; Solanaceous Alkaloids; Thromboplastin; Umbilical Veins