Effects Of Hirudo Active Extract On The Release Of Cultured Human Umbilical Vein Endothelial Cells Induced By Thrombin

Objective:To investigate the anti-coagulation mechanism of the hirudo extract on the release of thromboxaneB2(TXB2),endothelin-1(ET-1),nitrogen monoxide(NO),6-keto-prostaglandinF1α(6-keto-pGF1α),Tissue factor pathway inhibitor(TFPI)and Tissue factor(TF) induced by thrombin from human umbilical vein endotheliai cells (HUVECs).Methods:The RPMI1640completed culture medium containing10%FCS was used for culture HUVECs in vitro.HUVECs were randomly divided into5groups:blank control group(equal volume RPMI-1640),thrombin stimulated group(10kU/L),high dose hirudo extract group(0.6mg/mL+10kU/L thrombin),Medium dose hirudo extract group(0.3mg/mL+10kU/L thrombin),low dose Hirudo extract group(0.3mg/mL+10kU/L thrombin).These5groups were cultured for12hours each.The supernatant fluid and the thawed frozen liquid was observed.The content of TXB2and6-keto-pGF1α in the culture supernatant of HUVECs were measured with enzyme linked immunosorbent assay (ELISA),the content of ET-1in stromal supernatant was measured with radioimmunoassav(RIA).the activity of TFPI in the supernatant fluid and the activity of TF in the thawed frozen liquid were measured with Chromophore substrate method.the content of NO in stromal supernatant was measured with nitrate reductase method. Results:1. The morphology of HUVECs in thrombin stimulated group were changed compared with the blank control group.All the cells presented a monolayer arrangement. Thrombin stimulated group treatment slightly widened the intercellular space.The growth of HUVECs in each dose group of hirudo extract were similar to the blank control group. The experimental results demonstrate that thrombin can induced the HUVECs damage and the hirudo active extract can protective HUVECs induced by thrombin.2. Compared with the blank control group,thrombin promoted the release of TXB2(P<0.01)and markedly inhibit the release of6-keto-pGF1α(P<0.01)on VEC.we found that the content of TXB2in each dose group of hirudo extract was significantly lowered than the thrombin stimulated group(P<0.01)and the content of6-keto-pGF1α in each dose group of hirudo extract was higher than the thrombin stimulated group(P<0.01).The experimental results demonstrate that the hirudo extract could inhibit platelet aggregation.3. Compared with the blank control group, thrombin promoted the expression of TF(P<0.01) and inhibit the release of TFPI(P<0.01).The hirudo extract can inhibit the expression of TF (P<0.01)and promoted the release of TFPI(P<0.01) compared with the thrombin stimulated groupfrom HUVECs.The experimental results demonstrate that the hirudo extract has the effect of anticoagulation. 4.Hirudo extract in each dose group can substantially promote the release of NO(P<0.01) and inhibit the release of ET(P<0.01)compared with the thrombin stimulated group inHUVECs.Conclusion:1.The hirudo extract can protective HUVECs induced by thrombin.2.The hirudo active extract has the obvious effect of anticoagulation,the mechanism of anticoagulation maybe through inhibit the coagulation pathway actuating by thrombin.