Effect Of Naotaifang's Extract On Human Umbilical Vein Endothelial Cells-Apoptosis, Disorder Of Anticoagulant And Regulating Fibrinolysis Induced By Tumor Necrosis Factor-alpha

OBJECTIVE: To investigate the effect of Naotaifang's extract(NTE) on the cultured human umbilical vein endothelial cells(HUECs)including apoptosis, the disorder of anticoagulant and regulating fibrinolysis which induced by a concentrated (l00ng/ml) recombinant human tumor necrosis factor-alpha (rhTNF-a).METHODS: Cultured cells were divided randomly into 6 groups(including blank control group, NTE group, TNF group and three treatment groups with different concentrations of NTE) and incubated for 24 hours after addition of different treatments. The supernatant was used to determine von Willebrand Factor(vWF) content, tissue plasminogen activator (tPA ) activity and plasminogen activator inhibitor (PAI) activity. HUEC lysate was used to determine the procoagulant activity. Moreover, for qualitative determination of EC apoptosis, acridine orange (AO)/ethidium bromide (EB) fluorescence staining was used and the apoptotic rate was computed.RESULTS: Compared with the blank control, incubation with a concentration of rhTNF- a (l00ng/ml) for 24 hours decreased the amount of cultured cells and changed their normal morphology. The NTE in a final concentration of 0.6mg/ml an 0.2mg/ml inhibited the effects of TNF a .The rhTNF- a increased the amount of apoptotic HUECs with AO/EB fluorescence staining. NTE could decrease the rate of apoptotic cells.(3)rhTNF- a promoted the release of vWF and NTE in high concentration and medium concentration inhibited the effect (p<0.05).Moreover, treatment with rhTNFa increased the activity of PAI and enhanced the expression of coagulative activity. NTE in these three concentrations showed a protective effect against these changes. Cells treated with the NTE alone didn't show any visible morphologic abnormal and functional change except anticoagulant effect. CONCLUSION: The VEC injured by a concentration of pro-inflammatory cytokine TNF- a is mainly apoptosis and the apoptotic cells became procoagulant and fibrinolysis inhibited. These changes facilitate the thrombsis and may play an important role in the cascade of "ischemia-inflammation-thrombosis" in human cerebral infarction. To protect the endothelial cells from apoptosis and regulate it's funtion on anticoagulation and fibrinolysis are the possible therapeutic mechanism of NTE.