Effects Of Nicotine And Simvastatin On The Expression Of T-PA And PAI-1 In Cultured Human Umbilical Vein Endothelial Cells And Its Related Mechanism

BackgroudCigarette smoking was considered as a dependent danger factor to the development of cardio- vascular disease .It could damage the vascular endothelial cells, lead to endothelial cells dysfunction, as well as trigger fibrinolysis imbalance, then increasing the risk of thrombosis. As we all know, fibrinolytic system relies heavily on the activity of tissue-type plasminogen activator ( t-PA )and tissue-type plasminogen inhibitor-1(PAI-1) released by vascular endothelial cells. There has many reports about the disfunction of fibrinolysis studies associated with diabetes , coronary heart disease .But studies about disfunction of fibrinolysis associated with cigarette smoking was reported few. Nicotine, one of the main harmful substances of smoking, can damage the fibrinolytic function of endothelial cells, and its related mechanism was entirely unclear .Protein kinase C (PKC) is an important biological intracellular signal transduction pathway in the body, it widespread participate the secretion ,proliferation and differentiation of cells, its get to pay close attention increasingly .Now, the studies about nicotine decrease the fibrinolytic of endothelial cells associate with PKC is few reported. Simvastatin as a represent of liqid- lowering statin drug, it has many new function ,for example ,the protection of endothelial cells, inhibit inflammation, promote fibrolysis ,etc. Simvastatin can wether or not improved fibrinolytic activity induced by nicotine and its related mechanism has not been reported.ObjectiveTo study the effect of nicotine and different concentrations of staurosporine on the expression of t-PA and PAI-1 protein and mRNA in human umbilical vein endothelial cell (HUVECs), To explore the mechanism of fibrinolytic function stimulated by nicotine . Using different concentration of simvastatin to pretreatment the HUVECs, and observed the influence of simvastatin to the expression of t-PA,PAI-1 protein and mRNA stimulated by nicotine.MethodsExperimental Group:HUVECs were cultured in 25cm2 culture flasks and randomly divided into①the control group②100μmol/L nicotine group③STS intervention group. The cells were treated with STS at concentration of 20,50,100μmol/L for 30min,and then exposed to nicotine at concentration of 100μmol/L.④Simvastatin intervention group .The cells were treated with simvastatin at concentration of 1,10,100μmol/L for 2h,and then exposed to nicotine at concentration of 100μmol/L.The cells and supernatants of each group were collected after 24 hours cultured .The expression of t-PA and PAI-1 protein were measured by ELISA and the RNA expression of t-PA and PAI-1 was determined by RT-PCR.Results1.Compared with control group〔0.099±0.013(,38.660±1.749)ng/ml〕, the PAI-1 mRNA and protein expression of nicotine group〔0.969±0.112,(66.051±2.890)ng/ml〕were increased significantly(P<0.01, respectively).Compared with control group(1.011±0.132),the t-PA mRNA(0.099±0.011)expression of nicotine group were decreased significantly(P<0.01).However, the expression of t-PA protein was no significantly different(P>0.05).2.Compared with nicotine group , the PAI-1 mRNA and protein expression of different concentrations of STS intervention group〔0.588±0.077,(61.922±5.821)ng/ml〕,〔0.530±0.070,(57.976±8.009)ng/ml〕,〔0.222±0.029,(43.291±1.878)ng/ml〕were decreased significantly ,but still higher than control group(P<0.01, respectively). Compared with nicotine group, the t-PA mRNA expression of 20μmol/L STS group(0.184±0.023)were increased not significantly(P>0.05);The t-PA mRNA expression of 50,100μmol/L STS group(0.293±0.037),(0.412±0.053)were increased significantly ,but still lower than control group(P<0.01, respectively).The expression of t-PA protein was no significantly different (P>0.05).3.Compared with nicotine group , the PAI-1 mRNA and protein expression of different concentrations of simvastatin intervention group〔0.581±0.095,(60.788±2.500)ng/ml〕,〔0.511±0.084,(54.955±3.971)ng/ml〕,〔1.074±0.099,(16.190±2.149)ng/ml〕were decreased significantly ,and the most notably effect of simvastatin was at the concentrations of 100μmol/L ,almost the same as that in the control group(P>0.05). Compared with nicotine group, the t-PA mRNA expression of 1μmol/L simvastatin group(0.187±0.025)were increased not significant(P>0.05);The t-PA mRNA expression of 10,100μmol/L simvastatin group(0.278±0.038),(0.332±0.046)were increased significantly ,but still lower than control group(P<0.01, respectively). The expression of t-PA protein was no significantly different (P>0.05).Conclusions1.Nicotine through increased the expression of PAI-1 protein and mRNA in HUVECs,inhibits the fibrinolytic function of endothelial cells.2.Activation the PKC can partly disorder the fibrinolytic function of endothelial cells that induced by nicotine.3.Simvastatin can improves the fibrinolytic function of endothelial cells that induced by nicotine...