Effects Of Cigarette Smoke Extract And Simvastatin On The Expression Of T-PA And PAI-1 In Cultured Human Umbilical Vein Endothelial Cell

Background:Smoking,as a common risk factor for respiratory disease,the majority of research concentrated in the tracheal.Many studies have found that smoking can cause vascular endothelial damage,and endothelial damage are risk for pulmonary disease and coronary heart disease,therefore,the study of smoking and lung disease has great significance happened.There are two systems,coagulation and fibrinolysis,in the normal presence of human blood.The balance of their state can play a role in maintaining normal blood flow.Whether the coagulation system activated or the fibrinolytic system inactivated can trigger the formation of thrombosis. As a main component of fibrinolytic system,tissue-type plasminogen activator(t-PA) and tissue-type plasminogen inhibitor-1(PAI-1) determined the expression of fibrinolytic system.A variety of factors can affect the vascular endothelial cell fibrinolytic activity,but the study of cigarette smoke on fibrinolytic activity of vascular endothelial cell are less reported.Simvastatin as a new type of lipid-lowering statin drugs,recent years found that it has many new functions, for example,the protection of endothelial cells,inhibit inflammation,anticoagulation,etc. Simvastatin can significantly improved fibrinolytic activity which mediated by inflammatory factors,but there has not been reported if it can improve fibrinolytic activity which inhibited by cigarette smoke extract(CSE).To study the expression of tissue plasminogen activator(t-PA) and tissue plasminogen activator inhibitor-1(PAI-1) under different cigarette smoking extract(CSE) conditions(different concentrations and different time points).At the same time,simvastatin stimulated the vascular endothelial cells which pretreated by CSE,and measured the expression of t-PA,PAI protein and mRNA.Thereby proving that smoking is can damage endothelial cells of fibrinolytic function,and simvastatin has a protective effect on cigarette smoke extract-induced endothelial cell damage fibrinolytic function.Methods:In aseptic conditions,taking healthy full-term delivery of maternal newborn umbilical cord,digestion,isolated and cultured the endothelial cells(HUVECs),the experiment was divided into two parts:(1)The fibrinolytic balance under different CSE conditions:choose the 4th to 6th HUVECs, inoculated in 24 well culture plates,divided into three groups:①control group;②the different concentrations(5%,10%,20%) at the same time point CSE Group:After 6h,collecting the cell supernatant of each group;③the same concentration of CSE group at different time points:5% CSE stimulated HUVECs,the cell supernatant were collected at different time points(4h,6h,8h, 12h,24h) separately,using enzyme-linked immunosorbent assay analysis of double-antibody sandwich(ELISA) method determined the t-PA and PAI-1 antigen concentration of each group;(2) Effect of CSE on the fibrinolytic system of vascular endothelial cell by simvastatin Pre-treatment:choose the 4th to 6th HUVECs,inoculated in 24 well culture plate,divided into control group,tobacco extract group and simvastatin group.After 24h,collecting the cells and supernatants of each group and using enzyme linked immune- osorbent assay double antibody sandwich assay(ELISA) detected in the supernatant ofchain reaction(RT-PCR) assay detected in the t-PA and PAI-1 mRNA expression of each group cell.Results:(1) After 6h,compared with control group,with different concentrations(5%,10%,20%) the same time point CSE group,the concentration of CSE group PAI-1 levels were significantly higher(P＜0.05),but each group has no statistically significant.10%and 20%CSE group t-PA content decreased significantly,while the 5%CSE group t-PA content is not statistically significant;the same concentration at different time points CSE group,5%CSE Group PAI-1 concentration higher than the control group,and showed time-dependent,t-PA content is not statistically significant.(2) Compared with control group,the PAI-1 protein content(8.8030±0.4745) and mRNA (1.8155±0.0412) expression of CSE group were increased(P＜0.01);with the CSE group,PAI-1 protein content(5.4875±0.3166) and mRNA(1.3975±0.0297) expression of simvastatin group were decreased(P＜0.01),but still higher(P＜0.05);each group t-PA protein content and its mRNA expression was not significant(P＞0.05).Conclusion:(1) CSE can directly damage endothelial cells and inhibit its fibrinolytic activity,thereby, increasing the incidence of thrombosis'disease.(2) Simvastatin plays a protective role on the fibrinolysis of endothelial cell which pre-treatment by CSE.