Effect Of COX-2 On The Vascular Endothelial Cell Apoptosis Induced By Cigarette Smoking Extract

Objective:To probe the effect of cyclooxygenase-2 on vascular endothelial cell apoptosis induced by cigarette smoking extract.Methods:The human vascular endothelial cells(ECV-304)were cultured in vitro,those at exponential growth phase being studied in experiment.An inflaming commercial cigarette without filtration was sucked continuously by the vacuum aspiration driving device.Mainstream of smoke went into 1640 medium serum-free,from the other outlet of the T-tube(one cigarette was used per 20 ml of 1640 medium serum-free)to generate a complex CSE-1640 medium solution.The resulting solution were diluted with 1640 medium serum-free and used immediately in one hour after being sterilized by filtration.The cells were interfered by different concentrations of CSE solution.The research was completed through three steps:(1)the human vascular endothelial cells were divided into four groups according to the concentrations of CSE,including control group,0.5%CSE-interfered group,1%-CSE-interfered group and 5%-CSE-interfered group.Having been interfered for 12h persistently,the cell apoptosis rate was tested by Hoechst Staining method and Flow Cytometry,the expression of COX-2 protein by lmmunocytochemistry and Western Blot.(2)The endothelial cells were exposed to 5%CSE for Oh, 3h,6h,9h,12h,and 24h,then the apoptosis was tested by Flow Cytometry, and expression of COX-2 by Western Blot.(3)Endothelial cells were treated by 5%CSE and different concentrations of selective COX-2 inhibitor,celecoxib(0μM/L,2.5μM/L,5μM/L,10μM/L,20μM/L,50μM/L concentrations included)together for 9h,then Flow Cytometry were used to test the apoptosis,and Western Blot to test the expression of COX₂ protein after interfered.Data were expressed as means±SD((?)±s). Statistical analysis was performed using One—Way ANOVA.A value of P＜0.05 was taken as significant.All calculations were made using statistical software(SPSS 13.0).Results:After exposure to different concentrations of CSE for 12h,the apoptosis rate of the endothelial cell in 5%CSE interfering group was maximum,as compared with other groups,significant difference being found(P＜0.05).COX-2 expression increased after interfered by CSE,and reached the highest level in 5%CSE interfering group as well.Compared with groups,there being significant difference(P＜0.05).Besides,both the apoptosis rate of endothelial cell and COX-2 expression level in it increased with the intervention time going on,the apoptosis rate achieved the peak after being interfered by 5%CSE for 24h,while COX-2 expression level got to the crest after interfered 9h by 5%CSE.The results also revealed that the selective COX-2 inhibitor celecoxib can inhibit of secreting COX-2 in the endothelial cell,induced by CSE,and with stepping up of the inhibitor concentration,more and more COX-2 was inhibited,and we found the expression of COX-2 in 5%CSE+50μM/L celecoxib-interfered group was minimum.Finally,we also found that,the selective COX-2 inhibitor,celecoxib could induce endothelial cell apoptosis at the concentration≥10μM/L(P＜0.05).Conclusions:Cigarette Smoking Extract can induce endothelial cell's apoptosis,and increase the expression of COX-2 protein in it,moreover presenting concentration and time dependent effects.Celecoxib,the selective COX-2 inhibitor,can reduce the expression COX-2 protein in endothelial cells,while promoting the cells apoptosis at higher concentrations.So COX-2 may play an important role in the development of CSE-associated apoptosis of endothelial cells.