Ceramide Enhanced The Expression Of Cox-2 And Vessel Vasoconstriction By ER Stress In VSMCs

Objective:(1)To investigate the effects of pretreated ceramide on vessel vasoconstrictionthrough vascular smooth muscle in rats.(2)To investigate the role endoplasmic reticulum stress plays in the effect of ceramide on vascular smooth muscle Cox-2 expression and vessel vasoconstriction.Methods :Ceramide pretreated SD rats aorta vessel for different concentrations(0?M,1.0?M,5.0?M,10.0?M)and different time(0h,3h,6h,12h),then observed and recorded vascular smooth muscle contraction by iWorx LabScribe2 Data Recording.With the methods of Western blotting,test the protein expression level of Cox-2,Cox-1,Bip in ceramide pretreated primary VSMCs at different concentrations(0?M,0.5?M,1.0?M,5.0?M,10.0?M)and different time(0h,3h,6h,12 h,24h,48h).Then detected the expression of Cox-2,Cox-1,Bip in the cells treated with Cox-2 inhibitor Celecoxib and NS398,and the inhibitor of ER stress 4-PBA,TUDCA.Verify ceramide effect on the expression of Cox-2,Cox-1,Bip by using luciferase gene reporter assay in A10 cells.Finally,figured out the mRNA expression level of Cox-2,Cox-1,Bip in pretreatment of ceramide in VSMCs.Results:(1)Ceramide promoted the PE dependent construction in vascular rings in a dose dependent manner.And at 6h,ceramide treatment enhanced vasoconstriction to the highest level induced by PE.And Cox-2 inhibitor Celecoxib and NS398 can decrease vasoconstriction enhanced by ceramide,which suggested that Cox-2 was involved in ceramide promoted vasoconstriction.Endoplasmic reticulum stress inhibitors 4-PBA and TUDCA can also reduce the contraction of vascular ring,implying that endoplasmic reticulum stress also engaged in vasoconstriction.(2)Ceramide promoted the expression of Cox-2 and Bip in VSMCs in a dose dependent manner.Ceramide enhanced expression of Cox-2 and Bip in different time.At 6h,the expression of Cox-2 and Bip reached to the highest level.There was no change of Cox-1 expression with ceramide treatment in both dose and time manner.The inhibitor of ER stress 4-PBA,TUDCA decreased the effect of ceramide on the expression of Cox-2 and Bip in VSMCs,which implied ER stress may participate in the effect of ceramide on VSMCs.(3)Luciferase gene reporter assay showed ceramide increased the expression of Cox-2 and Bip in A10 cell and endoplasmic reticulum inhibitors 4-PBA,TUDCA can inhibit ceramide induced Cox-2 and Bip expression level.(4)RT-PCR showed that ceramide enhanced the mRNA expression level of Cox-2 and Bip in a dose-and time-dependent manner.At 3h,Cox-2 and Bip mRNA expression reached the highest level.4-PBA,TUDCA can reduce the mRNA expression of Cox-2 and Bip,but showed no effect on Cox-1.Conclusions:(1)Ceramide affected vascular ring through vascular smooth muscle contraction,and Cox-2 participated in this vasoconstriction process.(2)Ceramide can induce vasoconstriction through a possible mechanism that ceramide regulated the expression of Cox-2,and ER stress played a significant role in the process,which may form ceramide/endoplasmic reticulum stress/Cox-2 axis.