| Chronic kidney disease is one of the world's most important public health problems.The most serious consequence of CKD is the loss of kidney function,eventually leading to end-stage renal disease(ESRD).While the loss of kidney function may give rise to a series of complication,for as cardiovascular disease,which is the NO.1 killer of ESRD.The uremic toxins is considered by some scholars for one of the most important risk factor to rising cardiovascular morbidity and mortality in patients with ESRD.Inflammation is proved to induced endothelial cell damage,and PGE2 may be the significant inflammatory factor,whose expression is regulated through cox-2/mPGES-1/PGE2 pathway.Our previous studies have found that uremic toxins could cause mesangial cell proliferation by this pathway,also in acute kidney injury,COX-2/mPGES-1/PGE2 pathway plays critical roles in podocyte injury.Based on this,we speculate that uremic toxins may induce MAECS injury by this signal pathway.Objective:The role of uremic toxins on mouse vascular endothelial cells injure by the COX-2/mPGES-1/PGE2 pathway.Methods:1.The apoptosis rate of mouse vascular endothelial cells was measured by TUNEL staining.The expression of Caspas 3 and Bax was detected by quantitive real time PCR.The mouse vascular endothelial cells were treated by IS and UA at different dose for different time,then using the quantitative real-time PCR(RT-PCR)and Western blot to determine the expression of COX-2 and mPGES-1,and the concentration of PGE2 in the medium was determined by enzyme immunoassay.2.To evaluate the role of COX-2 in IS/UA-induce MAECS proliferation,a specific COX-2 inhibitor(NS-398)was applied to the cells before IS/UA administration,then persuade MAECS with IS/UA for 24 h,and expression of COX-2 was also detected by real-time PCR and western blot.The apoptosis rate of mouse vascular endothelial cells was measured by TUNEL staining.The expression of Caspas 3 and Bax was detected by quantitive real time PCR.The concentration of PGE2 in the medium was determined by enzyme immunoassay.Results:1.The apoptosis rate of mouse vascular endothelial cells was raised in cells which are stimulated with IS compare with control group(increased about 1.65 times),the expression of Caspas 3 and Bax which were considered as apoptosis markers was upregulated at the mRNA and protein level(the expression of Caspas 3 increased about 3.54,1.7 times,respectively,and the expression of Bax increased 2.47,1.8 times,respectively).After treating with UA,the apoptosis rate of cells was raised compare with control group(increased about 3.42 times),and the protein level of Caspas 3 and Bax were increased(increased 1.94,1.9 times,respectively).2.After treating with IS for 24 hours,the RNA level of COX-2 and mPGES-1 were increased(increased about 1.8 times),and the protein expression of COX-2?mPGES-1 increased(increased about 1.73,2.6 times,respectively).Also after treating with UA,,the protein level of COX-2 and mPGES-1 were increased(increased about 1.73,2.6 times,respectively),as with increasing PGE2 level.3.The specific COX-2 inhibitor(NS-398)which was applied to the cells can downregulate COX-2 mRNA and protein level(decreased approximately 40%),and compare with the treating with IS group,inhibited the apoptosis of mouse vascular endothelial cells induced by uremic toxins after TUNEL staining(decreased approximately 33.3%),the expression of Caspas 3 and Bax at RNA level were also reduced(decreased 29.7%,30%,respectively),and the protein expression were reduced(decreased 70%,41%,respectively).While after treating with UA,compare with UA group,inhibited the apoptosis of mouse vascular endothelial cells induced by uremic toxins after TUNEL staining(decreased approximately 34%),and the protein expression of Caspas 3 and Bax were reduced(decreased 41%,45%,respectively).Well the level of PGE2 was abolished by COX-2 inhibition.Conclusions:Uremic toxins induces the mouse vascular endothelial cells injure,possibly by regulating the COX-2/mPGES-1/PGE2 pathway. |
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