The Effect Of Uric Acid On The Vascular Endothelial Cells By Oxidative Stress And Its Cell Injury

ObjectiveTo observe the effect of oxidative stress, cell apoptosis and necrosis in human vascular endothelial cells(HUVEC) stimulated by uric acid(UA).MethodsHUVEC were cultured in vitro,stimulated by UA of different concentrations(0,0.05,0.10,0.20,0.30g/L) with different time (6,12,24,48h).Flow Cytometry was used to analyze cell apoptosis, necrosis and level of ROS in HUVEC;Spectrophotography was used to test the concentration of superoxide anion(O2-) in HUVEC; ELISA was used to test the expression of NADPH,XO,eNOS,COX-2and LOX in the supernatant fluid of HUVEC culture.DPI(an inhibitor of NADPH), Allo(an inhibitor of XO),eNOSR(an inhibitor of eNOS) and UA were co-cultured with HUVEC,and then to test the expression of NADPH,XO,eNOS,COX-2and LOX.Results1.HUVEC apoptosis significantly increased,after stimulated by UA of different concentrations(0,0.05,0.10,0.20,0.30g/L) with different time (6,12,24,48h),and it gradually increased following increased concentration or extended time of UA;HUVEC necrosis significantly increased as cultured with UA of0.30g/L or48h. 2.Both the expression of ROS and O2-in HUVEC significantly increased after stimulated by UA of different concentrations (0,0.05,0.10,0.20,0.30g/L) with different time (6,12,24,48h), and it gradually increased following increased concentration or extended time of UA; the expression of ROS and O2-in HUVEC had significant increase as cultured in the0.30g/L or48h of UA. The express of ROS and O2-was positively related to cell apoptosis.3.After cultured with different concentrations of UA(0,0.05,0.10,0.20,0.30g/L) for24h,the produce of NADPH,XO and eNOS significantly increased following increased UA, and they were positively related to cell apoptosis;but the express of COX-2and LOX had no obvious change,and were no related to cell apoptosis.4. As HUVEC had been co-cultured with different oxidase inhibitors and UA for24h, the expression of NADPH,XO and eNOS was activated, DPI inhibited the level of NADPH and eNOS,Allo inhibited the level of XO and eNOS,eNOSR inhibited level of eNOS,and both of DPI,Allo and eNOSR can decreased express of ROS and O2-.The express of ROS and O2-had significantly decreased in the group of UA+DPI+Allo,comparing with the group of UA+Allo,but had no significant difference comparing with the group of UA+DPI.Comparing with the group of UA+DPI or UA+eNOSR, the express of ROS and O2-had significantly decreased in the group of UA+DPI+eNOSR. Conclusion1.UA induced HUVEC injury in a concentration-depended and time-depended manner.2.UA induced HUVEC injury through activating oxidative stress and mainly by activing NADPH and eNOS.