Effects Of Advanced Glycation End Products(AGEs) On The NO And Its Mechanism

Objective: This study was performed to investigate the effects of advanced glycation end products(AGEs)modified protein on cultured human umbilical vascular endothelial cells( HUVECs). For this purpose, we examined the concentration of the NO, MDA, SOD ,NOSin the cellular cultured medium and the expression of the DDAH2 in the HUVECs. Methods: Human umbilical vein endothelial cells (HUVEC) - derived cell line (ECV304) grown on gelatin-coated glass bottom microwell dishes were allowed to reach confluence and subjected to AGE-HSA treatments. AGE-modified human serum albumin(AGE-HSA) was prepared by incubation 1.75mg/ml human serum albumin with 100mmol/l of D-glucose for 8 weeks. Human umbilical vein endothelial cells were incubated with AGE-HSA in different concentrations (25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml) and timing(24h, 48h). The supernatant was stored at -20°.Then 20ul of 5mg/ml MTT were added and the optical density ( OD ) at each concentration was determined in order to calculate the proliferation-inhibiting rate of cells. NO, SOD levels and MDA contents in the supernatant were determined by using spectrophotometer. Using immunocytochemistry, we detected the expression of DDAH2 in the Human umbilical vein endothelial cells and the effect of the RAGE on the Human umbilical vein endothelial cells.Results:1 Effects of AGEs on the growth of Human umbilical vein endothelial cells.1.1 The proliferation-inhibiting rates was no statistically significant between the two groups of AGEs25ug/ml, 50ug/ml(P>0.05). The proliferation- inhibiting rates (AGEs100ug/ml, 200ug/ml, 400ug/ml) was significantly higher than those in the AGEs25ug/ml, 50ug/ml(P<0.05).The proliferation- inhibiting rates was no statistically significant between the two groups of AGEs100ug/ml, 200ug/ml(P>0.05). The proliferation-inhibiting rates was no statistically significant between the two groups of AGEs200ug/ml, 400ug/ml (P>0.05). The proliferation-inhibiting rates was statistically significant between the two groups of AGEs 100ug/ml, 400ug/ml(P<0.05).1.2 The proliferation-inhibiting rates was no statistically significant,when the human umbilical vein endothelial cells were cultuered in the presence of the AGEs 25ug/ml at the 24h and 48h(P >0.05). The proliferation-inhibiting rates was no statistically significant,when the human umbilical vein endothelial cells were cultuered in the presence of the AGEs50ug/ml at the 24h and 48h(P >0.05). The proliferation-inhibiting rates was statistically significant, when the human umbilical vein endothelial cells were cultuered in the presence of the AGEs100ug/ml , 200ug/ml , 400ug/ml at the 24h and 48h(P <0.05).2 Effects of AGEs on the NO levels of Human umbilical vein endothelial cells.2.1 The human umbilical vein endothelial cells were cultured in vitro with AGE modified human serum albumin for the 24h at the concentraiton of 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml. The NO levels in the 25ug/ml AGEs was significantly higher than those in the 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml AGEs(P <0.05). The NO levels in the 50ug/ml AGEs was significantly higher than those in the 100ug/ml, 200ug/ml, 400ug/ml AGEs(P <0.05). The NO levels in the 100ug/ml AGEs was significantly higher than those in the 400ug/ml AGEs(P <0.05). The NO levels in the 200ug/ml AGEs was no significantly higher than those in the 400ug/ml AGEs(P >0.05).2.2 The human umbilical vein endothelial cells were cultured in vitro with AGE modified human serum albumin for the 24h and 48h at the concentraiton of 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml AGEs. The NO levels in the 24h were significantly higher than those in the 48h at at the concentraiton of 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml AGEs(P <0.05).3 Effects of AGEs on the activity of the SOD of Human umbilical vein endothelial cells.3.1 The human umbilical vein endothelial cells were cultured in vitro with AGE modified human serum albumin for the 24h at the concentraiton of 25ug/ml, 50ug/ml, 100ug/ml, 00ug/ml, 400ug/ml. The activity of the SOD was no statistically significant between the two groups of AGEs-HSA 25ug/ml, 50ug/ml(P >0.05). The activity of the SOD was statistically significant at the three groups of AGEs-HSA 100ug/ml, 200ug/ml, 400ug/ml(P <0.05).3.2 The human umbilical vein endothelial cells were cultured in vitro with AGE modified human serum albumin for the 24h and 48h at the concentraiton of 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml AGEs. The activity of the SOD was no statistically significant between the 24h and the 48h(P >0.05).4 Effects of AGEs on the contents of the MDA of Human umbilical vein endothelial cells.4.1 The human umbilical vein endothelial cells were cultured in vitro with AGE modified human serum albumin for the 24h at the concentraiton of 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml. The contents of the MDA was no statistically significant between the two groups of AGEs-HSA 25ug/ml . 50ug/ml(P >0.05). The contents of the MDA was no statistically significant between the two groups of AGEs-HSA 50ug/ml, 100ug/ml(P >0.05). The contents of the MDA was statistically significant at the three groups of AGEs-HSA 100ug/ml, 200ug/ml, 400ug/ml(P <0.05).4.2 The human umbilical vein endothelial cells were cultured in vitro with AGE modified human serum albumin for the 24h and 48h at the concentraiton of 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, 400ug/ml AGEs. The contents of the MDA was no statistically significant between the 24h and the 48h(P >0.05).Conclusion:The inhibitory action of AGEs to the secretion of in vitro culture allantoic veins endothelial cell NO, It maybe implemented by cutting down DDAH-2 expression and oxidative stress.1 AGEs can promote anthropo-umbilical vein endothelial cell apoptosis in vitro culture, and to be the dependence of density and time.2 AGEs can have the inhibition to the endothelial cell of anthropo-allantoic veins in vitro culture, and offer the concentration and time dependence. 3 For AGEs of in vitro human umbilical vein endothelial cells in the activity of SOD inhibition of concentration dependence. But in two time points 48h, 24h, there is no statistical significance .