| Purpose:A large number of previous studies have demonstrated that advanced glycation end products(AGEs)can cause endothelial cell dysfunction and apoptosis.AGEs lead to endothelial cell damage mainly by binding to cell surface receptor RAGE,thereby activating a series of signal molecules in the cell,especially by causing the up-regulation of NF-?B expression to cause subsequent inflammatory reaction damage.The endogenous secretory receptor for advanced glycation end products(esRAGE)is a variant of the RAGE gene that can traps extracellular RAGE ligands and blocks the activation of RAGE,thereby protecting endothelial cell.In this study,human umbilical vein endothelial cells(Huvecs)were over-expressed in esRAGE through gene recombination.The protective effect of esRAGE on endothelial cell apoptosis induced by AGEs and its mechanism were studied by measuring the expression of NF-?B,apoptosis-related protein bcl-2?Bax and apoptosis.Methods:(1)Construction of esRAGE over-expressed Huvecs by lentiviral vectors: Huvecs were infected with esRGAE over-expressed lentivirus,the expression of the marker protein GFP(green fluorescent protein)was observed under a fluorescent microscope;qRT-PCR was used to detect the RNA expression level of esRAGE in endothelial cells before and after viral infection;ELISA was used to determine the expression level of esRAGE protein in the culture supernatant of endothelial cells before and after viral infection,in order to determine whether the esRAGE overexpressed lentivirus successfully transferred to Huvecs.(2)Effects of different concentrations of AGEs on Huvec apoptosis: Human umbilical vein endothelial cells were incubated with three kinds of gradients(50ug/ml,100ug/ml,200ug/ml)of AGEs and BSA for 24 h respectively.Hoechst-pi double staining method for staining cells,Ipwin32 software counts apoptotic cells and total cells and calculates the rate of apoptosis.(3)Effects of over-expression of esRAGE on the function of human umbilical vein endothelial cells: The experiment was divided into four groups: control group,AGEs-group,AGEs+control-virus-group,AGEs+Lv-esRAGE-(esRAGE overexpressed Lentivirus)group.QRT-PCR and western-blot were used to detect the RNA and protein expression levels of pro-inflammatory cytokines NF-?B and apoptosis-related protein bcl-2?Bax respectively.Results:1.Human umbilical vein endothelial cell line over-expressed esRAGE was constructed successfully: a.The expression of GFP in cells infected by lentivirus was more than 80%;b.The RNA expression level of Human umbilical vein endothelial cell over-expressed esRAGE was 1532 times that of normal cells;the esRAGE protein concentration in the supernatant of over-expressed esRAGE group and the control group was 233.80ng/ml±12.764 vs 0.18 ng/ml±0.005,respectively,P<0.001.2.After co-incubation with three concentrations(50ug/ml,100ug/ml,and 200ug/ml)of AGE-BSA for 24 h,the endothelial cells apoptosis rate increased with the increase of AGEs concentration,and the apoptosis rates were:(5.334 +0.188)% vs(10.989 +1.277)% vs(23.273 +2.124)%,respectively,p<0.001.It is suggested that AGE-BSA can induce apoptosis of endothelial cells obviously,and the apoptosis rate has a concentration-dependent increase.Therefore,200ug/ml AGEs was used as the apoptosis-inducing concentration of endothelial cells.while in the BSA group,the apoptosis rate of endothelial cells incubated with three concentrations(50ug/ml,100ug/ml,200ug/ml)of BSA for 24 h was(2.062+0.355)% vs(2.312+0.244)% vs(2.287+0.163)% respectively,there was no statistical difference compared with the blank control group((2.058 + 0.210)%),P = 0.632.3.Effects of over-expression of esRAGE on the function of human umbilical vein endothelial cells and its mechanisms.3.1 Effects of over-expression of esRAGE on apoptosis of human umbilical vein endothelial cells: Over-expression of esRAGE significantly reduced AGEs induced apoptosis of endothelial cells: The rate of apoptotic cells in the AGEs+Lv-esRAGE group((2.151 ± 0.002)%)was significantly lower than that in the AGEs group((23.273 ±0.021)%),P<0.001.The apoptotic rate in the AGEs+control virus group((23.667 ±0.031)%)was not significantly different from that in the AGEs group((23.273 ±0.021)%),p=0.839.3.2 The effect of over-expression of esRAGE on the expression of apoptosis-related factors in human umbilical vein endothelial cells: AGEs incubated up-regulated apoptosis-related factor Bax expression and down-regulated anti-apoptosis factor bcl-2 expression levels.Over-expression of esRAGE can down-regulate the expression of Bax induced by AGEs and up-regulate the expression of bcl-2.Compared with the control group,the expression of Bax RNA was up-regulated in the AGE-BSA group(1.003±0.075 vs 1.346±0.095,p=0.001).There was no significant difference between the AGE-BSA group and the AGE-BSA+control virus group(1.346±0.095 vs 1.279±0.014,p=0.322).The AGE-BSA+Lv-esRAGE group had a lower Bax RNA expression level compared with the AGE-BSA group(0.981±0.033 vs 1.346±0.095,p<0.001).Compared with the control group,the expression of Bax protein was up-regulated in the AGE-BSA group(0.581±0.202 vs 0.856±0.033,P=0.048).There was no significant difference between AGE-BSA group and AGE-BSA+control virus group(0.856±0.033 vs 1.013±0.091,p=0.221).Compared with AGE-BSA group,the expression of Bax protein was down-regulated in AGE-BSA+Lv-esRAGE group(0.432±0.077 vs 0.856±0.033,P=0.007).There was no statistically significant difference in bcl-2 RNA expression levels between the four groups.Compared with the control group,the expression level of bcl-2 protein was down-regulated in the AGE-BSA group(0.720 ± 0.086 vs 0.411 ± 0.023,p=0.002).There was no significant difference between the AGE-BSA group and the AGE-BSA+control virus group(0.411±0.023 vs 0.383±0.074,p=0.704).The AGE-BSA+Lv-esRAGE group compared with the AGE-BSA group showed an up-regulation of bcl-2 protein expression(0.755±0.081 vs 0.411±0.023,p=0.001).3.3 Effect of over-expression of esRAGE on the expression of NF-?B in human u-mbilical vein endothelial cells: Incubation of AGEs promoted the expression of pro-inflammatory cytokines NF-?B.Over-expression of esRAGE decreased the expression of NF-?B.Compared with the control group,the NF-?B RNA expression level was up-regulated in the AGE-BSA group(1.004±0.080 vs 1.300±0.127,p=0.016);the protein expression level was up-regulated(0.470±0.025 vs 0.926±0.170,P=0.018).There was no significant difference between AGE-BSA group and AGE-BSA+control virus group,RNA(1.300±0.127 vs 1.260±0.119,p=0.690);protein(0.926±0.170 vs 0.985±0.230,p=0.709).The AGE-BSA+Lv-esRAGE group compared with AGE-BSA group,the expression level of NF-?B RNA was down-regulated(0.918±0.041 vs 1.300±0.127,p=0.004);the protein expression level was down-regulated(0.410±0.109 vs 0.926±0.170,P=0.01).Conclusion:1.Human umbilical vein endothelial cells can be used as esRAGE gene vectors,and esRAGE gene-transfected Huvecs can secrete esRAGE.2.AGEs can significantly induce Huvec apoptosis,significantly increase the expression level of pro-apoptotic factor Bax,and down-regulate the expression level of anti-apoptosis factor bcl-2.Over-expression of esRAGE can significantly reduce the apoptosis of endothelial cells induced by AGEs,down-regulate the expression level of Bax and up-regulated the expression level of bcl-2.3.AGEs can induce inflammatory reaction in Huvec and significantly increase the expression level of NF-?B in Huvec.Over-expression of esRAGE can significantly reduce AGEs induced inflammatory responses and significantly reduce the expression of NF-?B in Huvec.4.EsRAGE has a protective effect on AGEs induced apoptosis of endothelial cells,and its protective mechanism is mainly through down-regulating the expression of pro-apoptotic factors and inflammatory factors and up-regulating the expression of anti-apoptosis factors. |
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