| Objective: The aim of this study was to investigate the apoptosis within ECV-304 cell, the expression of nuclear factor-B (NF-κB), vascular cell adhesion molecule-1 (VCAM-1) and receptor for AGEs (RAGE) in ECV-304 cells after advanced glycation end products injured and interfered by Glucagon -like peptide 1 (GLP-1) in different concentration and action time, and to discussion the possible mechanism of protective effect by GLP-1 on endothelial cells in diabetes.Methods:1. AGEs was prepared by human serum album and D-glucose for 90 days at temperature of 37℃.2. ECV-304 cells were incubated in different culture environments with 5.5mmol/L glucose DMEM, 33.3mmol/L glucose DMEM and 5.5mmol/L glucose DMEM + AGEs-HSA, respectively. The apoptosis and the expression of NF-κB, VCAM-1 and RAGE were subsequently determined.3. ECV-304 cells were interfered by AGEs-HSA and GLP-1 with concentration of 0.03nmol/ml in 8, 12 and 24 hours respectively, then the cell supernatant and cell were collected, the apoptosis and the expression of NF-κB, VCAM-1 and RAGE were detected.4. The ECV-304 cell were interfered by AGEs-HSA and GLP-1 with the concentration of 0.03 nmol/ml and 0.3 nmol/ml for 8 hours, then the supernatant and cells were collected, and the apoptosis and the expression of NF-κB, VCAM-1 and RAGE were determined.5. Experimental apoptosis was detected by MTT, NF-κB and VCAM-1 within cell culture supernatant were detected by ELISA detection kit, expression of RAGE within cells were detected by PCR kit. The SPSS 13.0 statistical analysis was performed to analyze the experimental results.Results:1. The cell activity in AGEs group were remarkably decreased compared with control group and high glucose(33.3mmol/L) group(OD:0.065±0.009 vs 0.465±0.073 vs 0.671±0.002),The cell apoptosis were increased and had significant difference.(P<0.05), the expressions of NF-κB in AGEs group were significant higher than that in control group and high glucose group(NF-κB:341.830±11.324 ng/ml vs 197.578±6.863 ng/ml vs 65.776±5.293 ng/ml, P<0.05;VCAM-1:8.362±0.184 ng/ml vs 6.515±0.077 ng/ml vs 4.393±0.211 ng/ml, P<0.05 ; RAGE : 0.376±0.023 vs 0.298±0.024 vs 0.161±0.026, P<0.05).2. After the intervention of AGEs-HSA and GLP-1 (0.03nmol/ml), The cell activity in interfering group 1(5.5mmol/L glucose DMEM + AGEs-HSA +GLP-1 0.03nmol/ml 8H) were remarkably increased compared with AGEs group (OD:0.507±0.089 vs 0.065±0.009),the cell apoptosis were significantly decreased and had significant difference. (P<0.05), and the expression of NF-κB, VCAM-1 and RAGE in interfering group 1 were significant reduced compared with AGEs group (NF-κB:196.829±15.502 ng/ml vs 341.830±11.324 ng/ml,P<0.05;VCAM-1:6.646±0.567 ng/ml vs 8.362±0.184 ng/ml,P<0.05;RAGE:0.165±0.009 vs 0.376±0.023,P<0.05).3. After intervention of AGEs-HSA and GLP-1 with the concentration of 0.03 nmol/ml, The cell activity in interfering group 1 were remarkably increased compared with interfering group 3(5.5mmol/L glucose DMEM + AGEs-HSA +GLP-1 0.03nmol/ml 24H)(OD:0.507±0.089 vs 0.237±0.035),the cell apoptosis in interfering group 1 were significantly reduced and had significant difference (P<0.05); and the expression of NF-κB, VCAM-1 and RAGE in interfering group 1 were also significant decreased (NF-κB: 196.829±15.502 ng/ml vs 287.113±14.165 ng/ml,P<0.05;VCAM-1:6.646±0.567 ng/ml vs 7.484±0.341 ng/ml,P<0.05;RAGE: 0.165±0.009 vs 0.248±0.011,P<0.05).4. After intervention of AGEs-HSA and GLP-1 with the concentration of 0.03nmol/ml and 0.3nmol/ml for 8 hours; The cell activity in interfering group 1 were remarkably decreased compared with interfering group 4(5.5mmol/L glucose DMEM + AGEs-HSA +GLP-1 0.3nmol/ml 8H)(OD:0.507±0.089 vs 0.567±0.050),the cell apoptosis were significant decreased accompanied with increasing of concentration of GLP-1 and had significant difference(P<0.05). The expressions of NF-κB, VCAM-1 and RAGE in interfering group 4 were significant decreased compared with interfering group 1(NF-κB:159.340±9.737 ng/ml vs 196.829±15.502 ng/ml,P<0.05;VCAM-1:6.027±0.628ng/ml vs 6.646±0.567 ng/ml,P<0.05;RAGE:0.143±0.012 vs 0.165±0.009 P<0.05).5. The reduction of RAGE were correlated with NF-κB in each interfering group (r=0.803,P<0.01).Conclusion:1. AGEs-HSA and high glucose could increase the apoptosis and the expression of NF-κB, VCAM-1 and RAGE of ECV-304 cells,destroy endothelial cell function and hence cause diabetic vascular disease. They are important factors for caused diabetic vascular disease.2. GLP-1 would reduced the apoptosis and the expression of NF-κB,VCAM-1 and RAGE within ECV-304 cells after advanced glycation end products damaged, and improve endothelial function.3. The apoptosis and expressions of NF-κB, VCAM-1 and RAGE within ECV-304 cells for 24 hours, however, were increased accompanied with 8 hours intervention of GLP-1. The Protective effects of GLP-1 with 8 hours was better than 24 hours.4. With the increasing of concentration of GLP-1, the apoptosis and expressions of NF-κB, VCAM-1 and RAGE were decreased. In our experiment concentration range, higher concentrations of GLP-1 had better protective effective in diabetic endothelium than that of lower concentration, which can effectively reduce the apoptosis and the expressions of NF-κB, VCAM-1 and RAGE.5. The mechanism of GLP-1 for protection endothelial cells may performed by reduced the expression of RAGE through decreasing of NF-κB levels.
|
PDF Full Text Request