The Effects Of Glucagon Like Peptide-1 Against The Injury Of Oxidative Stress Induced By High Glucose And Its Mechanism

Objective To investigate the effects glucagon like peptide-1(GLP-1) on oxidative stress of the neonatal SD rat cardiomyocytes, which was induced by high glucose, and explore the mechanism of GLP-1 when it played the role against the injury of oxidative stress.Methods Primary 72～96 hours cardiomyocytes, which were obtained from neonatal rats by enzymatic digestion method and had been indentified by immumofluorescence method were used in experiment. Divided them into 5 groups:groupⅠ:normal glucose group :cultured with 5.5mmol/L DMEM, groupsⅡ:cultured with 25mmol/L DMEM, groupⅢ:high glucose+GLP-1 group:cultured with 25mmol/L DMEM containing 10nmol/L GLP-1, groupⅣ:high glucose +GLP-1+LY49002 group:cultured with 25mmol/L DMEM including 10nmol/L GLP-1 and 20μmol/L LY294002, groupⅤ:high osmolarity control group:cultured with 5.5mmol/L DMEM containing 19.5mmol/L mannitol.72 hours latter, supernatant and cells were collected for detection. The content of LDH was detected by chemistry chromatometry. The content of MDA was detected by TBA colouration method. The content of SOD was detected by xanthine oxidase method. The rate of cardiomycocytes apotosis was detected by agarose gel electrophoresislevels and Annexin V-FITC/PI flow cytometry.Results(1) The identification of cardiomyocytes:most cells displayed green fluorescence under fluorescent microscopy, which accords with the characteristics of cardiomyocytes.(2) Comparison of the levels of MDA in different groups:Compared with group H group N was increased (31.28±4.60 vs 3.80±1.62, P＜0.05), group HG was decreased (10.41±2.72 vs 31.28±4.60, P＜0.05); compared with group HG group HGL was increased (41.84±7.23 vs 10.41±2.72, P＜0.05); there is no statistically significant between group S and group N (4.31±2.36vs3.80±1.62), P＞0.05).(3) Comparison of the levels of SOD in different groups:Compared with group H group N was decreased (30.40±2.62 vs 58.51±6.58); group HG was increased (46.36±2.85 vs 30.40±2.62); compared with group HG group HGL was decreased (32.12±3.19 vs 46.36±2.85); there is no statistically significant between group S and group N (57.82±3.97 vs 58.51±6.58, P＞0.05).(4) NADPH P47 phox subunit mRNA:analysis of PCR products of agarose gel electrophoresis:the degree of brightness of GAPDH straps were coincident in each group. Strap of NADPH P47 phox subunit area:compared with group H, group H was brighter than N while group HG was darker. Group HGL was brighter than HG. There is no significant variant of brightness between group S and N.(5) Comparison of the levels of ROS in different groups:cells of ROS positive displayed green fluorescence under fluorescent microscopy. Group N and S almost showed no positive cells.Group H showed obvious cells displaying green fluorescence. HG group showed less cells and less degree of green fluorescence than H.HGL showed cells displaying green fluorescence and more degree.(6) The results of flow cytometry of ROS positive rate in cells:coincidence with the tendency showed under fluorescent microscopy. Group H vs N (36.97±2.87 vs 1.83±1.2),Group HG vs H (19.04±3.79 vs36.97±2.87), Group HGL vs HG (58.65±8.19 vs 19.04±3.79, P＜0.05), Group H vs S (1.83±1.2 vs 1.13±0.21, P＞0.05).(7) The results of flow cytometry:group H and S showed no apoptosis of cells and most cells distributed B3 district; the rate of apoptosis in group H is 11.2±1.68 respectively, there is statistically significant compared with group N, P＜0.05; the rate of apoptosis in group HG is 6.27±1.00, the rate of apoptosis decreased compared with group H, the apoptosis rate of group HGL is increased compared with group HG, P＜0.05.Conclusion(1) High glucose can induce the injury of oxidative stress and apoptosis of cardiomyocytes;(2) GLP-1 can reverse the injury and apoptosis and play a protective role on the injury and apoptosis of cadiomyocytes;(3) LY294002, the inhibitor of PI3K-Akt channel, can inhibit the protective role of GLP-1.