Effect Of Glucose In Different Concentrations On Nitric Oxide And Endothelial Nitric Oxide Synthase Produced By Endothelial Cells

Objective To investigate the effect of glucose in different concentrations on nitric oxide, superoxide anion and endothelial nitric oxide synthase produced by endothelial cells in vitro.Methods PIEC were incubated in RPMI-1640 containing 10%fetal bovine serum (FBS),penicillin 100U/ml and streptomycin 100μg/ml.The 6th passage of PIEC were digested with 0.25%trypsin,counted with cytometer chamber and inoculated on 24 well culture plates.Cells densities were 4×10⁵/well.In 24 hours,adhesion was accomplished and the cells grew well.When 70%-80%cells confluenced,serum concentration in the medium was changed to 2%to keep the cells in the quiescent state.Then the cells were divided into the control group,common group and the elevated glucose group randomly,separately added different concentrations of glucose(5.5 mmol/L,11.1 mmol/L, 22.2 mmol/L).Cells were grown to confluence in humidified air(5%CO₂)at 37℃and were administered for 24h,72h and 1 week for detection,5 wells repeated per group.In order to reduce the influence on the results of cell number,cells were passaged and counted 24 hours before time-point.We evaluated NO production by measuring levels of nitrite(NO₂^-),the oxidized product of NO,by Griess reaction.The concentration of nitrite was measured by colorimeter.In order to eliminate the interference of nitrite coming from culture medium, blank control group were set up.Nitrite could reduce WST-1 to produce chromatic dissolved materials;eNOS could catalytically generate NO and NO could produce colored compounds with nucleophile,chemical chromatometry were used to detect absorption value under the certain wave length(450nm and 530 nm),which could reflect superoxide anion and eNOS activity.Results The common group for 24 hours increased the activity of eNOS (0.95±0.27U/L P＜0.05),the production of superoxide anion(1.55±0.37 P＜0.05) and NO(77.80±9.05μmol/L P＜0.05) compared with control,the elevated glucose group increased the activity of eNOS(1.38±0.12 U/L P＜0.05),the production of NO (91.07±11.36μmol/L P＜0.05) and superoxide anion(2.64±0.11 P＜0.01),too.For 72 hours the common group increased the activity of eNOS(1.07±0.13 U/L P＜0.05) and production of NO(89.84±8.03μmol/L P＜0.05) and superoxide anion(4.86±0.42 P ＜0.01),the elevated glucose group increased the activity of eNOS(1.55±0.10 U/L P＜0.01) and production of NO(105.54±6.41μmol/L P＜0.05) and superoxide anion (5.65±0.76 P＜0.01).The exposure to high level of glucose for 1 week significantly decreased the activity of eNOS and production of superoxide anion and NO compared with control.Exposured to 11.1 mmol/L glucose the activity of eNOS is 0.47±0.06 U/L,the production of NO is 41.59±8.21μmol/L and superoxide anion is 2.11±0.56(P＜0.05).Exposured to 22.2 mmol/L glucose the activity of eNOS is 0.31±0.16 U/L,the production of NO is 31.05±5.22μmol/L and superoxide anion is 1.75±0.47(P＜0.05).With the time,both common group and elevated glucose group increased the production of NO initially and then decreased as well as superoxide anion release,and elevated the activity of eNOS at first,then depressed(P＜0.05).Conclusion This study demonstrates that elevated concentrations of glucose increase the production of NO initially and then decrease as well as superoxide anion release,and elevate the activity of eNOS at first,then depresse,dose-dependently.