| aims: Diabetic cardiovascular complications begin with endothelial cell dysfunction, andendothelial dysfunction is closely associated with the oxidative stress induced by highblood glucose. Here we investigate the effects and mechanism of glucagon-like peptide-1(GLP-1) and Exendin-4on apoptosis, nitric oxide (NO) bioavailability and mechanismssuch as oxidative stress, phosphatidyl inositol-3-kinase/Akt (PI3K/Akt) signal pathwayand advanced gyration end-products (AGES) metabolic process in human umbilical veinendothelial cells (HUVECs) cultured in high glucose.Materials and methods:(1)HUVECs isolated from human umbilical vein were culturedin M199medium contain endothelial cell growth supplement (ECGS) and randomlydivided into control group,high glucose group and treatment group (high glucose+100nM GLP-1/100nM Exendin-4/200nM Exendin-4).(2) Endothelial dysfunction is characterized by apoptosis acceleration and NObioavailability reduction. The toxicity was evaluated by lactate dehydrogenase (LDH)method. Cell survival and apoptosis rate of HUVECs was observed by4’,6-diamidino-2-phenylindole (DAPI) method and flow cytometry. The cellimpermeablefluorescence indicator DAF-FMDA method was used to detect NO. The mRNA expressionof endothelial nitric oxide synthase (eNOS) was detected by Real Time PCR. The proteinexpression of Cleaved Caspase-3and eNOS was analysed by western blot.(3) Intracellular reactive oxygen species (ROS) production and activity of superoxidedismutase (SOD) reflects the oxidative stress state in the cell. Fluorescenceenzyme-labeled instrument was used to detect the production of ROS. The activity of SODwas detected by xanthine oxidase method.(4)To investigate the mechanism involoved in protection of GLP-1analogue fromendothelial dysfunction, relevant index about oxidative stress, PI3K/Akt signal pathwayand AGES metabolic process were detected. The expression of GLO-1mRNA wasdetected by Real Time PCR. The expression of GLO-1, Akt and p-Akt protein wasanalysed by western blot. The concentration of GLO-1, AGES and advanced glycation end-products receptor (RAGE) was measured by enzyme linked immunosorbent assay(ELISA).Results:(1) GLP-1and Exendin-4could reduce toxicity of high glucose. We found thatafter high glucose trestment, the tatio of LDH leakage was markly increased to181.07%3.35%of the control group. However, the ratio of LDH leakage significantlydecreased in cells pre-treated with100nM GLP-1and100nM,200nM Exendin-4wasdecreased to134.94%1.78%(P＜0.01),136.56%2.18%(P＜0.01) and134.74%1.56%(P＜0.01), respectively.(2) When HUVECs were cultured in high glucose, NO production was reduced by aboutthirty-five percent (P＜0.01) which contributing to endothelial cell dysfunction. AfterGLP-1and Exendin-4pretreatment, Up regulation of NO production (39.33±2.49,40.31±2.22,39.90±2.22vs19.21±1.10, P＜0.01), mRNA expression of eNOS(104.01%±4.72%,101.95%±5.35%,105.16%±5.95%vs48.16%±2.54%, P＜0.01) andprotein expression of eNOS (0.71±0.01,0.72±0.01,0.72±0.02vs0.56±0.01, P＜0.01)means endothelial function was improved.(3) Comparing with control group, ROS production was significantly increased inHUVECs cultured in30mM D-glucose for seven days (300.004.73VS137.587.37, P＜0.01). ROS which beyond the antioxidant capacity make a contribution to intracellularOxidative stress state. After GLP-1(100nM) and Exendin-4(100nM or200nM)pretreatment, ROS was suppressed (156.689.40,158.1411.05,156.529.73vs291.1412.21, P＜0.01), and activity of SOD was increased (39.202.21U/mgprotein,38.171.59U/mgprotein,39.442.65U/mgprotein vs23.811.41U/mgprotein, P＜0.01).(4) GLP-1and Exendin-4could improve the cell and karyon morphology of HUVECscultured in high glucose, which was detected by DAPI, reduce apoptosis detected by flowcytometry (28.28±2.06%,28.64±1.83%,28.60±1.86%vs39.13±1.53%, P＜0.01), anddown regulate protein expression of Cleaved Caspase-3(0.33±0.02,0.32±0.02,0.32±0.02vs0.3842±0.01155, P＜0.01).(5) GLP-1and Exendin-4may regulate metabolic pathway of AGES. mRNA expression ofGLO-1up regulated from80.03%±2.34%to101.11%±2.60%,101.95%±3.32%or 108.93%±7.15%(P＜0.01). Protein expression of GLO-1detected by ELISA up regulatedrespectively from1.51±0.11ng/mgprotein to about2.34±0.17ng/mgprotein (P＜0.01) andfrom1.08±0.028to about1.34±0.03(P＜0.01), while hazardous substance like AGES wasreduced (P＜0.01).(5) GLP-1and Exendin-4pretreatment protect endothelial cell from dysfunction whileactivating PI3K/Akt signaling pathways, such as stimulating protein expression of p-aktprotein.Conclusion: In vitro studies with human umbilical vein endothelial cells showed thatGLP-1and analogue had a direct protective effect on endothelial cell function such asapoptosis process and NO bioavailability under high glucose condition. Further analysisrevealed that oxidative stress scavenger, glyoxalase-1and intracellular signaling by thePI3K/Akt survival pathway may be involved in this protective effect. |
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