Objective: To investigate the changes in reactive oxygen species (ROS) and dimethyl- arginine dimethylaminohydrolase-asymmetric dimethylarginine (DDAH-ADMA) system in the process of endothelial cell senescence after exposure to high glucose.
Methods: The human umbilical vein endothelial cells (HUVECs) were cultured with different concentrations of glucose, e.g. 5.5 mmol/L (normal level), and high levels as 11.0, 22.0 and 33.0 mmol/L, for 48 hours, respectively. Subsequently, SA-β-gal staining was used to evaluate senescence of cells. Telomerase activity was detected by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). The intracellular ROS level was measured by flow cytometry. The ADMA concentration and DDAH activity were determined with high-performance liquid chromatography.
Results: Compared with normal glucose concentration group, after the endothelial cells were treated with high glucose concentration (11.0-33.0 mmol/L) for 48 hours, the number of SA-β-gal positive cells was increased significantly [(7.00±1.73)%, (12.67±2.03)%, (16.00±2.26)% vs. (4.00±1.33)%, P>0.05, P<0.05, P<0.05] and the telomerase activity was inhibited dramatically [(91.32±4.01)%, (78.44±3.78)%, (56.04±3.35)% vs. 100%, all P<0.05]. The ROS level (mfi) was increased in all high glucose groups (159.84±27.52, 188.99±18.77, 244.56±20.96 vs.117.11±18.76, P<0.05 or P<0.01). At the same time, the ADMA (μmol/L) production was increas ed (0.78±0.14, 0.88±0.18, 1.08±0.15 vs. 0.70±0.12, P>0.05, P<0.05, P<0.05), and DDAH activity was decreased [(91.32±4.01)%, (78.44±3.78)%, (56.04±3.35)% vs.100%, all P<0.05].
Conclusion: High glucose can accelerate endothelial cells senescence in dose-dependent manner and the underlying mechanism may be related to an increased oxidative stress and change in DDAH-ADMA system.