Protective Effect Of Erythropoietin On Human Umbilical Vein Endothelial Cells Apoptosis Induced By Intermittent High Glucose

Object Through the apoptosis model of human umbilical vein endothelial cells induced by intermittent high glucose, and explore the pathological effects of intermittent hyperglycemia on diabetes vascular complication. to determine whether erythropoietin can prevent human umbilical vein endothelial cells from damage by intermittent high glucose -induced apoptosis in cultured human umbilical vein endothelial cell and then explore the mechanisms of its anti-apoptotic effect., as well as offering the clues for searching the drugs at preventing diabetes-associated vascular complications.Methods Human umbilical vein endothelial cells (HUVECs) were seeded at equal density in Petri dishes and allowed to attach overnight. Then they were exposed to the experimental conditions for 7 days. Therefore, Three groups were formed, each one receiving the following fresh medium every 24h: (1) continuous normal glucose medium (5.6 mmol/L), (2) normal (5.6 mmol/L)and high glucose(20 mmol/L) media alternating every 24h, (4) Epo was added to the second group media. morphological change was observed with fluorescence microscope. its apoptosis rates and cycle were analyzed by flow cytometry. Gel electrophoresis was used to detect HUVEC DNA fragmentation. Intracellular ROS was detected by Spectrofluorophotometer.Results1) The apoptosis indexes of normal glucose, intermittent high glucose, intermittent high glucose plus 100u/ml Epo were (5.71254±0.37583)%,(22.13754±1.50612)%,(16.31254±0.09910)% respectively. Compared with cells exposed to normal glucose, the apoptosis index of intermittent high glucose was higher(P=0.000). Compared with cells exposed to intermittent high glucose The apoptosis index of cells exposed to intermittent high glucose with EPO was lower(P=0.029).2) The apoptosis of HUVECs shown by micromolecular weight DNA fragment gel electrophoresis after 7 days cultured with intermittent high glucose, DNA electrophoresis showed typical "lad-der" band.3) Annexin V-FITC/PI staining: The data of FCM suggested intermittent high glucose could induce apoptosis. The cells exposed to normal glucose, intermittent high glucose, intermittent high glucose plus 100u/ml Epo for 7 days produced apoptotic rate (6.3125+0.21002) %, (18.2750±0.45277)%, (15.0250±0.26049)%,respectively. Compared with cells exposed to normal glucose, the apoptosis rate of intermittent high glucose was higher(P=0.000). Compared with cells exposed to intermittent high glucose The apoptosis rate of cells exposed to intermittent high glucose with EPO was lower(P=0.032).4) After exposed to intermittent high glucose, the G0/G1 phase cells increased than that of the normal glucose cells(P=0.000), whereas S phase cells decreased (P=0.013), Compared with cells exposed to intermittent high glucose, The G0/G1 phase cells decreased(P=0.023) and S phase cells increased in cells exposed to intermittent high glucose plus 100u/ml EPO (P=0.015).5) After HUVECs were exposed to intermittent high glucose for 7 days, Compared with cells exposed to normal glucose, the production of ROS in intermittent high glucose were increased (P=0.000). There was a significant decrease of ROS production in intermittent high glucose compared with it in intermittent high glucose plus Epo 100u/ml (P=0.017).Conclusions1) Intermittent high glucose further induced the apoptosis of HUVECs in vitro;2) intermittent high glucose could inhibit the proliferation of HUVECs, which was related with blockage of cell cycle;3) Erythropoietin had protective effect on human umbilical vein endothelial cells apoptosis induced by intermittent high glucose. The anti-apoptotic effect of Epo may relate to decrease the production of ROS and attenuate S phase blockage.