Effects Of Intermittent High Concertration Glucose On Survival,Apoptosis And The Changes Of ICAM-1 And TNF-α In Cultured Human Umbilical Vein Endothelial Cells

Objective:It was studying the impact of intermittent high concertration glucose and constant high concertration of glucose on survival and apoptosis of HUVECS and the effects on expression of ICAM-1 and TNF-α.At the same time,we explored its impossible mechanism.Methods:We divided HUVECS into three groups:(1)continuous normal concertration glucose medium(5.5mmol/L).(2) continuous high concertration glucose(25mmol/L).(3) high concertration glucose(25mmol/L)3h normal concertration glucose(5.5mmol/L)2h media altermating three times at day,high concertration glucose(25mmol/L)at night.The former two groups were received the following Fresh medium every 24h.After 3d,by MTT,AnnxenV-FITC,we detected the rate of cell apoptosis and survival and explored the function of intermittent high concertration glucose(5.5/25mmol/L) and constant high concertration glucose(25mmol/L) on HUVECS survival.Morphologieal change was observed with microscope.Its survival rates was detected by MTT and its apoptosis rates and cycle were analysed by flow cytometry.The level of ICAM-1 and TNF-αwas measured by ABC-enzyme linked immunosorbent assay(ABC-ELISA).Results:1)The morphology changes of HUVECS were same in normal concertration glucose group after 24h,48h,72h.The changes of normal concertration glucose and high concertration glucose conditions weren't obvious after 24h.There were a few apoptosis cells in intermittent glucose group after 24h.The apoptosis rate of intermitten high glucose was higher than constant high glucose after 48h.These changes were very obviously after 72h.HUVECS had changed from full to narrow and these changes were depended on the longer time.2)The rate of survival on HUVECS by MTT:In constant high concertration glucose conditions,cell survival rate is lower than that in the normal concertration condition,and under intermittent high concertration glucose condition,cell survival rate is lower than that under the constant high concertration glucose condition.Following longer time,the effect is more obvious.high concertration glucose,intermittent high concertration glucose were 6.6%,7.9%, 18.8%respectively.Com pared with cells exposed to normal concertration glucose and constant high concertration glucose respectively,the apoptosis rate of intermittent high concertration glucose was higher(P=0.000).Compared with normal glucose,the apoptosis rate of constant high concertration glucose was lower(P=0.000).After 72h,the G₀/G₁ Phase cell of intermittent high concertration glucose increased than the normal concertration glucose cells and constant concentration high glucose(P=0.000),whereas S and G₂/M phase cells decreased (P=0.013).Compared with normal high concertration glucose,the G₀/G₁ phase cell of constant high concertration glucose were higher,but the change was unobviously(P>0.05).4)The level of ICAM-1 by ELISA:The level of ICAM-1 is 6.144±0.170ng/mL,7.074±0.203ng/mL,9.155±0.900ng/mL in normal concertration glucose,constant high concertration glucose,intermittent high concertration glucose respectively after 24h,compared with between intermittent high concertration glucose and other groups,they increased significantly(p<0.001) Between the normal concertration glucose and constant high concertration glucose,the change wasn't significant(P>0.05).After 48h and 72h,compared with three groups,the changes were significant(p<0.05).But compared with the constant high concertration glucose and intermittent high concertration glucose,the change wasn't significant (P>0.05).5) The level of TNF-αby ELISA:,compared with three groups,intermittent high concertration glucose and normal concertration glucose,constant high concertration glucose. After 24h,48h,72h,the changes were significant(p<0.05).Compared with the constant high concertration glucose and intermittent high concertration glucose,the change wasn't significant (P>0.05).Within the normal concertration glucose group,compared with 24h,48h,72h,the changes weren't significant(P>0.05).But within the constant high concertration glucose group and intermittent high concertration glucose group,the change was significant(p<0.05).Conclusion:Compared with the constant high concertration glucose,intermittent high concertration glucose has the more strong effects on apoptosis of HUVECS.Intermittent high concertration glucose could inhibit the proliferation of HUVECS,which was related with blockage of cell cycle.Compared with the constant high concertration glucose,intermittent high concertration glucose can strengthed the expression of ICAM-1 and TNF-αon HUVECS.