High Glucose Induces Endoplasmic Reticulum Stress In COX-2 Dependent Manner In HUVECs

BackgroundDiabetes mellitus can cause a wide variety of cardiovascular diseases and vascular complications.Loss of the modulatory role of the endothelium may be a critical and initiating factor in the development of diabetic vascular disease.That hyperglycemia increases apoptosis in endothelial cells was first demonstrated by Barmgartner-Parzer et al in human umbilical vein endothelial cells(HUVECs).But the mechanism is still unclear.In addition to this mitochondrial apoptotic pathway, recent studies showed that the endoplasmic reticulum(ER)is another intrinsic apoptosis pathway.The ER is a central organelle entrusted with protein synthesis, folding and maturation.Perturbations of ER homeostasis affect protein folding and cause unfolded protein response(UPR)or ER stress.The chemical toxicants, oxidative stress and/or accumulation of misfolded proteins in the ER can all disrupt ER function,resulting in ER stress.Hyperglycemia induces mitochondrial ROS production,which can associate to the pathogenesis of diabetic vascular complications.So we hypothesized that ER stress might be involved in the high glucose induces apoptosis in HUVECs.Recently,it has been reported that inflammation is another key factor that involved in the vascular complications.And endothelial derived cyclooxygenase-2 (COX-2)is suggested as an important factor in the process of high glucose-induced inflammation.Whether COX-2 is also involved in the high glucose induced ER stress is still unknown. Objectives(1)To investigate the effect of high glucose on the expression of two ER stress proteins glucose-regulated protein 78(GRP78)and C/EBP homologous protein (CHOP).(2)To explore the role COX-2 in the high glucose-induced ER stress. Methods(1)Human umbilical vein endothelial cells(HUVECs)were cultivated and then were exposed to normal glucose(5.5mmol/L)and high glucose(30mmol/L)for 24, 36,or 48 h.(2)Cell viability was determined by MTT method.(3)Cell apoptosis was determined by PI staining flow cytometry.(4)The expression of COX-2, GRP78 and CHOP proteins were also evaluated by western blotting analysis.ResultsPartⅠEffect of high glucose on cell viability in HUVECsAfter incubation with high glucose(30 mmol/L)for 24,36 and 48h, respectively,the cell viability of HUVECs was measured by MTT.The results showed that the OD value decreased in a time-dependent manner from 24h to 48h. Compared with the control group,mannitol has no effect on the cell viability.PartⅡEffect of high glucose on the expression of GRP78 and CHOP proteinsThe results by Western Blotting show that after incubation with high glucose for 24h,36h and 48h,respectively,the expression of GRP78 protein increased at 24h and 36h,and then decreased at 48h.However,the expression of CHOP protein of HUVECs increased after incubated with high glucose for 36 to 48 h.Mannitol, functions as osmatic control,did not have the effect on the expression of GRP78 and CHOP proteins.PartⅢEffect of high glucose on COX-2 protein expressionThe results by Western Blotting show that after incubation with high glucose for 24h,36h and 48h,respectively,the COX-2 expression increased in a time-dependent manner.The expression of COX-2 for 48h is about 3.5 times higher than normal glucose group.PartⅣEffect of COX-2 selective inhibitor nimesulide on the expression of GRP78 and CHOP proteinsThe results by Western Blotting show that after incubation with high glucose for 48h,the expression of GRP78 decreased but expression of CHOP increased compared with those of normal glucose group.HUVECs co-incubated with nimesulide and high glucose,the expression of GRP78 increased but the expression of CHOP decreased compared with those of high glucose group.This indicates that COX-2 was involved in the changes of GRP78 and CHOP proteins expression induced by high glucose.PartⅤEffect of nimesulide on the high glucose-induced decrease of cell viability and increase of apoptosisAfter co-incubvation with high glucose(30 mmol/L)and nimesulide 25μmol/L)for 48h,the cell viability of HUVECs was measured by MTT method and the cell apoptosis was measured by Flow Cytometry(FCM).After incubation for 48h,the number of apoptotic cell increased and cell viability declined.Compared with high glucose group,the cell viability was higher and the number of apoptotic cell was lower in the group co-incubated with nimesulide and high glucose.ConclusionAfter incubated with high glucose for 48 h,the cell viability decreased and the number of apoptotic cells increased in HUVECs.The mechanism might be involved in the decrease in GRP78 protein expression and increase in CHOP protein expression.And the results also suggested that high glucose induced ER stress in COX-2 dependent manner.