Mechanisms Of NOX4 Mediated Apoptosis In Human Umbilical Vein Endothelial Cells Induced By High Glucose

Background:The diabetes mellitus is an important risk factor of the cardiovascular diseases, and hyperglycemia is thought to be one of some important causes of vascular complications. High glucose can change the profile and function of endothelial cells,which is the early marker of vascular complications.It is reported that high concentration of glucose contributes to endothelial damage induced mainly by oxidative stress. Reactive oxygen species (ROS) in human vascular system are mostly derived from nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). The seven NOX isozymes called NOX1, NOX2, NOX3, NOX4, NOX5,DUOX1 and DUOX2 have been found in different organs in recent years.Both NOX2 and NOX4 are expressed in endothelial cells,and the level of NOX4 mRNA in endothelial cells is twenty times than NOX2. Latest studies indicated that NOX4 is the most important enzyme for the ROS generation in vascular endothelial cells. It is reported NF-κB and p38MAPK sigal pathway play a central role in various cells apotosis. Our dates showed that high concentration of glucose could upregulate the expression of NOX4 and stimulate ROS production and apotosis in HUVECs. Furthermore, we transfected NOX4 siRNA into HUVECs, and observed the change of NOX4 and cell apoptosis. Finally we investigated the role of NF-κB and p38MAPK pathways involved in apoptosis of HUVECs induced by high glucose.Objectives: To investigate the change of NOX4 expression and cell apoptosis after transfection of NOX4 siRNA into HUVECs and elucidate the mechanisms of high glucose-induced apoptosis in HUVECs.Method: HUVECs were obtained by treatment of umbilical cord veins with collagenaseⅡas described previously. The apoptosis in HUVECs was detected by flow cytometry. The mRNA expression of NOX4 in HUVECs was measured by real-time PCR, and translocation of NF-κB in HUVECs was observed by immunofluorescence staining. Western blot was used to analyze the levels of IκB,P-IκB, p38MAPK and P-p38MAPK. The apoptosis of HUVECs was observed by Hoechst/PI staining.Results: High concentration of glucose induced apoptosis of HUVECs in a time-dependent. The mRNA expression of NOX4 was decreased significantly in NOX4 siRNA-transfected HUVECs compared with control group transfected with FAM siRNA (p<0.05,n=3). The treatment of NOX4 siRNA-transfected HUVECs with high glucose could not elevate the level of ROS and rate of apoptosis. Phosphorylation of p38 MAPK was induced by high glucose, and SB203580, an inhibitor of p38 MAPK, could prevent HUVECs from apoptosis. Moreover,high glucose could down-regulate IκB expression and up-regulate the level of P-IκB. NF-κB was translocated from cytoplasm to nuclear membrane in HUVECs treated by high glucose, and DPI, an inhibitor of NADPH oxidase, could reverse these effects.Conclusions: 1) High glucose induces apoptosis in HUVECs; 2) NOX4 mediates the apoptosis induced by high glucose; 3) NF-κB and p38MAPK pathways are involved in apoptosis of HUVECs.