Effect Of NR4A1 On Hydrogen Peroxide Induced Apoptosis Of Human Umbilical Vein Endothelial Cells And Its Mechanism

Objective:As a orphan nuclear receptor,NR4A1 is one of the important members of the NR4 A family,which can rapidly regulate the transcription of target genes and participate during cell proliferation and apoptosis,tumor occurrence,immune stress,energy metabolism and other life activities of the body.At present,many studies show that oxidative stress is an important factor leading to the occurrence and development of type 2 diabetes.It may increase the content of reactive oxygen species(ROS)and reactive nitrogen species(RNS)in vivo through various pathways such as mitochondrial electron transport chain and polyol pathway,and ultimately cause cell damage.Vascular endothelial cell apoptosis is an initial event of diabetic macrovascular disease.Studies have shown that NR4A1 can inhibit activation of Nuclear Factor Kappa B(NF-κB)which induced by tumor necrosis factor-α by increasing the expression of HUMAN NF-kappa-b inhibitor alpha(IκB-α).VCAM-1 and ICAM-1 were inhibited.In this study,we investigated whether NR4A1 could resist oxidative stress-induced endothelial cell apoptosis by regulating NF-κB pathway,and thus delay the progression of diabetic macrovascular disease.Methods:Part I :Human umbilical vein endothelial cells(HUVECs)were treated with different concentrations of H2O2 for different periods of time.Western blot detected the expression of NR4A1.The cells transfected with NR4A1 overexpression and control virus were screened,and the cells transfected with NR4A1 overexpression were named OV cells and the cells transfected with control virus were named NC cells.The cells were set as control group(NC),NR4A1 overexpression group(OV),NC+H2O2 group and OV+H2O2 group.Western blot was used to detect the protein level of Bcl-2 in each group,and TUNEL method was used to detect the apoptosis level of untreated NC group,H2O2-treated NC group and H2O2-treated OV group.Part II: The untreated NC cells,untreated OV cells,H2O2-treated NC cells and H2O2-treated OV cells were collected.The nuclear protein and cytoplasmic protein were extracted using the nuclear protein and cytoplasmic protein separation kit.The localization of P65 protein and the expression of IκBα protein was detected by Western blot.Results:Part I : The expression of NR4A1 was up-regulated after the establishment of oxidative stress model in HUVECs cells,suggesting that NR4A1 plays a role in oxidative stress in HUVECs cells.The apoptosis rate of THE H2O2 group was much higher than that of the control group,and the expression of apoptotic protein Bcl-2 was inhibited,indicating that oxidative stress could lead to apoptosis in HUVECs cells.The apoptosis rate of OV cells under oxidative stress model was lower than that of NC cells under the same treatment,and the expression of Bcl-2 was up-regulated compared with that of NC+H2O2 group.The results showed that NR4A1 could resist apoptosis induced by oxidative stress in HUVECs cells.Part II : Compared with the control group,the expression of P65 protein in the nucleus of NC treated with H2O2 was significantly up-regulated,while the expression of P65 protein in the cytoplasm was significantly down-regulated,the difference was statistically significant,and the expression of IκBα was decreased,suggesting that oxidative stress can reduce the expression of IκBα,leading to the transfer of NF-κB from the cytoplasm to the nucleus.This activates the NF-κB pathway,which may lead to apoptosis.We also observed that compared with NC+H2O2 group,the expression of P65 protein in the nucleus of OV+H2O2 group was significantly down-regulated,while the expression of P65 protein in the cytoplasm of OV+H2O2 group was significantly upregulated,with statistically significant differences,and the expression of IκBα was increased.These results suggested that NR4A1 could inhibit NF-κB nuclear translocation induced by oxidative stress by increasing the expression of IκBα,and possibly resist apoptosis induced by oxidative stress by inhibiting the activation of NF-κB pathway.Conclusions:NR4A1 can resist apoptosis induced by oxidative stress and inhibit NF-κB nuclear translocation induced by oxidative stress by increasing the expression of IκBα,suggesting that NR4A1 may play a role through the IκBα/NF-κB signaling pathway...