Apelin Alleviates High Glucose-induced Apoptosis Via ERK1/2 Signal Pathway In Human Umbilical Vein Endothelial Cells

ObjectiveThe aim of this study was to investigate the effect of Apelin on the high glucose-induced apoptosis in human umbilical vein endothelial cells（HUVECs）.Furthermore,we explored if the extracellular regulated protein kinases1/2（ERK1/2）and silent mating type information regulation 2 homolog 1（SIRT1）were involved in the the mechanism.Methods1.Immunohistochemistry was used to detect the expression of angiotensin type 1 receptor（APJ）on human renal vascular endothelial cells.2.The model of HUVECs with high glucose damage was established,and the intervention experiment of Apelin was carried out.The cell viability of HUVECs under different concentrations of Apelin at 16 hours,24 hours and 48 hours was detected by cell counting kit-8.The apoptosis of HUVECs under different concentrations of Apelin at 48 hours was detected by flow cytometry.3.The expression of SIRT1 mRNA was detected by RT-PCR and the expression of ERK1/2 and SIRT1 protein was detected by Western blot.By using the lentiviral cell transfection skill,the APJ low expressing cells constructed by small interfering RNA（Si-APJ）was selected as the experimental group,while the steady-state cells constructed by empty plasmid（Vector）were selected as the control group.4.The ERK1/2 inhibitor U0126 and SIRT1 inhibitor EX527 were added respectively to carry out signaling pathway experiments.The apoptosis rate was detected by flow cytometry to compare whether the inhibitors reversed the protective effect of Apelin on HUVECs in high glucose environment.Results1.Immunohistochemistry results suggested that APJ receptors were both expressed in vascular endothelial cells and smooth muscle cells of human kidney tissues.2.Apoptosis experiments after Apelin intervention:compared with the control group,the apoptosis rate of the high glucose group increased,while the cell viability decreased（P<0.05）.Compared with the high glucose group,the apoptosis rate of high glucose+Apelin group decreased,while the cell viability increased,especially in the high glucose+10^-8mol/L Apelin group（P<0.05）.3.ERK1/2 signaling pathway:Compared with the control group,the phosphorylation rate of ERK1/2 in the high glucose group significantly decreased（P<0.05）;compared with the high glucose group,the phosphorylation ratio of ERK1/2 in the high glucose+Apelin group significantly increased.Compared with the high glucose+Apelin+Vector group,the phosphorylation rate of ERK1/2 in the high glucose+Apelin+Si-APJ group significantly decreased（P<0.05）.4.SIRT1 signaling:Compared with the control group,SIRT1 mRNA and SIRT1 protein expression levels in the high glucose group significantly decreased（P<0.05）;compared with the high glucose group,SIRT1 mRNA and SIRT1 protein expression levels in the high glucose+Apelin group increased（P<0.05）.Compared with the high glucose+Apelin+Vector group,the expression level of SIRT1 protein in the high glucose+Apelin+Si-APJ group significantly decreased（P<0.05）.5.ERK1/2 and SIRT1 signaling inhibition test:Compared with high glucose+Apelin group,the apoptotic rate of high glucose+Apelin+U0126 group significantly increased（P<0.05）,the apoptotic rate of high glucose+Apelin+EX527 group didn’t change（P>0.05）.Conclusions1.APJ receptor is expressed in both vascular endothelial cells and smooth muscle cells of human kidney tissue.2.Apelin promotes cell viability and inhibits apoptosis of HUVECs in high glucose environment.3.Apelin promotes the expression of ERK1/2,which may be one of the mechanisms that Apelin reduces high glucose-induced apoptosis of endothelial cells.4.Apelin can up-regulate the expression of SIRT1,but SIRT1 does not participate in the mechanism of Apelin to reduce the apoptosis of endothelial cells induced by high glucose.