The Study Of Mechanism In Dipeptidyl Peptidase-4 Inhibitor Mediated Endothelial Apoptosis Regulation

Background and ObjectivesDiabetes mellitus(DM)is a chronic metabolic disease,challenging human being health.Type 2 diabetes mellitus(T2DM)is one of the main risk factors of cardiovascular diseases.Uncontrolled DM leads to coronary heart disease,retina vascular disease,renal vascular disease and diabetic foot.More than half of the T2 DM patients die from cardiovascular complications,including myocardial infarction and stroke.Atherosclerosis,endothelial dysfunction and apoptosis play a significantly role in the pathological development of diabetic cardiovascular complications,vascular endothelial apoptosis contributes to the initial vessel lesion of vascular.Dipeptidyl peptidase4 inhibitor(DPP4i)is a sort of oral hypoglycemic agent which decrease blood glucose with low risk in hypoglycemia and weight gain and is clinical used in DM treatment.Recent researches found that DPP4i shows cardiovascular protection effect beyond its hypoglycemic function.DPP4i sitagliptin improved endothelial dysfunction,enhanced endothelial nitric oxide synthase(eNOS)phosphorylation and reduced atherosclerotic plaque area.DPP4i was even demonstrated to be effective in heart failure.However,the relation between DPP4i and endothelial apoptosis is still unknown,the involving molecular mechanism needs further study.AMP-activated protein kinase(AMPK)is a crucial cellular energy and stress sensor,which works as an important regulator in cellular energy metabolic regulation.Previous study found that the activation of AMPK is closely related to cardiovascular metabolic disease.In HUVEC,AMPK phosphorylation significantly prevents oxidative stress-induced endothelial apoptosis.Vascular pathology is attributed to AMPK dysregulation.The regulation of AMPK plays an essential role in cardiovascular metabolic disease.In this study,we sought to demonstrate the mechanism by which DPP4inhibitor regulates endothelial apoptosis and determine whether AMPK involves in this regulatory process.Methods1.The effect of DPP4i on ROS production,mitochondrial membrane potential(??m)and apoptosisHUVEC were divided into three groups: control group,HG(high glucose)group and DPP4i group.HUVEC were incubated with HG(33 mM)in HG group and sitagliptin(1 ?M)in DPP4i group for 48 h.Then a flow cytometry was used to determine the ROS generation,??m and apoptosis of endothelial cell.The H2 DCFDA fluorescent dye was used in ROS detection,JC-1 fluorescent dye for ??m measurement and Annexin V-FITC/PI double labeled fluorescent dye for apoptosis.2.The effect of DPP4i on AMPK activationA: The effect of sitagliptin on AMPK phosphorylation in time-dependent manner.HUVEC were incubated with sitagliptin(1 ?M)for 0.5 h,1 h,2 h,4 h and AMPK activator AICAR(100 ?M)for 0.5 h as positive control.Western blot was used to determine AMPK activation.B: The effect of AMPK inhibitor compound C on DPP4inhibitor-mediated AMPK phosphorylation.HUVECs were incubated with sitagliptin(1 ?M),compound C(10 ?M)or sitagliptin(1 ?M)plus compound C(10 ?M)for 2 h.AMPK activation was detected by western blot.3.The effect of AMPK on DPP4i-mediated endothelial regulationHUVEC were divided into five groups:control group,HG group,DPP4i group,AMPK inhibitor group,AMPK activator group.HUVEC were treated with HG(33 mM)in the presence of sitagliptin(1 ?M),AICAR(100 ?M)or sitagliptin(1 ?M)plus compound C(10 ?M)for 48 h.Then cellular ROS generation level,??m and endothelial apoptosis were measured by flow cytometry.Results1.HUVEC incubated with high glucose increased cellular ROS generation,??m collapse and endothelial apoptosis;2.DPP4i sitagliptin decreased HG-induced ROS production,improved ??m collapse and significantly prevented endothelial apoptosis;3.Sitaglipitn enhanced AMPK phosphorylation from 30 min to 2 hours.AMPK inhibitor compound C significantly inhibited AMPK activation;4.Compound C reversed sitaliptin-mediated prevention on ROS production,??m collapse and endothelial apoptosis.AMPK activator AICAR mimicked the beneficial effect of sitagliptin.Conclusions1.Results show that DPP4i sitagliptin significantly prevented HG-induced cellular ROS generation,??m collapse and endothelial apoptosis;2.Our study demonstrates a novel mechanism of sitagliptin-mediated AMPK activation in preventing endothelial apoptosis and indicates a therapeutic potential of sitagliptin for vascular complications related to endothelial apoptosis.