Protective Effect And Its Underlying Mechanism Of Liraglutide On Human Umbilical Vein Endothelial Cell Oxidative Stress Damage Induced By Palmitic Acid

The number of patients with type2 diabetes has been increasing recently.Micro and macro vascular diseases are the leading causes of death and disability in diabetic patients,in which,endothelial dysfunction plays an important role.Palmitic acid is the most kind of free fatty acid in the body and high concentration of palmitic acid can damage endothelial cells,promoting the atherogenic process.Oxidative stress is the common basis of the onset of diabetes and its complications and it may play an important role in the endothelial cell injury.Glucagon-like peptide-1(GLP-1)is a kind of incretin hormones secreted by the intestinal L-cells.It can regulate the glucose level by increasing insulin secretion and inhibiting glucagon secretion,besides,it can also reduce blood pressure,lipid levels,weight and urine protein,enhance left ventricular ejection fraction,protect vascular endothelial cell function and improve diabetic vascular complications.So far,the specific mechanism has been unclear.It was confirmed that GLP-1 can regulate the different signaling pathways to reduce oxidative stress damage in endothelial cells which was induced by culturing with high glucose or homocysteine or advanced glycation end products.The study has shown that GLP-1 can reduce palmitic acid induced endothelial cells apoptosis,but the specific mechanism was still not clear.The study aims to investigate the effects and possible mechanisms of liraglutide acting on the human umbilical vein endothelial cells injured by palmitic acid-induced oxidative stress.Objective: To explore the effect of liraglutide on oxidative stress induced by palmitic acid in human umbilical vein endothelial cells and to elucidate the possible mechanisms.Methods: Recovery human umbilical vein endothelial cells,incubated under palmitic acid as different concentrations(0.2 mmol/L,0.4 mmol/L,0.6 mmol/L)for 12 hours,24 hours,48 hours,respectively.The cell viability was measured by MTT assay.Human umbilical vein endothelial cells,which were cultured with palmitic acid for inducing the model of endothelial cells damage,were exposed to different concentration of liraglutide(10 nmol/L,100 nmol/L,200nmol/L,400nmol/L)for 24 hours.The optimum concentration of liraglutide were screen out according to the cell viablity.The appropriate concentration of liraglutide were screen out according to the cell viablity.The human umbilical vein endothelial cells were randomly divided into six groups: normal control group(NC),palmitic acid group(PA),liraglutide group(L),liraglutide+ palmitic acid group(L+PA),Exendin 9-39+ liraglutide+ palmitic acid group(EX),BAY11-7085+ liraglutide+ palmitic acid group(BAY).The human umbilical vein endothelial cells were pre-incubated with liraglutide/ glp-1 receptor antagonist(Exendin 9-39)/ NF-?B inhibitor(BAY11-7085)for 2 hours before incubation with palmitic acid and /or liraglutide for 24 hours.The ratio of cell apoptosis was detected by flow cytometry.Intracellular level of reactive oxygen species(ROS)was detected by dihydroethidium(DHE)fluorescent probe.Intracellular superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT)activity and the content malondialdehyde(MDA)levels were measured with commercial enzyme assay kits.The mRNA expression of Bax,Bcl-2,caspase 3,SOD 2,CAT and GPX-1 in each group were measured by quantitative real-time PCR.The protein levels expression of Bax,Bcl-2,caspase 3,NF-?B p65,p-NF-?B p65,JNK and p-JNK were evaluated by western blot analysis.Data were expressed as mean±SE.Significance of differences among groups was determined using Student's t-test or ANOVA.Results:1 The cell viability of HUVECsCompare with normal control group,the endothelial cells viability was gradually decreased along with the increase of concentration of palmitic acid and the time of culture,in addition,0.4 mmol/L and 0.6 mmol/L palmitic acid for 24 hours and 48 hours significantly decreased the cells viability.There was no significant difference in statistics in cells viability between 0.4 mmol/L and 0.6 mmol/L palmitic acid for 24 hours.Compare with palmitic acid group,the cells viability was gradually increased with pretreatment of Liraglutide(100 nmol/L,200nmol/L,400 nmol/L)(P<0.05),there were not statistically difference between the three groups.While there were no obviously incresed between palmitic acid group and Liraglutide(10 nmol/L)group.2 Apoptosis of HUVECsCompare with normal control group,apoptosis of human umbilical vein endothelial cells induced by 0.4 mmol/L palmitic acid for 24 hours was obviously incresed(P<0.05),the mRNA and protein expression levels of caspase-3 and Bax were up-regulated,but the mRNA and protein expression levels of Bcl-2 were decreased(P>0.05).Compare with palmitic acid group,pretreatment of human umbilical vein endothelial cells with Liraglutide(200 nmol/L)for 24 hours resulted in the decrease of apoptosis and the mRNA and protein expression levels of caspase-3 and Bax(P<0.05),while the expression of Bcl-2 was increased(P>0.05).3 Oxidative stress of HUVECsCompare with normal control group,palmitic acid group obviously increased intracellar ROS and MDA levels,decreased SOD,GSH-PX,CAT activity and down-regulated the mRNA expression of SOD2,GPX-1,CAT(P<0.05).Compare with palmitic acid group,Liraglutide not only prevented the incremental ROS and MDA levels,but also exhibited augmented SOD,GSH-PX,CAT activity and the mRNA expression(P< 0.05).4 NF-?B?JNK signaling pathwaysCompare with normal control group,the ratio of p-NF-?B/ NF-?B,pJNK/ JNK was increased in palmitic acid group(P<0.05),which was counteracted by Liraglutide.Compare with Liraglutide+ palmitic acid group,Exendin 9-39 attenuated the protective effect of Liraglutide in the palmitic acid induced oxidative stress(P<0.05).Compare with Liraglutide+ palmitic acid group,BAY11-7085 pretreatment up-regulated the mRNA expression of GPX-1 and CAT,increased GSHPX and CAT activity and reduced the ratio of p-NF-?B/ NF-?B and p-JNK/ JNK(P<0.05).While the activity and mRNA expression of SOD were no significant differences between BAY11-7085 and Liraglutide+ palmitic acid group(P>0.05).Conclusions:1 Palmitic acid could induce oxidative stress damage and apoptosis of human umbilical vein endothelial cells.2 Liraglutide could protect against the oxidative stress damage induced by palmitic acid and the protective effect of Liraglutide may be attributed to regulating NF-?B and JNK signal pathing.