Study Of The Mechanisms Of GLP-1 Receptor Agonist Inhibits ? Cells Apoptosis Induced By NOX2

Objectives: The beta cell apoptosis is an important pathological mechanism which induced development and progression of type 2 diabetes(T2DM).Glucagon like peptide 1(GLP-1)is a kind of new antidiabetic drugs,increasing evidences have showned that it could decrease beta cell apoptosis.But the underlying molecular mechanisms remain poorly understood.The purpose of this study is to observe the effects of GLP-1 on glycaemic control and islets function in obese patients with T2 DM,then animal and cell experiments will be performed to investigate the possible mechanisms which inhibit beta cell apoptosis.Contect and Methods:Part One: Clinical Trials A total of 35 obese type 2 diabetic patients were included in the prospective,12-weeks observational,self-controlled clinic study.They were subcutaneously injected with liraglutide once daily as an add-on therapy based on their previous oral antidiabetic agents except for thiazolidinediones(TZDs)and insulin.HOMA2-IR was calculated with serum glucose and C-peptide values using the validated calculator to evaluate changes of insulin resistance.HOMA2-%? index was used to evaluate changes of islets function.In addition,changes of general condition,glycemic control and the biochemical indicators were aslo observed.Part Two: Animal Experiments Male SD rats were used to establish T2 DM animal model.The animals were divided into three groups and each group has 12 rats.The experiment lasted 12 weeks.1.Observe influnces of GLP-1 receptor agonist on the behavior weight,food intake,blood sugar and biochemical indicators of rats;2.Hyperglycemic clamp tests were performed to evaluate the effection of GLP-1 receptor agonist on the secretion function of islets;3.Changes of islets' histomorphology were assessed by HE and immunofluorescence double staining;4.TUNEL,immunohistochemistry and Western blot were used to observate GLP-1 effects on pancreatic apoptosis;5.DCFH-DA fluorescent probe was used to detect effect of GLP-1 on Reactive oxygen species(ROS)in islets;6.Immunohistochemistry and Western blot were performed to observate level of key protein of AMPK alpha-NOX2-JNK1/2 signaling pathways.Part Three: Cell Experiment Application of INS-1 cell lines in combination with specific NOX2 and JNK inhibitors,intervention in 48 h,to investigate whether the GLP-1 receptor agonists inhibit beta cell apoptosis by AMPK alpha-NOX2-ROS-JNK signaling pathway;1.Annexin V-FITC/PI flow cytometry and Western blot were applied to detect effects of GLP-1 on beta cells apoptosis;2.DCFH-DA fluorescent probe was used to detect the impact of GLP-1 on ROS levels;3.Western blot was used to detect NOX2 AMKP alpha,p-AMKP alpha,JNK1/2,p-JNK1/2,Caspase 3,and Cleaved Caspase-3 protein expression level in INS-1 cells;ResultsPart one:Glycemin control,IR and beta cell function were significantly improved after 12 weeks GLP-1 receptor agonist treatment with additional benefits of decreasing patient's weight,SBP,DBP and blood lipid level;Part two:1.Behavior and hair color were improved,food intake was depressed and weight recovery in GLP-1 treatment rats;2.GLP-1 treatment significantly improved the glucose metabolism of T2 DM rats,at the same time reduce the cholesterol levels;3.First and second phase insulin secretionwere improved after GLP-1 receptor agonist treatment;4.GLP-1 obviously improved ? cells mass and reduced ratio of ?/? cells in T2 DM rat;5.TUNEL,immunohistochemistry and Western blot results showed that GLP-1 receptor agonist ameliorated apoptosis in pancreas and islets;6.ROS levels of islets were blunted by GLP-1 receptor agonist and specific NOX2 inhibitors treatment;7.Hyperglycemic state led to the decrease of p-AMKP ?,while NOX2 and p-JNK1/2 expression levels were improved,and GLP-1 receptor agonist could reverse these states;Part Three:1.Annexin ?/PI double dye flow cytometry instrument and Western blot test had shown GLP-1 receptor agonist could significantly inhibit INS-1 cells apoptosis induced by high glucose;2.Levels of NOX2 and ROS overproduction induce by high glucose were blunted by GLP-1 receptor agonist or specific inhibitors,without further decreases by a combination of these elements;3.? cells apoptosis were decreased after intervention with GLP-1 receptor agonist and specific inhibitors.At the same time,protein levels of p-AMKP? were elevated and p-JNK1/2 levels were decreased.There were no further synergistic effects were observed by a combind application of GLP-1 receptor agonist with specific inhibitors;Conclusion:1.GLP-1 receptor agonist could significantly improve the glucose metabolism of obese patients with T2 DM.Also,Insulin resistance and beta cell function could be improved.At the same time,weight and blood lipid levels of the patients got significantly improved.2.Islets function and pancreas histomorphology of T2 DM rats were improved,while ROS production and apoptosis of beta cells were markedly supressed by GLP-1 receptor agonists treatment.AMPK?-NOX2-ROS-JNK signaling pathway may be one of the important mechanisms in GLP-1 receptor agonists inhibit beta cell apoptosis.