The Research On The Relationship Between Exendin-4 And Apoptosis Of Pancreatic ? Cells

Research background:Type 2 diabetes mellitus is a common metabolic disease characterized by a lack of insulin or/and a deficiency of its function of insulin.With the development of economy and the improvement of the average life,the incidence of type 2 diabetes mellitus is increasing and to be a younger trend,which is becoming a serious threat to the health of human beings around the world.In China,the number of people with diabetes is growing at an alarming rate.Current endocrine therapy mainly includes diet control,drug intervention and reasonable exercise.However,oral hypoglycemic drugs are difficult to maintain stable blood glucose,and the compliance of insulin injection is poor,which makes the blood glucose control of most diabetic patients not ideal.So it is difficult to avoid all kinds of related complications,such as blindness,kidney failure,stroke,diabetic foot,etc.Due to its complex etiology and unclear mechanism,it is difficult to make a breakthrough in the medical treatment of type 2 diabetes.In recent years,more and more evidence shows that surgical treatment for diabetes has curative effect,can control the blood sugar,prevent the occurrence of diabetes complications,is a new kind of safe and effective treatment method and brings hope for diabetics.In 2011,the International Diabetes Federation formally recommended surgery as a treatment for obesity and type 2 diabetes.Although surgery is effective in treating diabetes,its therapeutic mechanism is still being studied.Among them,glucagon-like peptide-1 is considered to be one of the major gastrointestinal hormones in diabetic patients after surgery to regulate insulin control.The expression of glucagon-like peptide-1 was reduced in diabetic patients.In patients with diabetes after the operation,the glucagon-like peptide-1 was significantly higher than that in the control group,which suggested that glucagon-like peptide-1 may be an important factor in the control of postoperative blood glucose control in diabetic patients.Glucagon-like peptide-1 is secreted by L cells in the distal small intestine which can be rapidly inactivated in the body.Exendin-4,the analogue of the glucagon-like peptide-1,not only simulates the function of glucagon-1,but is not inactivated by dipeptidase-4,which is a new kind of short-effect hypoglycemic agent used in clinic in recent years.Exendin-4 is effective in controlling blood glucose,protecting the cells,reducing weight,protecting cardiovascular and improving non-alcoholic fatty liver disease.Research objectiveThis study aims to study the possible mechanism of exendin-4 to inhibit the apoptosis of the pancreatic islet,to verify the possible mechanism of surgical treatment for type 2 diabetes mellitus,and provide more theoretical basis for the treatment of diabetes mellitus.Research method:1.To determine the high glucose concentration and stimulation time of the insulinoma Rin-m5 f cells of rats by pre-experiment.The cells were divided into three groups: A,B and C,and the cells were inoculated into a 96-pore plate,and each group was set with 5 duplicate holes.Group A used the complete medium with glucose concentration of 11.1mmol/L.Group B alternatively used the complete medium with glucose concentration of 22.2mmol/L and 11.1mmol/L.Group C alternatively used the complete medium with glucose concentration of 33.3mmol/L and 11.1mmol/L,and replaced the medium every 24 hours.Three groups of cells were cultured under the same conditions for 3,5,and 7 days,to detect the proliferation activity of each group of cells by MTT assay to determine the high glucose concentration and stimulation time.2.To determine the stimulus concentration of Exenatide-4 by pre-experiment.Under the same concentration of high glucose stimulus,divided the cells into three groups.The concentration of Exenatide-4 was set at 50nmmol/L,100nmmol/L,150nmmol/L.Under the same conditions,cultured for 7 days,then to detecte the proliferation activity of the three groups of cells by MTT assay to determine the concentration of the Exenatide-4.3.To detect the effects of high glucose and Exenatide-4 on cells proliferation activity by MTT assay.The insulinoma Rin-m5 f cells of rats were divided into three groups: control group,high glucose group,high glucose + Exenatide-4 group.Cells were inoculated into a 96-pore plate,and each group was set with 5 duplicate holes.Control group used the complete medium with glucose concentration of 11.1mmol/L.High glucose group alternatively used the complete medium with glucose concentration of 33.3mmol/L and 11.1mmol/L.High glucose + Exenatide-4 group alternatively used the complete medium with Exenatide-4 and glucose concentration of 33.3mmol/L and 11.1mmol/L,and replaced the medium every 24 hours.Under the same conditions,cultured for 7 days,then to detecte the proliferation activity of the three groups of cells by MTT assay.4.The relative protein expressions of cells was detected by Western blottingmethod.Cells were divided into three groups: Control group,High glucose group,High glucose + Exenatide-4 group.Under the same conditions,cultured for 7 days,western blotting technique was used to detect the expressions of the molecules(BIP,CHOP)of cell endoplasmic reticulum stress(ERS)and the phosphorylation of c-jun N-terminal kinase(p-JNK),apoptotic molecule Caspase-3.5.To detecte the cell apoptosis by flow cytometryCells were divided into three groups: Control group,High glucose group,High glucose + Exenatide-4 group.Under the same conditions,cultured for 7 days.To detecte the cell apoptosis by flow cytometry.Experimental result:1.The cell proliferation activity in High glucose group was significantly lower than that in the control group,difference was statistically significant(p<0.05).The cell proliferation activity in High glucose + Exenatide-4 group was significantly increased than that in the High glucose group,difference was statistically significant(p<0.05).2.Expressions of CHOP,BIP,p-JNK,Caspase-3 proteins were lower in control group,while they were all significantly increased in high glucose group(p<0.05).Expressions of CHOP,BIP,p-JNK,Caspase-3 were obviously decreased in high glucose and Exendin-4 group compared with high glucose group(p<0.05).3.Compared with control group(17.51%),the apoptosis rate of high glucose group(31.25%)increased significantly(p<0.001).The apoptosis rate of high glucose and Exendin-4 group(19.52%)was significantly decreased compared with high glucose group(p<0.001).Conclusions:1.Prolonged high glucose stimulation can decrease the activity of the islet cells and induce the Endoplasmic Reticulum Stress in Rin-m5 f cells.2.Exenatide-4,the analogue of GLP-1,inhibits the cell endoplasmic reticulum stress and inhibits the apoptosis of the islet cells by regulating the cell JNK signaling pathway.