The Role Of PEDF In The Chronic High Glucose-induced Oxidative Stress Injury To Pancreatic Beta-cells

Objective:Chronic high glucose can damage tissues and organs of the body, including thepancreatic β cells. Recent years, the theory of glucose toxicity received extensive attention,indicating that long-term exposure to high concentrations of glucose can decrease theinsulin secretion of pancreatic β-cells, and it was irreversible damaged. Studies have shownthat the mechanism of it was high glucose-induced intracellular oxidative stress whichcaused pancreatic beta-cell injury. PEDF is an endogenous anti-oxidative stress factor. Anumber of studies in recent years at home and abroad have shown that it had a closerelationship with oxidative stress-related diseases, such as Metabolic Syndrome,Abdominal obesity, Diabetic nephropathy, etc. Basic studies have shown that PEDF mayplay protective effect against oxidative stress performance in a variety of cells throughdifferent ways. However, reports about the islet cells were rare. The aim of this study wasto identify and explore two things: first, the effect of different glucose concentration onINN-1E cell intracellular ROS generation、 cell activity、 apoptosis ratio andglucose-stimulated insulin secretion; second, whether the PEDF could improve theoxidative damage of INS-1E cells induced by high glucose conditions.Materials andethods:1. The day after they were seeded, INS-1E cells would be starved with no glucose mediafor2h, than they were divided into5.6mmol/l group(Control group),16.7mmol/l groupand30mmol/l group which were adjusted osmotic pressure by mannitol, written as5.6G,16.7G and30G respectively. Than the intracellular ROS levels、cell viability、apoptosisratio and GSIS were detected by DCFH-DA probe、MTT、PI/Hoechst and ELISArespectively after treating INS-1E cells with that three different glucose concentrations.2. The mRNA levels of PEDF in rat kidney, pancreatic islets, acinar and INS-1E cells weredetected by RT-PCR.3. Building PEDF plasmid, transfected INS-1E cells with PEDF plasmids and detect the protein expression to identify whether the transfection succecess or not.4. The day after they were seeded, cells were divided into3grope:5.6G,16.7G and30G.and the cells with the same glucose concentration were grouped into GS(glucose+scramble) and GP（glucose+PEDF), depending on the transfected plasmid.24h aftertransfection, when PEDF have the highest protein expression, making2h starvation forcells. and than, incubate cells with that three different glucose concentration mediarespectively. Doing the same as above to detect and compare levels of ROS, the cellactivity, apoptosis ratio and the GSIS difference in different groups of cells.(scramblealternative to the empty plasmid vector)Result1. After48h dealed with high glucose medium, levels of ROS, apoptosis ratio increasedsignificantly while cell activity and insulin secretion reduced in the INS-1E cell2. In the rat kidney, islet, pancreatic acinus and the INS-1E cells, a great difference occursin mRNA levels of PEDF. What’s more, mRNA in the INS-1E cell comes into minimal.3. Transfecting INS-1E cells by2ug PEDF, Overexpression is effective.4. Compared with16.7GS and the30GS group, ROS and apoptosis rate in corresponding16.7GP and30GP group of cells significantly reduced, while cell viability and the GSISsignificantly improved. Thus is full of statistically significant. IN5.6mM glucoseconcentration, PEDF transfer had no significant effect on the above indicators.ConclusionThis section initially revealed that high glucose would cause high levels of ROS in theINS-1E cells along with increased apoptosis and decreased cell viability and insulinsecretion with a concentration dependent manner. Overexpression of PEDF cansignificantly reduce the generation of ROS and apoptotic ratio, increase cell viability andthe GSIS function in INS-1E cells under high glucose condition(16.7G,30G).PEDF canimprove the oxidative stress injury of the cells.