Intervention Of Exendin-4 On β-cell Lipotoxic Oxidative Stress And Relationship Of TLR4-NF-κB Signaling Pathway

Objective:Glucagon-like peptide-1 analogue exendin-4 has multiple effects onβ-cells.In this study,to investigate the relationship between the protective effects of exendin-4 on lipotoxicity induced oxidative stress inβ-cells and TLR4/NF-κB signaling pathway.Methods:1.Cell experiment:（1）βTc6 Cells were cultured in vitro and intervened with 0.5 mmol/L palmitic acid（PA）for 24 h,then intervened with exendin-4 at a concentration of 25,50,100 nmol/L for 72hours to observe the effect of exendin-4 on PA-inducedβ-cells.The expression of TLR4,NF-κB subunit P65,NADPH oxidase subunit p47 phox protein and the ROS levels were detected.（2）Transfection ofβ-cells with RNAi lentiviral vector（siRNA）or TLR4 overexpression lentiviral vector to regulate TLR4 gene expression;β-cells were intervened with0.5mmol/L PA for 24 hours and then intervened with 100 nmol/L exendin-4 for 72 hours to study the relationship between TLR4 expression level and Exendin-4 intervention in PA-inducedβ-cell oxidative stress.Cell apoptosis,insulin secretion,the expression of P47 phox protein and ROS levels were detected.（3）Using the above methods to regulate the expression of TLR4 inβcells;β-cells were intervened with 100 umol/L H₂O₂ for 24 hours and then intervened with 100 nmol/L exendin-4 for 72 hours to study TLR4 is involved in the process of exendin-4 restrained H₂O₂-inducedβ-cells oxidative stress.Cell apoptosis,insulin secretion,the expression of P47 phox protein and ROS levels were detected.（4）Theβ-cells were intervened with TLR4 agonist（LPS）and NF-κB agonist（TNF-α）to observe the intervention of exendin-4 on oxidative stress induced by TLR4 agonists or NF-κB agonists.Cell apoptosis,insulin secretion,the expression of P47 phox protein and ROS levels were detected.2.Animal experiment:Wild-type rats and TLR4 knockout rats induced by high fat diet were given subcutaneous injection of exenatide for 16 weeks to observe the effect of exenatide intervened oxidative stress induced by high fat diet and the relationship with TLR4expression.Body weight and body length were recorded during the study.The levels of TCHO,TG,LDL,FFA,INS,FBG of blood were measured.Cell apoptosis,insulin secretion,the expression of TLR4,NF-κB subunit P65,P47 phox protein and ROS levels were detected.3.Techniques:The expression of TLR4,NF-κB subunit P65 and NADPH oxidase subunit P47 phox protein were evaluated by western blotting.Cell apoptosis was detected by TUNEL assay.Insulin secretion and FFA were detected by ELISA assay.The intracellular ROS levels were assessed using a ROS detection assay kit.The levels of TCHO,TG,LDL and FBG of blood were measured by an automatic biochemical analyzer.Results:1.Exendin-4 inhibited the expression of TLR4,NF-κB subunit P65,NADPH oxidase subunit p47 phox and decreasing the intracellular level of ROS in a concentration-dependent manner（P<0.05）.2.Silencing of TLR4 expression enhanced the protective effects of exendin-4 on PA intervened cells,reduced cell apoptosis,improved insulin secretion and weaken oxidative stress（P<0.05）.While TLR4 overexpression attenuated this protective effect（P<0.05）.3.Exendin-4 has a protective effect on H₂O₂ intervened cells,reduced cell apoptosis,improved insulin secretion and weaken oxidative stress（P<0.05）.Silencing of TLR4expression enhanced the protective effect of exendin-4 on H₂O₂ intervention cells（P<0.05）.Overexpression of TLR4 attenuated the protective effect of exendin-4（P<0.05）.4.Exendin-4 inhibits TLR4 agonists and NF-κB agonists,reduced cell apoptosis,improved insulin secretion and weaken oxidative stress（P<0.05）.5.Exenatide intervened wild-type with high-fat diet group reduced Lee’s index,FFA,fasting blood glucose and HOMA-IR,increased insulin levels,inhibited cell apoptosis,protein expression of TLR4,NF-κB subunit P65,P47 phox and ROS in high-fat diet rats（P<0.05）.Compared with wild-type with high-fat diet group,TLR4 knock-out in high-fat diet group showed lowered weight,HOMA-IR,cell apoptosis,the expression of TLR4,NF-κB subunit P65and the levels of oxidative stress（P<0.05）.Simultaneously,knockout of TLR4 amplified the benign intervention of exenatide intervened obese rats（P<0.05）.Conclusions:Exendin-4 can antagonize lipotoxicity induced oxidative stress inβ-cells via inhibiting TLR4-NF-κB pathway.This study revealed a new pathway for exendin-4 to antagonize lipotoxic oxidative stress in β-cells.