Molecular Mechanism Of Liraglutide Against NLRP3 Inflammasome-dependent Diabetic Pyroptosis In Islet βTC-6 Cells’ Dysfunction

PurposeThe main pathogenesis of type 2 diabetes mellitus(T2DM)is insulin resistance(IR)and islet β cell failure.Islet tissue can secrete insulin,as the only hypoglycemic hormone in human body,and plays an important role in the occurrence and development of T2 DM.At present,there is little cognition and no unified conclusion about the pathophysiological mechanism of islet tissue and its key cells.Pyroptosis,as a kind of inflammatory necrosis,has been found to be involved in a variety of acute and chronic inflammatory injury processes so that it becomes an new and important topic about the mechanism exploration and therapeutic target of diseases.Our design of this study is based on the following two points.On the one hand,glycolipid toxicity runs through the whole course of T2 DM.As an adjunct product of pathogenic bacteria,lipopolysaccharide(LPS)plays as the post-effect of gut microbiota metabolites homeostasis disequilibrium and closely related with T2 DM.Previous studies have shown that glycolipid toxicity and LPS can induce pyroptosis by regulating inflammation in diabetic related diseases.However,no studies confirms whether T2 DM islet beta cells occur pyroptosis or not.On the other hand,liraglutide,as a novel hypoglycemic drug,glucagon-like peptide-1 receptor agonist(GLP-1RA),has recently been found to inhibit pyrin-domain NOD-like receptor family 3(NLRP3)-related apoptosis in mononuclear macrophages,although no anti-pyrin-1RA-related studies have been conducted on T2 DM islet beta cells.However,there is no doubt that the hypoglycemic drug GLP-1RA shows great anti-scorch potential.On the other hand,GLP-1 receptor agonist as a new hypoglycemic drug,its liraglutide has recently been found the inhibitory effect on NLRP3 inflammasome-related pyroptosis in monocytes and macrophages.Although there is no study definiting GLP-1RA’s anti-pyroptosis to T2 DM islet β cells,abselutely GLP-1RA has so much potential of anti-pyroptosis.Based on the above content,the purpose of this study is to explore the following four issues: 1)whether high glucose,high lipid and LPS can cause NLRP3-dependent pyroptosis in T2 DM pancreatic islet β cells and its molecular mechanism.2)whether pyroptosis injures pancreatic islet β cell’s insulin secretory function.3)to explore whetherliraglutide,a GLP1-RA,can inhibit the pyroptosis of islet β cells.4)whether liraglutide can improve the secretory function of pancreatic islet cells after pyroptosis injury.This study broadens the cognitive field for the pathological mechanism of islet β cells in T2 DM and adds new evidence for the clinical application of liraglutide.Methods1.To explore the optimal conditions for the establishment of pyroptosis model of islet βTC-6 cells induced by glucose,palmitic acid(PA)and LPS.Islet βTC-6 cells were treated with glucose and PA at different concentrations and for different duration.Also islet β cells were treated with LPS combined with ATP or not.The expression levels of NLRP3 and caspase-1 in islet β cells were detected by real-time fluorescent quantitative polymerase chain reaction(RT-PCR)and Western Blot.Glucose-Stimulated Insulin Secretion test(GSIS)under high glucose stimulation was performed on the interfered βcells.The insulin secretion in cell supernatant was determined by ELISA.To investigate the pyroptosis of pancreatic islet β cells induced by above three factors and the damage of islet β cell insulin secretion induced by pyroptosis.2.Basede on the results of the first part,glucose,PA and LPS with the best inducing conditions were used to induce pyroptosis in pancreatic islet βTC-6 cells and liraglutide and caspase-1 inhibitors were also used in β cells.Cells were stained with FITC-Annexin V/PI cell double-staining kit,and the percentage of pyroptosis cells was analyzed by the flow cytometry.The expression of NLRP3 and caspase-1 were detected by RT-PCR and Western Blot.The insulin level was measured by ELISA after GSIS test in islet β cells.Above all,we investigated the liraglutide’s anti-pyroptosis effect in isletβ cells which was stimulated by glucose,PA and LPS and also evaluated the protective effect of liraglutide on islet secretion function.Results1.Glucose,PA and LPS treatment under different conditions could induce different degrees of NLRP3 and caspase-1 expression in islet βTC-6 cells,and different degrees of pyroptosis in islet β cells were accompanied by different degrees of decline in cell insulin secretion function.2.Ttreatment with glucose,PA and LPS,it showed that the percentage of pyroptosis cells in islet βTC-6 cells increased in flow cytometry analysis,the mRNA and protein expression of NLRP3,caspase-1 and GSDMD also increased,and the concentration of insulin in cell supernatant decreased by ELISA.After using liraglutide and caspase-1 inhibitors,the percentage of pyroptosis cells decreased significantly,the expression levels of NLRP3,caspase-1 and GSDMD decreased,and the insulin secretion increased.Conclusion1.High glucose,high fat and LPS can induce NLRP3-dependent pyroptosis of isletβTC-6 cells and impair the function of insulin secretion of β cells.2.Liraglutide can inhibit the NLRP3-dependent pyroptosis induced by high glucose,high fat and LPS,and improve the islet secretion function impaired by pyroptosis in islet βTC-6 cells.