Effects Of Interleukin-1 Receptor Blocker And Liraglutide On Human Islet Amyloid Polypeptide Related Inflammation And Apoptosis Of Islet Cells

Objective: Islet amyloid and inflammation are closely related,both of which contribute to beta-cell injury in type 2 diabetes.Islet amyloid polypeptide,also called amylin,is the main component of islet amyloid.Amylin can aggregate to oligomers and trigger a localized inflammatory response,especially interleukin-1?(IL-1?).IL-1? is one of the important inflammatory factors,which invlove in beta-cell function and apoptosis.Interleukin-1 receptor blocker(IL-1Ra)can block the binding of IL-1? and its receptor and therefore protect cells from the toxicity of hyperglucose and hyperlipid.However,there are very few studies about the role of amylin in the islet inflammation.Whether blocking IL-1 receptor can protect islet cells from amylin related toxicity is still unclear.Besides,some studies have shown the anti-inflammation ability of liraglutide,which can reduce the amyloid plaque load and inflammation in AD mice.But the reserches about whether liraglutide can protect islet cells from amylin toxicity are few.Therefore,we evaluated whether IL-1 signal pathway played an important role in amylin related islet injury and whether liraglutide could reduce the islet toxicity of amylin.Methods: Pancreatic islets were isolated from adult Wistar rats.The cell viability was identified.The experiment groups included normal islets,10 ?mol/L amylin incubated islets,10 ?mol/L amylin and 10 ug/mL IL-1Ra coincubated islets or 10 ?mol/L amylin and 100 nmol/L liraglutide coincubated islets.The incubation time was 24 hours.Annexin V/propidium iodide(PI)double-staining was used to detect the cell death and quantitative reverse transcription(RT-PCR)was used to detect the apoptosis of islet cells.The expression of inflammation,especially IL-1?,was measured using ELISA and western blot.Thioflavin T binding assay and electron microscopy assay were used to test the effect of IL-1Ra and liraglutide on amylin aggregation.Results: 1.Cell death of islets in various groups.The annexin V/ PI double-staining tests showed that islets treated with amylin were associated with a 22.9-fold increase in cell death compared with that of the non-treated control islets.This indicated that amylin amyloid was toxic to islets.Compared with the amylin group,IL-1Ra and liraglutide significantly reduced cell death.2.The relative expression levels of apoptosis relatedgenes in various groups.RT-PCR showed that when treated with amylin,the expression of Bax increased and Bcl-2 reduced.IL-1Ra downregulated the expression of Bax and upregulated expression of Bcl-2 mRNA.Bcl-2 mRNA was upregulated in liraglutide group.3.The relative expression and secretion of inflammatory factors in various groups.Compared with the non-treated control islets,the expression of IL-1?,tumor necrosis factor(TNF)and monocyte chemoattractant protein-1(CCL-2 mRNA)were increased in the hIAPP group.The IL-1? protein was also increased in the hIAPP group.Compared with the hIAPP-treated control islets,the expression of IL-1?,TNF were decreased significantly in IL-1Ra group.The IL-1? protein was also decresed in IL-1Ra group.Compared with the hIAPP-treated control islets,the expression of IL-1?,TNF and CCL-2 were decreased in liraglutide group.The IL-1?protein in culture was also decresed in liraglutide group.4.The fibrillation of amylin in various groups.When amylin alone was incubated in vitro,a time-dependent increase in ThT fluorescence with time was observed and followed a sigmoidal curve.The fluorescence of amylin reached a maximum.Co-incubation with IL-1Ra(molecular concentration ratio 1:1),the fibrillation curve of amylin did not change.Co-incubation with liraglutide(molecular concentration ratio 1:10)delayed the process of amylin fibrillation.The thioflavin T fluorescence did not increase until 2.5h.When Co-incubation with liraglutide(molecular concentration ratio 1:1),the effect became markedly obvious and the curve of amylin fibrillation was low without obvious peak.Electron microscopy tests showed that compared with 20 ?M amylin alone group,the fibrils decresed obviously in 2 ?M liraglutide and 20 ?M amylin group.When co-incubation with 20 ?M liraglutide,the fibrils were nearly undetectable.Conclusions: 1.Amylin promoted the expression of islet inflammatory factors,as well as apoptosis.2.IL-1Ra reduced islet inflammation and apoptosis from amylin toxicity and could not inhibit amylin aggregation,implying the protective role of IL-1 signal pathway.3.Liraglutide protected islets from inflammation and apoptosis caused by amylin.4.Liraglutide affected the aggregation of amylin.