The Role And Mechanism Of IL-11-Mediated Islet Fibrosis In β-cell Dysfunction

Background:Islet β-cell dysfunction is a major manifestation of type 2 diabetes mellitus(T2DM)in its later stages.Chronic inflammatory response,immune cell infiltration and fibrotic changes can cause β-cell apoptosis and insufficient insulin secretion.However,most studies have focused on the effects of the inflammatory response on islet β-cells function,and have rarely addressed the role of fibrosis.Currently,the phenomenon of islet fibrosis is gradually attracting the attention of scholars.Fibrosis mainly destroys normal tissues through the synthesis of excess collagen by fibroblasts or fibroblast-like cells,while the orderly arrangement of β cells is necessary for the normal secretion of insulin.In recent years,the effect of interleukin(IL)11 on fibrosis and its mechanism of action have been gradually uncovered,and a variety of inflammatory factors,high glucose and high lipids may induce IL11 secretion and thus affect the occurrence of fibrosis.The downstream pathways of IL11-induced fibrosis mainly include extracellular regulated MAP kinase(ERK)and c-Jun-amino-terminal kinase(JNK),and the direct or indirect effects of IL11 were found to be harmful to parenchymal cells.However,the effects of IL11 on islet fibrosis and islet β-cell function have not been reported.Objectives:1.To analyze the key pathogenic genes and the possible pathogenesis of isletβ-cell dysfunction by bioinformatics analysis.2.To observe islet fibrosis alterations in in vivo studies,and to detect the main sources of IL11 in vivo and ex vivo.3.To explore whether IL11 induces endothelial-to-mesenchymal transition(End MT)in islet endothelial cells,leading to fibrogenic changes by differentiating into fibroblast-like cells.4.To detect whether IL11 affects β-cell function and activity directly or indirectly through islet endothelial cells.Methods:1.The database GSE41762 containing human islet sequencing information was obtained in the Gene Expression Omnibus(GEO)platform,and samples with incomplete clinical characteristics were excluded.Weighted gene coexpression network analysis(WGCNA)was used to obtain the gene modules most closely associated with T2 DM and compare them with body mass index(BMI)and glycated hemoglobin(Hb A1c).We also analyzed the gene functions and signaling pathways of the enriched modules of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)to analyze which pathological mechanisms are associated with islet β-cell dysfunction.In addition,the most critical core genes were screened by combining WGCNA,protein-protein interaction(PPI)and receiver operating characteristic curve(ROC).Finally,the data set GSE50397 was used for validation.2.In vivo study,High-Fat-Diat(HFD)diet induced T2 DM rat model,Lee’s index assessed obesity model.Glucose oxidase was used to detect fasting blood-glucose(FBG),and triglyceride(TG),total cholesterol(TCH)and low-density lipoprotein(LDL)were analyzed by enzyme colorimetric assay.Enzyme linked immunosorbent assays(ELISA)were performed to detect serum insulin,free fatty acids(FFA),IL6,transforming growth factor beta(TGF-β)and IL11.Immunofluorescence analysis of islet β-cell content and collagen marker actin alpha 2(ACTA2)was performed.HE and Masson staining was performed to observe islet structure and fibrosis levels.Immunohistochemical detection of IL6,TGF-β and IL11 in islets.In vitro studies,palmitic acid(PA),high glucose(HG),IL6 and TGF-β interfered with islet endothelial cells and β cells,and IL11 synthesis and secretion levels were detected by q RT-PCR and ELISA.3.IL11 at 10ng/ml,30ng/ml and 50ng/l was applied to intervene in islet endothelial cells,and WB was used to detect endothelial cell marker protein platelet and endothelial cell adhesion molecule 1(CD31),fibrosis marker protein ACTA2 and Collogen I;microscopic observation of morphological changes of islet endothelial cells;scratch assay to detect the effect of IL11 on the migration of islet endothelial cells.Finally,the phosphorylation levels of ERK and JNK were observed by WB.4.After applying 50 ng/ml of IL11 to the islet endothelial cells,the supernatant was collected to prepare conditioned medium(CM),and the β-cells were stimulated with IL11 and CM respectively.Basal insulin secretion(BIS)and glucose stimulated insulin secretion(GSIS)detect the insulin of β-cell;Td T-mediated d UTP nick-End labeling(TUNEL)assay and flow cytometry forβ-cell apoptosis.Results:1.The genes with the top 5000 absolute deviation values were used for WGCNA construction,showing a total of 18 gene co-expression modules.Among them,the yellow module was the most correlated with T2 DM and showed significant positive correlation with both Hb A1 c and BMI.Enrichment analysis showed that the genes in the yellow module mainly affected T2 DM through extracellular matrix collagen synthesis,chemotaxis and inflammatory response;the signaling pathways mainly involved mitogen-activated protein kinase(MAPK)(including ERK and JNK)and inflammatory interactions.A combined WGCNA,PPI and ROC screen yielded core genes IL6,IL11 and prostaglandin peroxidase 2(PTGS2),which were significantly upregulated in the T2 DM group.In the validation dataset,IL6,IL11 and PTGS2 remained significantly upregulated in the T2 DM group,and IL6 and PTGS2 showed significant positive correlations with Hb A1 c and BMI,but IL11 was only positively correlated with Hb A1 c and weakly correlated with BMI.In the ROC analysis,all three had some value in distinguishing between T2 DM and non-diabetes(ND).2.In vivo study,serum FBG,TCH,TG,LDL and FFA were all upregulated in the HFD group compared with the NC group,while insulin levels were mildly downregulated and accompanied by a decrease in islet β-cells.HE and Masson results showed that the islets in the HFD group were structurally incomplete,with a large number of collagen fibers and extremely irregular cell arrangement.Immunofluorescence detection revealed an increase in the intra-islet fibrotic protein ACTA2.In addition,IL6 and TGF-β were upregulated in the serum of rats in the HFD group while IL11 was not significantly altered,but the expression levels of IL6,TGF-β and IL11 were significantly increased in the islets.In vitro study,IL11 m RNA synthesis was upregulated in islets endothelial cells under the intervention of high glucose and high lipids,but IL6 and TGF-β had no effect on them,and there was no increase in IL11 secretion in culture supernatant;in addition,PA and TGF-β could effectively promote IL11 m RNA synthesis in β cells,and the detection of cell culture supernatant revealed that only PA significantly upregulated IL11 secretion.3.After IL11 intervention in islet endothelial cells for 48 h,WB results showed that with the increase of IL11 concentration,endothelial marker protein CD31 was mildly downregulated,and collagen ACTA2 and Collogen I were subsequently increased,among which the most significant was in the intervention group of50ng/ml.Microscopic observation showed that islet endothelial cells transformed from normal polygonal to long spindle shape into fibroblast-like cells after IL11 intervention,and the pro-chemotactic effect was enhanced.Finally,the downstream signaling pathways of IL11,ERK and JNK,were phosphorylated to some extent,but the phosphorylation of ERK was more significant.4.After IL11(50ng/ml)intervention on islet endothelial cells,CM was collected,and IL11(50ng/ml)and CM were used to intervene on β-cells,respectively.The results showed no differential change in BIS of β-cells in IL11 vs.NC group and CM-IL11 group vs.CM-NC group,while GSIS was significantly downregulated in both,but CM-IL11 vs CM-NC is more obvious.In addition,TUNEL assay showed an increase in apoptosis of β-cells under IL11 and CM-IL11 interventions compared to the control group.Flow cytometry assay showed that the indirect effect of IL11 significantly induced apoptosis in β-cells,while direct stimulation of IL11 only mildly affected apoptosis.Conclusion:Bioinformatic analysis revealed possible fibrotic alterations within human islets,which were closely associated with the expression of IL11.In a rat model of HFD,significant fibrotic alterations were observed within the islets,and high glucose,high lipids,IL6 and TGF-β may induce IL11 secretion.However,in vitro studies showed that IL11 mainly originated from β cells and was predominantly affected by high lipids.In addition,IL11 induces End MT to fibroblast-like transformation of islet endothelial cells,leading to an increase in fibrotic proteins,the activation mechanism of which may be related to phosphorylation of ERK.The pro-endothelial-to-fibroblast conversion by IL11 is accompanied by downregulation of islet β-cell function and the onset of apoptosis.In summary,revealing the pathogenesis of fibrosis from various aspects may lay the foundation for our research to inhibit fibrosis and thus improve islet β-cell function,and provide new treatments.