| Type1diabetes mellitus (T1DM), also known as insulin-dependent diabetes mellitus or juvenile diabetes, is generally believed to be caused by an interaction between the immune system and an intricate network of environmental and genetic factors. The key feature of T1DM is cell-mediated destruction of insulin-secreting β cells in the pancreatic islets.High-mobility group box1(HMGB1) was initially identified in1970s as a nuclear protein that is important for transcriptional regulation. HMGB1causes DNA bending and facilitates the binding of several regulatory protein complexes to DNA. In addition to its role in transcriptional regulation, HMGB1has been shown to activate proinflammatory responses following its release by necrotic cells into the extracellular environment. HMGB1is also implicated in the pathogenesis of a number of autoimmune diseases associated with inflammation and tissue injury such as rheumatoid arthritis and systemic lupus erythematosus (SLE). Several important receptors have been implicated in HMGB1signaling, including members of the toll-like family of receptors (TLRs) and the receptor for advanced glycation end products (RAGE). HMGB1may signal TLRs and RAGE to activate nuclear factor-κB (NF-κB), which induces the production of proinflammatory cytokines and the upregulation of leukocyte adhesion molecules, thereby promoting injury and inflammation.Although a cells are not affected during the development of diabetes, whereas β cells undergo extensive destruction, even though a cells form a ring and provide a natural physical barrier around the β cells. The non-obese diabetic (NOD) mouse develops a spontaneous form of autoimmune diabetes that mimics many features of the human disease, which is considered the excellent model in the world. HMGB1is involved in the pathogenesis of many autoimmune diseases. T1DM is also an autoimmune disease. However, whether HMGB1is involved in the pathogenesis of T1DM and its potential mechanism in T1DM have not been reported.In the present study, the distribution of HMGB1in the pancreas was examined by immunohistochemical staining in NOD mice, which demonstrated that the rate of HMGB1expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The results demonstrated that HMGB1might be involved in the pathogenesis of T1DM. We tested HMGB1receptors that are distributed on different pancreatic cell types in NOD mice, which confirmed that HMGB1interacts with TLR4in isolated islets and TLR4is mainly distributed on β cells rather than a cells. Then the genetic changes in NOD mice were detected with gene expression microarrays in the development of disease. At last the mechanism that HMGB1selectively damage TLR4signaling pathway of pancreatic β cells were clarified.Part ⅠDistribution and variation characteristics of HMGB1in the development of T1DM of NOD miceObjective To detect the expression of HMGB1in pancreas of NOD mice, the change in the pathogenesis and the distribution of HMGB1receptors on the islets.Methods NOD mice were used to establish the model of type1diabetes, the changes of blood glucose were tested weekly, the changes of islets were detected by hematoxylin and eosin (HE) staining, confocal laser scanning technology was used to detect the immunofluorescence of islets in non-diabetic and diabetic NOD mice, and the number of islets were counted. Immunohistochemistry was used to examine the relationship between HMGB1translocation and islet destruction by HMGB1staining. Confocal microscopy was used to further detect the expression of distinct HMGB1receptors on different pancreatic islets of NOD mice.Results (1) T1DM began as early as4weeks of age in female NOD mice, the incidence of T1DM increased significantly between14and26weeks of age, the cumulative incidence of T1DM was70%at the end of30week.HE staining showed that the size of pancreatic islet cells varied in non-diabetic NOD mice, islet cells piled together, the structure of islets was intact and full, abundant capillaries were seen between islet cells, mononuclear cells or lymphocytes were hardly seen in the islets; however, pancreatic islets were degenerated, necrosis or even disappeared in diabetic mice, islets became small and shrink, the number of islet cells dropped obviously, fibrous hyperplasia and hyalinization increased significantly at the same time;(2) Confocal immunofluorescence detection:the islets were intact, and insulin-positive islets with red fluorescence were widely distributed in the pancreas of non-diabetic NOD mice, however, insulin-positive islets with red fluorescence was hardly seen in the pancreas of diabetic NOD mice; The number of islets:there was a large number of islet cells in non-diabetic NOD mice while there was a small number of islet cells in the diabetic NOD mice, the number of islet cells decreased significantly from42.5±4.90(non-diabetic mice) to30.85±9.24(diabetic mice), the difference was statistically significant (P<0.01);(3) Immunohistochemical staining of HMGB1: HMGB1was mainly distributed in the nuclei of islet cells in the non-diabetic NOD mice. With the development of type1diabetes, the rate of HMGB1translocation from nuclei to the cytoplasmin rapidly increased from19.44±5.08to79.22±6.92, the difference was statistically significant (P<0.01);(4) The expression and cellular distributions of HMGB1receptors, including TLR2, TLR4, TLR9and RAGE, in the pancreatic islets of NOD mice were examined by immunofluorescence and visualized by confocal microscopy. Little or no expression of TLR2, TLR9or RAGE was observed in the pancreatic islets of4-week-old, non-diabetic NOD mice. In contrast, TLR4was highly expressed on cell of the islets (green) in4-week-old non-diabetic NOD mice;(5) Next, we investigated which of the pancreatic cell types were positive for TLR4receptors. We performed double-labeling for islet a cells and β cells separately with TLR4in4-week-old non-diabetic NOD mice. The cells expressing TLR4were insulin-positive cells (i.e., βcells), which comprise the majority of cells in the islet. The glucagon-positive cells (a cells) formed a ring around the islet; however, relatively few a cells expressed TLR4.Conclusion The expression of HMGB1significantly increased with the development of T1DM, suggesting that HMGB1may participate in the occurrence and development of T1DM by immune mechanisms; TLR4was highly expressed on cell of the islets while little or no expression of TLR2, TLR9or RAGE was observed in the pancreatic islets; HMGB1receptor TLR4was mainly distributed on pancreatic β cells rather than a cells, which was more sensitive to damage.Part IIGenetic changes in NOD mice were detected with microarrays in the development of T1DM Objective To explore the genetic changes in NOD mice, which were detected with gene expression microarrays in the development of T1DMMethods The pancreas of NOD mice was distended via bile duct puncture and retrograde injection of1.5-2ml of4℃collagenase type V. The gland was removed and placed into the D-Hank’s balanced salt solution containing collagenase type V. The pancreas was digested at37℃under continuous shaking. The digest was washed three times with RPMI1640containing5%fetal bovine serum (FBS) and purificated by Ficoll separating medium as described. The separated islets were further purified by handpicking using capillary pipettes and were identified by selectively staining crimson red with Dithizone (DTZ) for the purity of the islets; Total RNA were extracted from islets in non-diabetic and diabetic NOD mice separately, which was quantified by the NanoDrop ND-1000and RNA integrity was assessed by standard denaturing agarose gel electrophoresis; About5μg total RNA of each group was used for labeling and array hybridization. Scanned images were then imported into NimbleScan software (version2.5) for grid alignment and expression data analysis. Differentially expressed genes were identified through fold change filtering; Gene ontology (GO) analysis and pathway analysis were performed using the standard enrichment computation method; Hierarchical clustering was performed to show distinguishable gene expression profiling between two groups.Results (1) There were still a small number of pancreatic exocrine cells and ductal cells after the Ficoll purification. The separated islets were further purified by handpicking using capillary pipettes and were stained with DTZ for identification. The freshly isolated islet cells were round or oval, which formd a complete refraction under an inverted microscope. The islets were crimson red after stained with DTZ. Our results showed that about95%of the islet cells were crimson red, which demonstrated the high purity of the isolated islets. The three fairly sharp and intense bands such as28S,18S and5s ribosomal RNA bands could be seen in the agarose gel electrophoresis. The intensity of the28s rRNA band was about twice that of thel8s rRNA band. And the ratio of OD260/OD280was between1.9-2.0, which suggested the quality of total RNA was very well;(2) In this study, the mouse gene expression array testing44,170gene was manufactured by Roche NimbleGen company. The scanned image obtained above was imported to NimbleScan software (version2.5), the raw data was normalized and the genes of low level were cut off, which were used for grid alignment and expression data analysis.A total of1007differentially expressed genes were obtained, in which432genes were upregulated and575genes were downregulated;(3) The Box Plot was used for comparing the distributions of the intensities from all samples, which is a convenient way to quickly visualize the distributions of a dataset; The hybridization map of the diabetic NOD mice and non-diabetic NOD mice was completely overlapped and analyzed with specialized software. The Scatter-Plot was gained, which is a visualization method used for assessing the gene expression variation between the two compared groups;(4) The GO analysis covers three domains:biological process, cellular component and molecular function. Each major category could be divided into the lower level and the much lower level branch. The results showed that in biological processes,885(87.7%) metabolism-related genes were up-regulated;22(18.97%) metabolism-related genes and21genes (18.10%) on regulation of catalytic were down-regulated; The up-regulated genes were distributed more evenly in the cellular components, in which cells and cell components accounted for488(29.20%); Cytoskeleton and micotubule cytoskeleton genes were down-regulated, which take up57(78.08%);591(84.07%) up-regulated genes was associated with binding, while the down-regulated genes were mainly consisted of44(30.77%) hydrolase activity related genes and17(11.89%) enzyme regulatoractivity related genes;(5) After fold enrichment analysis, DNA fragments formed in the apoptosis of nucleus and catabolic process of DNA raised obviously in biological functions, while the positive regulation of chondrocyte differentiation down-regulated apparently; In the part of cellular components, myosin complex and endosome up-regulated evidently, while myosin complex II and cytoplasmic dynein complex down-regulated; In the part of molecular function, deaminase activity increased, while Rac GTPase activator activity down-regulated;(6) Mucin type O-Glycan biosynthesis pathway was significantly up-regulated in the development of T1DM in NOD mice. Vasopressin-regulated water reabsorption pathway was down-regulated; TIRAP (TIR domain-containing adapter protein) and downstream signaling molecules in TLR4signaling pathway associated with the study were activated.Conclusion Several signaling pathways were activated in the pathogenesis of T1DM, in which TIRAP and downstream signaling molecules in TLR4signaling pathway in the study were activated.PartⅢ HMGB1selectively damage TLR4signaling pathway of pancreatic β cellsObjective To explore the mechanism how HMGB1combined with TLR4and HMGB1participated in the damage of pancreatic β cells.Methods Isolated and purified islets of non-diabetic NOD mice were cultured in vitro; different doses of HMGB1were used for intervention, and the apoptosis of islet cells were detected by TUNEL method; To further research the binding capacity of HMGB1and its corresponding receptor, cells were preincubated with anti-TLR2,-TLR4,-TLR9or-RAGE antibodies separately and then treated with NHS-fluorescein-labeled HMGB1. After fixing, the binding capacity was recorded with confocal microscope. Pancreatic HMGB1and TLR4protein expression was evaluated by western blotting at various times in the natural history of diabetes in NOD mice; We assessed pancreatic HMGB1and TLR4mRNA expression from pancreas of NOD mice by qRT-PCR analysis during the development of diabetes in NOD mice; ELISA technology was used to detect the concentration of inflammatory cytokines from serum of peripheral blood in NOD mice, which helped us to know the changes in the level of systemic inflammatory response.Results (1) Apoptosis detection by TUNEL methods after HMGB1intervention:in the group of high concentration of HMGB1(15μg/mL), the number of TUNEL-positive nuclei of islet cells was (20.31±2.36), which was much more than the group of lower concentration of HMGB1(10μg/mL) group with the number of TUNEL-positive islets (5.56±1.71) and the group of concentration of HMGB1(5μg/mL) with the number of TUNEL-positive islets (2.86±1.36), the difference was statistically significant between the groups (P<0.01). And the concentration of HMGB1was proportional to the number of apoptotic islets, which indicated that HMGB1could promote apoptosis of islet cells;(2) HMGB1could combined with TLR4on islets specifically:pretreatment with anti-TLR2, anti-TLR9, anti-RAGE or IgG did not significantly influence HMGB1cell surface binding. However, anti-TLR4antibodies or unlabeled HMGB1decreased HMGB1cell surface binding, visualized by a reduction in cell-associated fluorescence intensity compared with IgG-treated controls. These results indicated that HMGB1could physically interact with TLR4in islet cells;(3) Expression changes of protein and mRNA in HMGB1and TLR4were detected throughout the course of diabetes. Pancreatic expression of both HMGB1and TLR4was low in young NOD mice (4-6weeks of age) by western blotting. In contrast, the pancreatic expression of HMGB1and TLR4was significantly upregulated in the early stage of diabetes (10-14weeks of age)(P<0.01). To our surprise, in older diabetic mice (28-32weeks of age), the protein expression of HMGB1and TLR4was markedly downregulated (P<0.01); We assessed pancreatic HMGB1and TLR4mRNA expression every3-5weeks during the development of diabetes in NOD mice. This analysis showed no marked changes in HMGB1mRNA expression in the first8weeks of life of NOD mice. However, HMGB1mRNA expression was significantly increased at10weeks of age, which had been increased to (8.83-11.59) times as compared with4weeks of age. Then it was followed by slow reduction in expression over time to reach levels similar to that at8weeks of age. The NOD mice displayed much homogeneity in the expression of TLR4mRNA during the development of diabetes. At6weeks of age, there was a distinct increase in TLR4mRNA expression compared with that at4weeks of age. This increase was transient and followed by a significant increase in TLR4mRNA expression between8and10weeks of age, which had been increased to (14.03-18.58) times as compared with4weeks of age. It was followed by a significant reduction in the values in later weeks. The data of Western Blotting and real-time quantitative PCR showed that the TLR4overexpression on pancreatic (3cells was closely related to the progress of T1DM and HMGB1expression.(4) The results of ELISA suggested that, the concentration of inflammatory cytokines IL-1β (P<0.01), IL-6(P<0.05) and interferon (IFN)-γ (P<0.01) increased in peripheral blood of diabetic NOD mice compared with the non-diabetic mice. These data suggested that the secretion of inflammatory cytokines increased in the diabetic NOD mice, which might be related to the activation of TLR-4signaling pathway. These data suggested that the secretion characteristics of the systemic inflammatory cytokine changed, the secretion of proinflammatory cytokines increased, and anti-inflammatory levels of T1DM were improved in vivo with the the development of T1DM, the overexpression of HMGB1and TLR4, and the activation of TLR4signaling pathway resulted from the interaction of HMGB1and TLR4.Conclusion TLR4is the main receptor on β cells and that HMGB1could signal via TLR4to selectively damage β cells rather than a cells during the development of T1DM; TLR4overexpression on pancreatic β cells was closely related to the progress of T1DM and HMGB1expression; TLR4was upregulated in pancreatic β cells, and played the corresponding function. The activation of TLR4signaling pathway could activate IL-1β, IL-6, IFN-γ and other downstream inflammatory factors, leading to the local inflammation-like reaction, the destruction of islet β-cells, the inadequate secretion of insulin, and the development of T1DM. |
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