| BackgroundHigh-mobility group box1(HMGB1), a highly conserved protein previously known as a DNA-binding protein involved in maintenance of nucleosome structure and regulation of gene transcription, was first identified as a chromosomal protein and later on also as a membrane-bound form on neuronal cells and was termed ’amphoterin’. Whereas HMGB1is actively released from the activated monocytes and macrophages, its release also occurs passively during the late phase of apoptosis as well as during necrosis.Recently,extracellular HMGB1has been found to act as a potent proinflammatory cytokine. Extracellular HMGB1has been identified as a crucial cytokine involved in many inflammatory conditions such as the inflammatory bowel disease,tumor and rheumatic diseases. Extracellular HMGB1stimulates the vascular endothelial cells, monocytes and maerophages to secrete inflammatory cytokines and adhesion molecules, which can recruit inflammatory cells to the injury site to produce a large number of cytokines, mediating a variety of acute and chronic inflammatory diseases. HMGB1is a regulatory factor in the process of maturation of dendritic cell and polarization of Thl cells, it also can induce TNF-a, IL-1, IL-6and many kinds of adhesion molecules such as VCAM-1and MCP-1in extracellular, and plays an important role on maintaining the balance between Thl and Th2type cytokines. As an endogenous immune adjuvant, HMGB1also can promote the development of diseases by inducing the immune response and interfering the removal of the apoptotic cells,in addition to directly stimulate the inflammatory and immune function.Previous studies have proved that there is a relationship between HMGB1and the autoimmune diseases, and HMGB1has been a therapeutic target in rheumatoid arthritis. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the involvement of multiple organ systems. It is characterized of immune complex deposition caused by autoantibodies, of which antinuclear antibodies is the representment,and multi-system damage, almost every system and every organ of the whole body may be damaged. The autoantibodies in SLE is mainly against the components of cell nucleus.And the presence of these autoantibodies is associated with many clinical manifestations,among which lupus nephritis is one of the most serious complications of SLE and associated closely with the illness and mortality.The effects of medical treatment in the past few decades have significantly increased. However, the high risk of end-stage renal disease and the serious side-effects of drugs prompt us to find more effective and less toxic drugs to treat LN.The pathogenesis of SLE and LN has long been a difficult problem in the field of rheumatology. Therefore, studying the pathogenesis of SLE and finding effective treatment for improving the life quality of patients are of great significance.The dysregulation of immune system,including the dysfunction of immune cell and the abnormal expression of many cytokines,plays a dominant role in the pathogenesis of SLE. In addition, the production of many autoantibodies in serum is the typical characteristic of SLE, especially the autoantibodies against the nucleosome and DNA,which are the compositions of nucleus.It is suggested that apoptosis is the source of self-antigens. Due to the defects of clearance mechanism in SLE, the apoptotic cells accumulate to become the later apoptotic cells and secondary necrosis. Recently,studies suggested that HMGB1is associated with nucleosomes released from the apoptotic cells, and that HMGB1contributes to the immnostimulatory effect of nucleosomes. In addition, it is found that the levels of nucleosome and HMGB1are significantly increased in serum of patients with SLE. HMGB1is considered to be one of the ingredients in DNA-containing immune complexes. However, the role of HMGB1in the pathophysiology of SLE is still not clearly understood. The elevated concentration of DNA-containing immune complexes in serum of the patients with SLE proves that HMGB1and RAGE activate the plasmacytoid dendritic cells and B lymphocytes to produce the antibodies against DNA,which can lead to the occurrence of autoimmune. Furthermore, HMGB1can activate the B lymphocytes.It has been detected that the levels of HMGB1and anti-HMGB1antibody are elevated in serum of the patients with SLE. So HMGB1is identified to be a new antigen.There are several researches on the relationship between HMGB1and the pathogenesis of SLE, however, the results are not the same.The roles of HMGB1in the pathogenesis of SLE are not yet clear.Whether can the HMGB1inhibition impact on SLE progression? Whether can HMGB1be served as a potential therapeutic target of SLE?ObjectiveUsing the lupus-prone model BXSB mice, we explored the potential of HMGB1inhibition as a treatment option for lupus-like disease and the underlying mechanisms. The anti-HMGB1monoclonal antibody (HMGB1mAb) was applied to inhibit the functions of HMGB1and the influences of HMGB1inhibition on BXSB mice were evaluated to explore the role of HMGB1in the pathogenesis of SLE,which supplied the new therapeutic strategies and experimental basis for SLE.MethodsKidney samples were collected at the time of harvest and fixed in10%buffered formalin, embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed.Image analysis was performed with ImageJ (ImageJ, NIH). We evaluated glomerular pathology by examining changes in each glomerulus identified in20glomerular cross sections (GCS) per kidney. For immunofluorescence staining, the kidney tissues were snap-frozen in liquid nitrogen and placed in optimum cutting temperature (OCT) compound. Frozen sections (5mm thick) were stained with fluorescein-conjugated anti-mouse IgG(1:50) or anti-mouse C3(1:30) after being blocked with10%fetal bovine serum.Slides were scored according to the intensity and coverage of immunofluorescence in the glomerulus. Levels of serum IL-1β,IL-6, IL-17, IL-18and IFNa were detected by using a mouse inflammation cytometric bead array kit and was analyzed on a FACSCalibur flow cytometer. Statistics were performed using SPSS17.0software. Data are presented as means±standard deviation (SD). Group comparisons were analyzed by unpaired two-tailed Student t test or Kruskal-Wallis test. The p<0.01were considered significant.Results1.HMGB1mAb reduced proteinuria and splenomegaly.Both groups of mice developed proteinuria from16weeks of age. In contrast, mice treated with HMGB1mAb developed statistically attenuated proteinuria compared to controls starting from8weeks after treatment (p<0.01). In addition, splenomegaly was observed in adult BXSB mice and treatment with HMGB1mAb significantly decreased the sp1enomegaly, as indicated by the SI(p<0.01).2.HMGB1mAb reduced the serum levels of anti-dsDNA autoantibody and circulating IgGThere was a remarkable rise in serum anti-dsDNA antibody levels in the control group at week26, which was obviously inhibited by HMGB1mAb treatment(p<0.01). Similarly, HMGB1mAb-treated group showed significantly lower circulating IgG compared to the control group at week26(p<0.01).3.HMGB1mAb alleviated renal pathology and renal immune complex deposition.H&E-stained kidney sections were assessed by histological scoring for overall glomerular inflammation. BXSB mice in the control group exhibited lupus-like diffuse glomerulonephritis. HMGB1mAb-treated mice, however, exhibited a significant improvement in glomerular histology in the kidneys(p<0.01).Glomerular IgG and C3deposition was significantly reduced in HMGB1mAb-treated mice compared with controls (p<0.01).4.HMGB1mAb reduced proinflammatory cytokines. HMGB1mAb-treated and control-treated BXSB mice were bled at the end of the study.The HMGB1mAb-treated mice showed lower serum levels of IL-1β,IL-6, IL-17,IL-18and IFNa than the control-treated mice(p<0.01).5.HMGB1mAb reduced the caspase-1activity. We found that HMGB1mAb significantly reduced the caspase-1activity in the kidneys of lupus-prone BXSB mice as compared with the control group(p<0.01).Conclusions In this study, we showed that administration of HMGB1mAb significantly ameliorated the severity of murine lupus in BXSB mice, as reflected by reduced proteinuria and glomerulonephritis. Further studies suggest that this therapeutic effect was closely associated with reducing casapase-1activity and multiple proinflammatory cytokines. These data support the HMGB1played a role in the pathogenesis of murine LN.In the present study, we found that HMGB1mAb treatment attenuated the circulating anti-dsDNA and immune complex deposition. These results suggest that HMGB1plays a functional role in the presence of in immune complexes.In summary, our study demonstrates that HMGB1blockade confers benefit on murine lupus-like disease. Our analysis demonstrates the therapeutic efficacy of HMGB1blockade in the lupus-prone BXSB mice. The effects involve inhibition of autoimmunity, caspase activity and inflammatory responses in murine lupus-like disease. Taken with the current data, HMGB1blockade might be a potential target for treating human SLE. |
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