The Role Of NLRP3 And HMGB1 In The Pathogenesis Of Idiopathic Inflammatory Myopathy And Its Proteomics Research

Backgrounds:Idiopathic inflammatory myopathy (IIM) is a group of autoimmune diseases,which mainly include polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis. Although much evidence showed that IIM is mediated by immunity, more specific pathogenesis is still unclear. Research shows that NLRP3 inflammasome, a multiprotein complex comprised of NLRP3 (NOD-like receptor protein 3), ASC (apoptosisassociated speck-like protein containing a CARD) and the aspartate protease Caspase-1 is involved in the innate immune response. However, it is still to be further studied that whether NLRP3 inflammasome is related to the degeneration and necrosis of muscle fibers in muscle tissue of PM/DM patients, and what role it plays in the pathogenesis of IIM. High mobility group box1 (HMGB1), as an important inflammatory substance, was confirmed to participate in the pathogenesis of NLRP3 inflammasome pathway together with the downstream material IL-18 and IL- 1? of caspase-1, by which being regulated in the animal model of pulmonary interstitial fibrosis, but there are no related studies in IIM.Proteomics is widely used in disease research to find possible biomarkers of a disease that provide the best diagnostic gold standard for the disease. Many related studies show that it is highly possible that there are particularly relevant protein biomarkers in the blood and muscle tissue of IIM patients, which is still being tried to explore. In non-quantitative proteomics techniques,SWATH-MS (Sequential Window Acquisition of all theoretical fragment ions - Mass Spectrometry) is a relatively stable and widely used technique with the characteristic of high throughput. In the field of medicine, SWATH-MS has showed preliminary application results in clinical diagnosis, disease pathogenesis and drug development. Therefore, it is of great practical significance to directly study the proteomics of diseased muscle tissue of IIM patients.Objectives:1. To explore the expression of NLRP3 and HMGB1 in PM/DM patients and EAM mice.2. To intervene EAM mice through immunity and observe the expression of NLRP3 and HMGB1 to understand its therapeutic effect.3. To find possible specific protein markers of IIM through proteomics research of PM/DM patients' muscle.Materials and Methods:1.12 BALB/c mice were chosen, guinea pig skeletal muscle homogenate and Freund's adjuvant suspension were used to prepare the model. Then the clinical manifestation,serum CK and pathological changes of muscle were observed.2.18 BALB/c mice were selected and divided into EAM treatment group, EAM group and control. We treated EAM treatment group with BAY 11-7082, and observed their clinical manifestations and the mRNA and protein expression of muscle tissue NLRP3 and HMGB1.3.The expression of NLRP3 and HMGB1 in muscle tissue of six patients with PM,ten patients with DM and ten healthy people through muscle biopsy (control) was observed by immunohistochemical staining.4.The muscle tissue proteomics of three patients with dermatomyositis, three patients with polymyositis, three patients with immune-mediated necrotizing myopathy and three patients with Duchenne muscular dystrophy were detected by SWATH-MS technique and the differential proteins of the results were analyzed and compared.Results:1. The clinical manifestations of EAM group were obvious with the increase of the level of serum CK and the typical myositis changes as pathology, suggesting that it was an ideal EAM animal model.2. Compared with the EAM group and the control group, the clinical manifestations of the EAM treatment group were significantly improved (1.73 ±0.1 vs 1.55±0.1,P <0.01),the limb muscle strength being raised (11.0±0.7g/g vs 8.1 ±0.2g/g vs 13.4±0.7g/g, P <0.01) and the mRNA and protein expression of NLRP3 and HMGB1 in muscle tissue being decreased (P <0.01).3. Compared with the control group, the expression of NLRP3 in the muscle tissue of PM patients was all positive, while the positive rate of NLRP3 expression in the muscle tissue of DM patients was 80%. The expression of HMGB1 in the muscle tissue of PM patients was all positive, while the positive rate of HMGB1 expression in the muscle tissue of DM patients was 30%.4. Compared with DMD group, there were 302 kinds of differential proteins detected in PM group and 484 in DM group. There were 176 kinds of dilferential proteins detected in PM group and 261 in DM group compared with NM group. In order to find the differential proteins related to inflammatory immunity, further analysis revealed that six kinds in DM group and ten kinds in PM group were associated with inflammation. Among them, heat shock protein 90-?, double chain glycoprotein in DM group and apoptosis-associated speck-like protein containing a CARD in PM group were related to the reaction pathway of NLRP3 inflammasome.Conclusions:1. NLRP3 and HMGB1 participate in the pathogenesis of IIM.2.Treatment may be effective by intervening the expression of NLRP3 and HMGB1.3.There may be biomarker proteins related to IIM.