RESUMO
Connective tissue growth factor (CTGF) has been recently acknowledged as an ideal biomarker in the early disease course, participating in the pathogenesis of pannus formation in rheumatoid arthritis (RA). However, existing approaches for the detection of or antagonist targeting CTGF are either lacking or unsatisfactory in the diagnosis and treatment of RA. To address this, we synthesized and screened high-affinity single-stranded DNA aptamers targeting CTGF through a protein-based SELEX procedure. The structurally optimized variant AptW2-1-39-PEG was characterized thoroughly for its high-affinity (KD 7.86 nM), sensitivity (minimum protein binding concentration, 2 ng), specificity (negative binding to other biomarkers of RA), and stability (viability-maintaining duration in human serum, 48 h) properties using various biochemical and biophysical assays. Importantly, we showed the antiproliferative and antiangiogenic activities of the aptamers obtained using functional experiments and further verified the therapeutic effect of the aptamers on joint injury and inflammatory response in collagen-induced arthritis (CIA) mice, thus advancing this study into actual therapeutic application. Furthermore, we revealed that the binding within AptW2-1-39-PEG/CTGF was mediated by the thrombospondin 1 (TSP1) domain of CTGF using robust bioinformatics tools together with immunofluorescence. In conclusion, our results revealed a novel aptamer that holds promise as an additive or alternative approach for CTGF-targeting diagnostics and therapeutics for RA.
Assuntos
Aptâmeros de Nucleotídeos , Artrite Experimental , Artrite Reumatoide , Neovascularização da Córnea , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/uso terapêutico , Artrite Experimental/diagnóstico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Humanos , Camundongos , PannusRESUMO
Resveratrol/RES (3,5,4'-trihydroxy-trans-stilbene) is a natural compound found in many food items and red wine, which exhibits pleiotropic biological effects. Several preclinical studies evaluating the efficacy of RES in animal models of rheumatoid arthritis (RA) have been conducted, but the diversity of the experimental conditions and of their outcomes preclude definitive conclusions about RES's efficacy. We, therefore, performed a meta-analysis to assess its efficacy in mitigating experimental RA. We searched three databases until January 2021 and used the random-effects model for drawing inferences. Eighteen studies involving 544 animals were used in this study. Pooled analysis showed that experimental RA causes paw swelling (Hedge's g = 9.823, p = 0.000), increases polyarthritis score and arthritis index, and RES administration reduces paw volume (Hedge's g = -2.550, p = 0.000), polyarthritis score, and arthritis index besides amelioration in the histopathological score and cartilage loss. RA is accompanied by increased oxidative stress due to high malondialdehyde (MDA) level (p < 0.001) and low superoxide dismutase (SOD) activity (p = 0.002), and RES reduced MDA level (p < 0.001) and increased SOD activity (p < 0.001). Experimental RA exhibited an increase in pro-inflammatory cytokines viz. tumor necrosis factor (TNF)-α (p < 0.001), interleukin (IL)-6 (p = 0.002), and IL-1 (p < 0.001); however, insufficient quantitative data precluded us from assessing changes in the anti-inflammatory cytokine, IL-10. In experimental RA, RES decreased TNF-α (p < 0.001), IL-6 (p < 0.001) and IL-1 (p = 0.001) and increased IL-10. This meta-analysis suggests that RES can be a clinically effective therapy for RA, pending clinical trials.
Assuntos
Antioxidantes/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Resveratrol/farmacologia , Animais , Antioxidantes/uso terapêutico , Artrite Experimental/diagnóstico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Resveratrol/uso terapêutico , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: To investigate the effect of matrine on rats with collagen-induced arthritis (CIA) and its regulatory effect on receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) expression. METHODS: Wistar rats (n = 6) and CIA rats (n = 30) were randomly divided into six groups: healthy, CIA control, low/medium/high matrine (25, 50, or 100 mg/kg, once per day for six weeks), and methotrexate (MTX) (2 mg/kg, once per week for six weeks). The degree of joint damage was evaluated by X-ray and HE staining. Bone marrow suppression was assessed by routine blood analysis. In addition, the levels of serum RANKL and OPG in the rats were measured by ELISA. RESULTS: The level of joint swelling and degree of joint damage assessed by ankle swelling measurements, AI score, X-ray, and HE staining were alleviated in the CIA rats treated with MTX or different doses of matrine. Furthermore, no obvious inhibitory effect was observed on the bone marrow of the CIA rats, regardless of the dose of matrine or treatment with 2 mg/kg MTX (P > 0.05). The levels of OPG in serum and the ratio of OPG/RANKL were higher, and RANKL expression was lower in the low/medium/high matrine group compared with that of the CIA control group. The serum levels of OPG and OPG/RANKL ratio increased with the matrine dose, while the opposite was observed for RANKL expression. CONCLUSION: Matrine treatment was associated with a lower degree of bone destruction, increased OPG expression and OPG/RANKL ratio, and decreased RANKL expression in CIA rats. Thus, matrine may represent a novel drug candidate for the treatment of RA.
Assuntos
Alcaloides/farmacologia , Artrite Experimental/etiologia , Artrite Experimental/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoprotegerina/genética , Quinolizinas/farmacologia , Ligante RANK/genética , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/tratamento farmacológico , Biomarcadores , Biópsia , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Osteoprotegerina/sangue , Osteoprotegerina/metabolismo , Ligante RANK/sangue , Ligante RANK/metabolismo , Radiografia , Ratos , MatrinasRESUMO
Induction of severe inflammatory arthritis in the collagen antibody-induced arthritis (CAIA) murine model causes extensive joint damage and pain-like behavior compromising analysis. While mild models are less severe, their reduced, variable penetrance makes assessment of treatment efficacy difficult. This study aimed to compare macroscopic and microscopic changes in the paws, along with central nervous system activation between a mild and moderate CAIA model. Balb/c mice (n=18) were allocated to control, mild, and moderate CAIA groups. Paw inflammation, bone volume (BV), and paw volume (PV) were assessed. Histologically, the front paws were assessed for joint inflammation, cartilage damage, and pre/osteoclast-like cells and the lumbar spinal cord and the periaqueductal gray (PAG) region of the brain for glial reactivity. A moderate CAIA dose induced (1) significantly greater local paw inflammation, inflammatory cell infiltration, and PV; (2) significantly more osteoclast-like cells on the bone surface and within the surrounding soft tissue; and (3) significantly greater glial reactivity within the PAG compared with the mild CAIA model. These findings support the use of a moderate CAIA model (higher dose of monoclonal antibodies with low-dose lipopolysaccharide) to induce more consistent histopathological features, without excessive joint destruction.
Assuntos
Artrite Experimental/patologia , Reabsorção Óssea/patologia , Cartilagem Articular/patologia , Modelos Animais de Doenças , Edema/patologia , Animais , Anticorpos Monoclonais/administração & dosagem , Artrite Experimental/induzido quimicamente , Artrite Experimental/diagnóstico , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/patologia , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/diagnóstico , Cartilagem Articular/efeitos dos fármacos , Edema/induzido quimicamente , Edema/diagnóstico , Feminino , Membro Anterior/efeitos dos fármacos , Membro Anterior/patologia , Histocitoquímica , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Substância Cinzenta Periaquedutal/patologia , Índice de Gravidade de Doença , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologiaRESUMO
The development of collagen type II (CII)-induced arthritis (CIA), a model of rheumatoid arthritis, in rats housed in cages with bedding composed of Celliant fibres containing ceramic particles, which absorb body heat and re-emit the energy back to the body in the form of infrared radiation (+IRF rats), and those housed in cages with standard wooden shaving bedding (-IRF control rats) was examined. The appearance of the first signs of CIA was postponed, while the disease was milder (judging by the arthritic score, paw volume, and burrowing behaviour) in +IRF compared with -IRF rats. This correlated with a lower magnitude of serum anti-CII IgG antibody levels in +IRF rats, and lower production level of IL-17, the Th17 signature cytokine, in cultures of their paws. This could be partly ascribed to impaired migration of antigen-loaded CD11b + dendritic cells and their positioning within lymph nodes in +IRF rats reflecting diminished lymph node expression of CCL19 /CCL21. Additionally, as confirmed in rats with carrageenan-induced paw inflammation (CIPI), the infrared radiation from Celliant fibres, independently from immunomodulatory effects, exerted anti-inflammatory effects (judging by a shift in pro-inflammatory mediator to anti-inflammatory/immunoregulatory mediator ratio towards the latter in paw cultures) and ameliorated burrowing behaviour in CIA rats.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Autoimunidade/efeitos da radiação , Roupas de Cama, Mesa e Banho/veterinária , Raios Infravermelhos/uso terapêutico , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/radioterapia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/radioterapia , Carragenina/administração & dosagem , Carragenina/imunologia , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/imunologia , Abrigo para Animais , Humanos , Masculino , Ratos , Índice de Gravidade de DoençaRESUMO
The scavenger receptor CD163 is highly expressed in macrophages in sites of chronic inflammation where it has a not yet defined role. Here we have investigated development of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) in CD163-deficient C57BL/6 mice. Compared to wild-type mice, the CIA in CD163-deficient mice had a several-fold higher arthritis score with early onset, prolonged disease and strongly enhanced progression. Further, the serum anti-collagen antibody isotypes as well as the cytokine profiles and T cell markers in the inflamed joints revealed that CD163-deficient mice after 52 days had a predominant Th2 response in opposition to a predominant Th1 response in CD163+/+ mice. Less difference in disease severity between the CD163+/+ and CD163-/- mice was seen in the CAIA model that to a large extent induces arthritis independently of T-cell response and endogenous Th1/Th2 balance. In conclusion, the present set of data points on a novel strong anti-inflammatory role of CD163.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Receptores de Superfície Celular/deficiência , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Artrite Experimental/sangue , Artrite Experimental/diagnóstico , Artrite Experimental/patologia , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/patologia , Colágeno Tipo II/imunologia , Citocinas/sangue , Citocinas/imunologia , Citocinas/metabolismo , Progressão da Doença , Humanos , Articulações/imunologia , Articulações/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Índice de Gravidade de Doença , Líquido Sinovial/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Equilíbrio Th1-Th2 , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1ß production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Macrófagos/imunologia , Piruvato Quinase/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/patologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/patologia , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/metabolismo , Camundongos , Naftoquinonas/administração & dosagem , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Ratos , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
Diacerein (DIA) is a slow-acting drug for osteoarthritis (OA). Oral DIA administration, however, exerts side effects including diarrhea and urine discoloration. We fabricated DIA-loaded poly(d,l-lactide-co-glycolide) nanoparticles (DIA/PLGA NPs) that allow sustained release of DIA. In vitro, rat synoviocytes were used to investigate the cytotoxicity and anti-inflammatory effects of DIA-loaded NPs. In vivo, monosodium iodoacetate (MIA)-induced OA rats were divided into seven groups that included non-treated healthy control rats and rats injected with MIA alone or in combination with NPs, DIA(5%) solution, DIA(1%)/NPs, DIA(5%)/NPs, or oral DIA. The in vitro studies revealed that DIA/PLGA NPs dose-dependently suppressed mRNA levels of pro-inflammatory cytokines and enzymes, including interleukin-1 (IL-1), IL-6, tumor necrosis factor-α, matrix metalloproteinase-3 (MMP-3), MMP-13, cyclo-oxygenase-2, and a disintegrin and metalloproteinase with thrombospondin motifs-5 in synoviocytes. The in vivo studies demonstrated that intra-articular treatment of OA rat models with DIA-loaded PLGA NPs markedly decreased mRNA levels of these pro-inflammatory factors and increased those of anti-inflammatory cytokines (IL-4 and IL-10). Micro-computed tomography and histological evaluations indicated that intra-articular injection of DIA-loaded NPs was effective in protecting against cartilage degradation. Administration of DIA/PLGA NPs via intra-articular injection is promising for inhibiting inflammation and protecting against cartilage degradation in OA.
Assuntos
Antraquinonas/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Artrite Experimental/tratamento farmacológico , Portadores de Fármacos/química , Osteoartrite/tratamento farmacológico , Administração Oral , Animais , Antraquinonas/efeitos adversos , Antraquinonas/farmacocinética , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacocinética , Artrite Experimental/induzido quimicamente , Artrite Experimental/diagnóstico , Artrite Experimental/patologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Diarreia/induzido quimicamente , Diarreia/prevenção & controle , Relação Dose-Resposta a Droga , Liberação Controlada de Fármacos , Humanos , Injeções Intra-Articulares , Ácido Iodoacético/toxicidade , Masculino , Nanopartículas/química , Osteoartrite/induzido quimicamente , Osteoartrite/diagnóstico , Osteoartrite/patologia , Projetos Piloto , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Cultura Primária de Células , Ratos , Sinoviócitos , Microtomografia por Raio-XRESUMO
Rheumatoid arthritis (RA) is considered a systemic chronic inflammatory joint disease characterized by chronic synovitis and cartilage and bone destruction. Interleukin-33 (IL-33) is a proinflammatory cytokine which is highly expressed in the synovium of RA patients and the joints of mice with collagen-induced arthritis (CIA) and exacerbates CIA in mice. However, the role of the IL-33-neutralizing antibody in the murine model of CIA remains unclear. In the present study, CIA mice were given intraperitoneally with polyclonal rabbit anti-murine IL-33 antibody (anti-IL-33) or normal rabbit IgG control after the first signs of arthritis. Administration of anti-IL-33 after the onset of disease significantly reduced the severity of CIA and joint damage compared with controls treated with normal rabbit IgG. Anti-IL-33 treatment also significantly decreased the serum levels of interferon-γ(IFN-γ),IL-6, IL-12, IL-33, and tumor necrosis factor-α (TNF-α). Moreover, anti-IL-33 treatment significantly downregulated the production of IFN-γ, IL-6, IL-12, IL-33, and TNF-α in ex vivo-stimulated spleen cells. Together, our results indicate that the IL-33-neutralizing antibody may provide a therapeutic strategy for RA by inhibiting the release of proinflammatory cytokines.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Interleucina-33/antagonistas & inibidores , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Colágeno/administração & dosagem , Colágeno/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Humanos , Injeções Intraperitoneais , Interleucina-33/metabolismo , Articulações/imunologia , Articulações/patologia , Masculino , Camundongos , Índice de Gravidade de DoençaRESUMO
To prevent spinal progression in ankylosing spondylitis, initiating TNF-inhibitor treatment as early as possible is suggested. However, the outcomes are inconsistent in previous clinical studies. Here, we investigated the effect of TNF inhibition alone on spinal progression when used during arthritis development in a murine model. We injected 8-week-old SKG mice with curdlan (curdlan group). We injected adalimumab at 3 and 9 weeks after the first curdlan injection (ADA group). The clinical scores of peripheral arthritis decreased in the ADA group at 3 weeks after first adalimumab injection. Using positron emission tomography-magnetic resonance imaging and histologic examination, spinal inflammation was observed in the curdlan group, and was significantly deceased in the ADA group. However, spinal osteoblast activities by imaging using OsteoSense 680 EX and bone metabolism-related cytokines such as receptor activator of nuclear factor-kappa B ligand, osteoprotegerin, Dickkopf-1, and sclerostin levels except IL-17A level were not different between the two groups. We conclude that treating TNF inhibitor alone reduced peripheral arthritis score and spinal inflammation in curdlan-injected SKG mice but did not decrease the spinal osteoblast activity, suggesting little effect on spinal ankylosis.
Assuntos
Adalimumab/administração & dosagem , Artrite Experimental/tratamento farmacológico , Coluna Vertebral/efeitos dos fármacos , Espondilite Anquilosante/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Progressão da Doença , Feminino , Humanos , Injeções Intraperitoneais , Camundongos , Índice de Gravidade de Doença , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/imunologia , Coluna Vertebral/patologia , Espondilite Anquilosante/diagnóstico , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/patologia , Tomografia Computadorizada por Raios X , beta-Glucanas/administração & dosagem , beta-Glucanas/imunologiaRESUMO
We investigated the effects of ibandronate, a bisphosphonate; eldecalcitol, an active vitamin D3 analogue; and combination treatment with both agents on secondary osteoporosis and arthritis using rats with adjuvant-induced arthritis. Arthritis was induced in 8-week-old male Lewis rats. Rats were randomized into four treatment groups and an untreated normal control group: ibandronate, eldecalcitol, ibandronate + eldecalcitol, vehicle, and control. Paw thickness was measured to evaluate arthritis. Joint destruction was evaluated histomorphometrically by the ankle joint stained with Fast Green and safranin O. The femur and lumbar spine were scanned using dual-energy X-ray absorptiometry, and the distal femur was scanned using micro-computed tomography for bone mineral density (BMD) and trabecular microstructural evaluations. Ibandronate and/or eldecalcitol increased BMD in both the lumbar vertebrae and femur and improved several microstructural parameters (bone volume/total volume, structure model index, trabecular number, and trabecular separation of the distal femur). In addition, there was an additive effect of combination treatment compared with single treatments for most trabecular parameters, including BMD and bone volume. However, ibandronate and/or eldecalcitol did not inhibit arthritis and joint destruction. Combination treatment with ibandronate and eldecalcitol may be effective for secondary osteoporosis associated with arthritis.
Assuntos
Artrite Experimental/diagnóstico , Artrite Experimental/etiologia , Ácido Ibandrônico/farmacologia , Osteoporose/diagnóstico , Osteoporose/etiologia , Vitamina D/análogos & derivados , Microtomografia por Raio-X , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Biópsia , Modelos Animais de Doenças , Imageamento Tridimensional , Articulações/diagnóstico por imagem , Articulações/patologia , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Fenótipo , Proteoglicanas/metabolismo , Ratos , Vitamina D/farmacologiaRESUMO
Nanobody against V-set and Ig domain-containing 4 (Vsig4) on tissue macrophages, such as synovial macrophages, could visualize joint inflammation in multiple experimental arthritis models via single-photon emission computed tomography imaging. Here, we further addressed the specificity and assessed the potential for arthritis monitoring using near-infrared fluorescence (NIRF) Cy7-labeled Vsig4 nanobody (Cy7-Nb119). In vivo NIRF-imaging of collagen-induced arthritis (CIA) was performed using Cy7-Nb119. Signals obtained with Cy7-Nb119 or isotope control Cy7-NbBCII10 were compared in joints of naive mice versus CIA mice. In addition, pathological microscopy and fluorescence microscopy were used to validate the arthritis development in CIA. Cy7-Nb119 accumulated in inflamed joints of CIA mice, but not the naive mice. Development of symptoms in CIA was reflected in increased joint accumulation of Cy7-Nb119, which correlated with the conventional measurements of disease. Vsig4 is co-expressed with F4/80, indicating targeting of the increasing number of synovial macrophages associated with the severity of inflammation by the Vsig4 nanobody. NIRF imaging with Cy7-Nb119 allows specific assessment of inflammation in experimental arthritis and provides complementary information to clinical scoring for quantitative, non-invasive and economical monitoring of the pathological process. Nanobody labelled with fluorescence can also be used for ex vivo validation experiments using flow cytometry and fluorescence microscopy.
Assuntos
Artrite Experimental/diagnóstico , Artrite Experimental/metabolismo , Macrófagos/metabolismo , Imagem Molecular/métodos , Receptores de Complemento , Anticorpos de Domínio Único , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Animais , Imunofluorescência , Corantes Fluorescentes/química , Imuno-Histoquímica , Macrófagos/imunologia , Masculino , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Estrutura Molecular , Receptores de Complemento/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Espectroscopia de Luz Próxima ao Infravermelho , Coloração e Rotulagem , Membrana Sinovial/imunologiaRESUMO
Objectives: The discovery of alternative and well-tolerated anti-arthritic drugs, especially from natural products, is becoming an area of active research. Pedunculoside (PE) is a novel triterpene saponin extracted from the dried bark of Ilex rotunda Thunb. Limited published papers have reported its pharmacological properties, including anti-inflammatory, anti-myocardial ischaemia, anti-liver injury, and hypocholesterolaemic activities. However, the effect of PE on rheumatoid arthritis (RA) remains unknown. Here, we investigated the anti-arthritic effect of PE in both in vitro and in vivo models. Method: The inhibitory effects of PE on proliferation, migration, and production of inflammatory mediators in primary fibroblast-like synoviocytes (FLSs) were examined by a 5-ethynyl-2'-deoxyuridine incorporation assay, wound-healing assay, and real-time polymerase chain reaction, respectively. Cellular signalling mechanisms were analysed by Western blot. The in vivo studies were performed using a collagen-induced arthritis (CIA) rat model. Multiple methods, including arthritis scoring, enzyme-linked immunoassay, radiography, and histopathological assessment, were used to evaluate the therapeutic effects of PE on CIA rats. Results: The in vitro studies revealed that PE significantly inhibited proliferation and migration of FLSs. PE also decreased the production of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, IL-8, and tumour necrosis factor-α (TNF-α). Western blot results suggested that PE suppressed TNF-α-stimulated activation of p38 and extracellular signal-regulated kinase. The in vivo studies showed that PE treatment significantly inhibited synovial inflammation and bone destruction in CIA rats. Conclusion: These results demonstrate that PE exerts an inhibitory role in FLSs and CIA rats, and therefore may have therapeutic value for the treatment of RA.
Assuntos
Artrite Experimental/tratamento farmacológico , Desoxicitidina/análogos & derivados , Glucose/análogos & derivados , Membrana Sinovial/patologia , Triterpenos/farmacologia , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/metabolismo , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Desoxicitidina/metabolismo , Medicamentos de Ervas Chinesas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Citometria de Fluxo , Glucose/farmacologia , Fenótipo , Radiografia , Ratos , Ratos Wistar , Membrana Sinovial/metabolismoRESUMO
C-telopeptide of type II collagen (CTX-II) has been shown to be a highly relevant biomarker of cartilage degradation in human rheumatic diseases, if measured in synovial fluid or urine. However, serum or plasma CTX-II have not been demonstrated to have any clinical utility to date. Here, we describe the GPDPLQ1237 ELISA which targets the EKGPDPLQ↓ neo-epitope, an elongated version of the CTX-II neo-epitope (EKGPDP↓), speculated to be a blood-precursor of CTX-II generated by the cysteine protease cathepsin K. Human osteoclast cartilage resorption cultures as well as oncostatin M and tumour necrosis factor α-stimulated bovine cartilage explant cultures were used to validate GPDPLQ1237 biologically by treating the cultures with the cysteine protease inhibitor E-64 and/or the matrix metalloproteinase (MMP) inhibitor GM6001 to assess the potential contributions of these two protease classes to GPDPLQ1237 release. Cartilage resorption-derived GPDPLQ1237 release was inhibited by E-64 (72.1% inhibition), GM6001 (75.5%), and E-64/GM6001 (91.5%), whereas CTX-II release was inhibited by GM6001 (87.0%) but not by E-64 (5.5%). Cartilage explant GPDPLQ1237 and CTX-II release were both fully inhibited by GM6001 but were not inhibited by E-64. No clinically relevant GPDPLQ1237 reactivity was identified in human serum, plasma, or urine from healthy donors or arthritis patients. In conclusion, the GPDPLQ1237 biomarker is released during osteoclast-derived cysteine protease- and MMP-mediated cartilage degradation in vitro, whereas CTX-II release is mediated by MMPs and not by cysteine proteases, as well as from MMP-mediated cartilage degradation under a pro-inflammatory stimulus. These findings suggest that GPDPLQ1237 may be relevant in diseases with pathological osteoclast activity and cartilage degradation. Further studies are required to validate the neo-epitope in human samples.
Assuntos
Artrite Experimental/diagnóstico , Cartilagem Articular/patologia , Colágeno Tipo II/análise , Epitopos/imunologia , Osteoclastos/metabolismo , Fragmentos de Peptídeos/análise , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/imunologia , Bovinos , Colágeno Tipo II/imunologia , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Ensaio de Imunoadsorção Enzimática , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Limite de Detecção , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/imunologia , Fragmentos de Peptídeos/imunologia , Ratos , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismoRESUMO
Objectives: To explore the molecular mechanisms in which vitamin D (VD) regulates T cells, especially Th17 cells in collagen-induced arthritis (CIA). Methods: DBA1/J mice induced for CIA were intraperitoneally treated with VD. CIA clinical symptoms and inflammatory responses including Th1/Th17/Tregs percentages were determined and compared. Mouse naïve CD4+ T cells transduced with miR-124 inhibitor or not were polarized to Th17 cells with or without VD. Subsequently, cellular differentiation and IL-6 signaling moleculars were analyzed. Results: VD treatment significantly delayed CIA onset, decreased incidence and clinical scores of arthritis, downregulated serum IgG levels and ameliorated bone erosion. VD downregulated IL-17A production in CD4+ T cells while increased CD4+Foxp3+Nrp-1+ cells both in draining lymph nodes and synovial fluid in arthritic mice. VD inhibited Th17 cells differentiation in vivo and in vitro and potentially functioning directly on T cells to restrain Th17 cells through limiting IL-6R expression and its downstream signaling including STAT3 phosphorylation, while these effects were blocked when naïve CD4+ T cells were transduced with miR-124 inhibitor. Conclusions: VD treatment ameliorates CIA via suppression of Th17 cells and enhancement of Tregs. miR-124-mediated inhibition of IL-6 signaling, provides a novel explanation for VD's role on T cells in CIA mice or RA patients and suggests that VD may have treatment implications in rheumatoid arthritis.
Assuntos
Artrite Experimental/etiologia , Artrite Experimental/metabolismo , Interleucina-6/metabolismo , MicroRNAs/genética , Células Th17/imunologia , Células Th17/metabolismo , Vitamina D/análogos & derivados , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/tratamento farmacológico , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/efeitos dos fármacos , Vitamina D/metabolismo , Vitamina D/farmacologia , Microtomografia por Raio-XRESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory arthropathy associated with articular damage and attendant comorbidities. Even although RA treatment has advanced remarkably over the last decade, a significant proportion of patients still do not achieve sustained remission. The cause of RA is not yet known despite the many potential mechanisms proposed. It has been confirmed that RA is associated with dysregulated immune system and persistent inflammation. Therefore, management of inflammation is always the target of therapy. Sinomenine (SIN) is the prescription drug approved by the Chinese government for RA treatment. A previous study found that SIN was a robust anti-inflammation drug. In this study, we screened the different secretory cytokines using inflammation antibody arrays and qRT-PCR in both LPS-induced and SIN-treated RAW264.7 cells followed by evaluation of the ability of SIN to modulate cytokine secretion in a cell model, collagen-induced arthritis (CIA) mouse model, and RA patients. Several clinical indexes affecting the 28-joint disease activity score (DAS28) were determined before and after SIN treatment. Clinical indexes, inflammatory cytokine secretion, and DAS28 were compared among RA patients treated with either SIN or methotrexate (MTX). To explore the mechanism of SIN anti-inflammatory function, RA-associated monocyte/macrophage subsets were determined using flow cytometry in CIA mouse model and RA patients, both treated with SIN. The results demonstrated that SIN regulated IL-6, GM-CSF, IL-12 p40, IL-1α, TNF-α, IL-1ß, KC (CXCL1), Eotaxin-2, IL-10, M-CSF, RANTES, and MCP-1 secretion in vivo and in vitro and reduced RA activity and DAS28 in a clinical setting. Furthermore, SIN attenuated CD11b+F4/80+CD64+ resident macrophages in the synovial tissue, CD11b+Ly6C+CD43+ macrophages in the spleen and draining lymph nodes of CIA mice. The percentage of CD14+CD16+ peripheral blood mononuclear cells was reduced by SIN in RA patients. These data indicated that SIN regulates the secretion of multiple inflammatory cytokines and monocyte/macrophage subsets, thereby suppressing RA progression. Therefore, along with MTX, SIN could be an alternative cost-effective anti-inflammatory agent for treating RA.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Morfinanos/uso terapêutico , Adulto , Idoso , Animais , Antirreumáticos/farmacologia , Artrite Experimental/sangue , Artrite Experimental/diagnóstico , Artrite Experimental/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Colágeno/administração & dosagem , Colágeno/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Diclofenaco/uso terapêutico , Progressão da Doença , Quimioterapia Combinada , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Metotrexato/uso terapêutico , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Morfinanos/farmacologia , Células RAW 264.7 , Índice de Gravidade de Doença , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Resultado do TratamentoRESUMO
Mineral oils are extensively used in our daily life, in food, cosmetics, biomedicine, vaccines and in different industrial applications. However, exposure to these mineral oils has been associated with immune adjuvant effects and the development of autoimmune diseases. Here we investigate the structural impacts of the hydrocarbon oil molecules on their adjuvanticity and autoimmunity. First, we showed that hydrocarbon oil molecules with small atomic differences could result in experimental arthritis in DA rats differing in disease severity, incidence, weight change and serum levels of acute phase proteins. Injection of these hydrocarbon oils resulted in the activation, proliferation and elevated expression of Th1 and especially Th17 cytokines by the T cells, which correlate with the arthritogenicity of the T cells. Furthermore, the more arthritogenic hydrocarbon oils resulted in an increased production of autoantibodies against cartilage joint specific, triple-helical type II collagen epitopes. When injected together with ovalbumin, the more arthritogenic hydrocarbon oils resulted in an increased production of αß T cell-dependent anti-ovalbumin antibodies. This study shows the arthritogenicity of hydrocarbon oils is associated with their adjuvant properties with implications to not only arthritis research but also other diseases and medical applications such as vaccines in which oil adjuvants are involved.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Artrite Experimental/imunologia , Autoimunidade , Hidrocarbonetos/efeitos adversos , Óleo Mineral/efeitos adversos , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Artrite Experimental/sangue , Artrite Experimental/diagnóstico , Artrite Experimental/patologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Colágeno Tipo II/imunologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Hidrocarbonetos/administração & dosagem , Hidrocarbonetos/química , Masculino , Óleo Mineral/administração & dosagem , Óleo Mineral/química , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Ratos , Índice de Gravidade de Doença , Vacinas/administração & dosagem , Vacinas/efeitos adversos , Vacinas/químicaRESUMO
Wenjinghuoluo prescription, a traditional Chinese medicine compound treatment of rheumatoid arthritis characterized by wind-cold-dampness arthralgia, contains five herbs, namely, C. cassia Presl., Cinnamomum cassia Presl., Paeonia lactiflora Pall., Saposhnikovia divaricate (Turcz.) Schischk., and Clematis chinensis Osbeck. We have reported that WJHL could inhibit the production of inflammatory mediators in immune cells. This study explored the effect and mechanism of WJHL on collagen-induced arthritis mice. WJHL could significantly improve clinical arthritic conditions; inhibit bone erosion and osteophyte formation in joints; decrease expression of proinflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-17); reduce protein expression levels of JAK2, p-JAK2, STAT3, p-STAT3 and gene expression levels of JAK2, STAT3, IL-17A, RORγt mRNA; elevate osteoprotegerin and Foxp3 mRNA levels and lower Th17 cell proportions in splenocytes. Results suggest that WJHL, specifically regulating the JAK2/STAT3 pathway and Th17 cells, may be a promising herbal medicine candidate for the treatment of RA.
Assuntos
Artrite Experimental/etiologia , Artrite Experimental/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/tratamento farmacológico , Biomarcadores , Biópsia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Imuno-Histoquímica , Imunomodulação/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Fenótipo , Fosforilação , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Microtomografia por Raio-XRESUMO
This article describes a rapid, simple and cost-effective technique that could lead to a screening method for colitis without the need for biopsies or in vivo measurements. This screening technique includes the testing of serum using Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy for the colitis-induced increased presence of mannose. Chronic (Interleukin 10 knockout) and acute (Dextran Sodium Sulphate-induced) models for colitis are tested using the ATR-FTIR technique. Arthritis (Collagen Antibody Induced Arthritis) and metabolic syndrome (Toll like receptor 5 knockout) models are also tested as controls. The marker identified as mannose uniquely screens and distinguishes the colitic from the non-colitic samples and the controls. The reference or the baseline spectrum could be the pooled and averaged spectra of non-colitic samples or the subject's previous sample spectrum. This shows the potential of having individualized route maps of disease status, leading to personalized diagnosis and drug management.
Assuntos
Colite/diagnóstico , Colite/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Análise Química do Sangue , Colite/patologia , Colo/metabolismo , Colo/patologia , Fezes/química , Feminino , Interleucina-10/genética , Interleucina-10/metabolismo , Lipocalina-2/metabolismo , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/metabolismoRESUMO
Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease and derived from immune granule cells. It has been suggested playing an important role in the development of rheumatoid arthritis. In this study, a coumarin based fluorescent probe (GF-AFC) was designed and synthesized to evaluate DPPI activity in serum or tissue homogenates of collagen-induced arthritis (CIA) rats, an inflammatory arthropathy model. It was revealed that the fluorescent intensity was significantly increased in a very short time after specific substrate GF-AFC reacted with the DPPI. The fluorophore (AFC) was released to shine after the cleavage reaction which was examined by 19F NMR spectroscopy. It has been shown that DPPI hydrolyzed the GF-AFC in a robust, linear, and time dependent manner at a significant high rate. A serum-based DPPI activity assay was validated by spiking and gradient dilution methods, there were no interferences or auto-fluorescence observed. The Coefficient of Variance calculated for serum-based DPPI activity assays indicates the good reproducibility. The good correlation has been seen between serum DPPI levels and the severity of arthritis during RA development in CIA rats. Our study has demonstrated a new serum based diagnostic assay for detecting DPPI activity using coumarin conjugated fluorescent (GF-AFC) as a substrate. The successful implementation of the case would provide beneficial experience in rheumatoid arthritis research.