Connective tissue growth factor-targeting DNA aptamer suppresses pannus formation as diagnostics and therapeutics for rheumatoid arthritis.

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.

The pharmacological assessment of resveratrol on preclinical models of rheumatoid arthritis through a systematic review and meta-analysis.

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.

Therapeutic Effect of Matrine on Collagen-Induced Arthritis Rats and Its Regulatory Effect on RANKL and OPG Expression.

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.

The impact of airborne endotoxin exposure on rheumatoid arthritis-related joint damage, autoantigen expression, autoimmunity, and lung disease.

Airborne biohazards are risk factors in the development and severity of rheumatoid arthritis (RA) and RA-associated lung disease, yet the mechanisms explaining this relationship remain unclear. Lipopolysaccharide (LPS, endotoxin) is a ubiquitous inflammatory agent in numerous environmental and occupational air pollutant settings recognized to induce airway inflammation. Combining repetitive LPS inhalation exposures with the collagen induced arthritis (CIA) model, DBA1/J mice were assigned to either: sham (saline injection/saline inhalation), CIA (CIA/saline), LPS (saline/LPS 100 ng inhalation), or CIA + LPS for 5 weeks. Serum anti-citrullinated (CIT) protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) antibodies were strikingly potentiated with co-exposure (CIA + LPS). CIT- and MAA-modified lung proteins were increased with co-exposure and co-localized across treatment groups. Inhaled LPS exacerbated arthritis with CIA + LPS > LPS > CIA versus sham. Periarticular bone loss was demonstrated in CIA and CIA + LPS but not in LPS alone. LPS induced airway inflammation and neutrophil infiltrates were reduced with co-exposure (CIA + LPS). Potentially signaling transition to pro-fibrotic processes, there were increased infiltrates of activated CD11c+CD11b+ macrophages and transitioning CD11c+CD11bint monocyte-macrophage populations with CIA + LPS. Moreover, several lung remodeling proteins including fibronectin and matrix metalloproteinases as well as complement C5a were potentiated with CIA + LPS compared to other treatment groups. IL-33 concentrations in lung homogenates were enhanced with CIA + LPS with IL-33 lung staining driven by LPS. IL-33 expression was also significantly increased in lung tissues from patients with RA-associated lung disease (N = 8) versus controls (N = 7). These findings suggest that patients with RA may be more susceptible to developing interstitial lung disease following airborne biohazard exposures enriched in LPS.

Critical roles of the E3 ubiquitin ligase FBW7 in B-cell response and the pathogenesis of experimental autoimmune arthritis.

Proper regulation of B-cell function is essential for effective humoral immunity and maintenance of immune tolerance. Here, we found that FBW7 (F-box/WD40 repeat-containing protein 7) is highly expressed in germinal centre B and B1 cells, and confirmed that it has an intrinsic role in maintaining homeostasis of mature B cells and B-1 cells. FBW7 deletion led to an impairment of antibody response, and although germinal centre formation was not affected, antibody class-switch recombination and affinity maturation processes were defective. Likewise, memory immune response was severely impaired. Moreover, FBW7 ablation ameliorated the pathogenesis of an autoimmune disease model, collagen-induced arthritis, by reducing the production of anti-collagen II autoantibodies. Taken together, these data suggest that FBW7 may be an attractive target for developing new therapeutics for the treatment of autoimmune diseases.

Effects of Mild and Moderate Monoclonal Antibody Dose on Inflammation, Bone Loss, and Activation of the Central Nervous System in a Female Collagen Antibody-induced Arthritis Mouse Model.

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.

Pathogenic Role of Circulating Citrullinated Antigens and Anti-Cyclic Monoclonal Citrullinated Peptide Antibodies in Rheumatoid Arthritis.

We examined whether it is possible to directly detect citrullinated antigens in the serum of rheumatoid arthritis (RA) patients using a monoclonal antibody (mAb) designed to be specific for citrullinated peptides. In order to confirm the potential of the mAb as a direct arthritis-inducing substance through experimental model of RA, a monoclonal antibody (mAb) 12G1 was generated using by immunization of mice with a challenging cyclic citrullinated peptide. Immunohistochemical analysis of RA-affected synovial tissue showed that our mAb 12G1 could indeed detect citrullinated proteins in target tissues. Subsequently, serum levels of citrullinated type II collagen and filaggrin were measured in healthy volunteers, patients with RA, ankylosing spondylitis (AS), and systemic lupus erythematosus (SLE) using a 12G1-based sandwich ELISA. This showed that citrullinated filaggrin showed 78.9% sensitivity and 85.9% specificity for RA diagnosis with a cutoff optical density (OD) value of 1.013, comparable with the results from a second-generation anti-citrullinated protein antibody (ACPA) test. Circulating citrullinated collagen and filaggrin were detected even in sera of RA patients who were negative for both rheumatoid factor (RF) and ACPA. ELISA results also showed that RF and ACPA titers showed significantly positive correlation with both citrullinated collagen and filaggrin OD values in sera of RA patients. 12G1 challenging aggravated the severity of murine arthritis. In summary, mAb 12G1 can directly detect citrullinated proteins in RA target tissue and in sera of RA patients and 12G1 showed direct arthritogenic potential in vivo. This, 12G1 might be useful for diagnosis of RA including seronegative RA and may help to elucidate the pathophysiological role of citrullination in RA.

Infrared radiation from cage bedding moderates rat inflammatory and autoimmune responses in collagen-induced arthritis.

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.

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis.

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation.

Ceria-based nanotheranostic agent for rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that affects 1-2% of the human population worldwide, and effective therapies with targeted delivery for local immune suppression have not been described. We address this problem by developing a novel theranostic nanoparticle for RA and assessed its therapeutic and targeting effects under image-guidance. Methods: Albumin-cerium oxide nanoparticles were synthesized by the biomineralization process and further conjugated with near-infrared, indocyanine green (ICG) dye. Enzymatic-like properties and reactive oxygen species (ROS) scavenging activities, as well as the ability to reprogram macrophages, were determined on a monocyte cell line in culture. The therapeutic effect and systemic targeting potential were evaluated in collagen-induced arthritis (CIA) mouse model using optical/optoacoustic tomographic imaging. Results: Small nanotheranostics with narrow size distribution and high colloidal stability were fabricated and displayed high ROS scavenging and enzymatic-like activity, as well as advanced efficacy in a converting pro-inflammatory macrophage phenotype into anti-inflammatory phenotype. When administrated into affected animals, these nanoparticles accumulated in inflamed joints and revealed a therapeutic effect similar to the gold-standard therapy for RA, methotrexate. Conclusions: The inflammation-targeting, inherent contrast and therapeutic activity of this new albumin-cerium oxide nanoparticle may make it a relevant agent for assessing severity in RA, and other inflammatory diseases, and controlling inflammation with image-guidance. The design of these nanotheranostics will enable potential clinical translation as systemic therapy for RA.

Mouse CD163 deficiency strongly enhances experimental collagen-induced arthritis.

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.

Upregulated PKM2 in Macrophages Exacerbates Experimental Arthritis via STAT1 Signaling.

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.

Inhibition of BMP3 increases the inflammatory response of fibroblast-like synoviocytes in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a persistent autoimmune disease. Fibroblast-like synoviocytes (FLS) are a key component of invasive pannus and a pathogenetic mechanism in RA. Expression of bone morphogenetic protein 3 (BMP3) mRNA is reportedly decreased in the arthritic synovium. We previously showed that BMP3 expression is significantly downregulated in the synovial tissues of RA patients and models of adjuvant-induced arthritis (AIA). In the present study, we explored the association between BMP3 and FLS migration and secretion of proinflammatory factors in RA. We found that inhibition of BMP3 expression using BMP3 siRNA increased the proinflammatory chemokines and migration of FLS stimulated with TNF-α. Inhibition of BMP3 expression also increased expression of IL-6, IL-1ß, IL-17A, CCL-2, CCL-3, VCAM-1, MMP-3, and MMP-9, but not TIMP-1, in AIA and RA FLS. Correspondingly, induction of BMP3 overexpression through intra-articular injection of ad-BMP3 diminished arthritis severity in AIA rats. We also found that BMP3 may inhibit activation of TGF-ß1/Smad signaling. These data indicate that BMP3 may suppress the proliferation and migration of FLS via the TGF-ß1/Smad signaling pathway.

Reduction of osteoarthritis severity in the temporomandibular joint of rabbits treated with chondroitin sulfate and glucosamine.

Osteoarthritis is a degenerative disease that causes substantial changes in joint tissues, such as cartilage degeneration and subchondral bone sclerosis. Chondroitin sulfate and glucosamine are commonly used products for the symptomatic treatment of osteoarthritis. The aim of the present study was to investigate the effects of these products when used as structure-modifying drugs on the progression of osteoarthritis in the rabbit temporomandibular joint. Thirty-six New Zealand rabbits were divided into 3 groups (n = 12/group): control (no disease); osteoarthritis (disease induction); and treatment (disease induction and administration of chondroitin sulfate and glucosamine). Osteoarthritis was induced by intra-articular injection of monosodium iodoacetate. Animals were killed at 30 and 90 days after initiation of therapy. The treatment was effective in reducing disease severity, with late effects and changes in the concentration of glycosaminoglycans in the articular disc. The results indicate that chondroitin sulfate and glucosamine may have a structure-modifying effect on the tissues of rabbit temporomandibular joints altered by osteoarthritis.

A comparative pilot study of oral diacerein and locally treated diacerein-loaded nanoparticles in a model of osteoarthritis.

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.

Blocking Interleukin-33 Alleviates the Joint Inflammation and Inhibits the Development of Collagen-Induced Arthritis in Mice.

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.

Effect of tumor necrosis factor inhibition on spinal inflammation and spinal ankylosis in SKG mice.

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.

Micro-CT imaging analysis for the effects of ibandronate and eldecalcitol on secondary osteoporosis and arthritis in adjuvant-induced arthritis rats.

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.

Pedunculoside attenuates pathological phenotypes of fibroblast-like synoviocytes and protects against collagen-induced arthritis.

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.

NIRF-Molecular Imaging with Synovial Macrophages-Targeting Vsig4 Nanobody for Disease Monitoring in a Mouse Model of Arthritis.

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.