Modulation of early osteoarthritis by tibiofemoral re-alignment in sheep.

OBJECTIVE: To investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA. METHOD: Normal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone. RESULTS: Multivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface-to-volume ratio (all P ≤ 0.0359 vs. normal). CONCLUSION: The data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

Clinical Scoring of Disease Activity in Animal Models.

Disease severity in murine arthritis models, such as collagen-induced arthritis (CIA), is commonly assessed by clinical scoring of paw swelling and histological examination of joints. Clinical scoring using a qualitative scoring system of paw inflammation (paw thickness, width, or volume) over time is the standard method used for subjective quantification of arthritis activity. To evaluate paw swelling status, a quantitative method using three-dimensional T2-weighted flash sequence magnetic resonance imaging (MRI) is introduced. The efficacy of a therapeutic approach can be semiologically quantified using a clinical scoring system and an index of paw inflammation in CIA mice.

A Model of Type II Collagen-Induced Spondylitis and Arthritis in F1 Hybrid Male Mice.

In this study, we tested a new model of ankylosing spondylitis in order to determine its histological and radiological features needed to investigate peripheral arthritis, spondylitis, and formation of the new bone tissues. F1 hybrid male mice (BALB/c×DBA/1), a progeny of spondylitis-susceptible BALB/c male mice and rheumatoid arthritis-susceptible DBA/1 female mice, were immunized intraperitoneally with bovine type II collagen (CII) mixed with adjuvant dimethyldioctadecylammonium bromide. Radiological and histological studies were performed at the peak of swelling, redness, and stiffness. The incidence of peripheral arthritis and spondylitis induced by CII in F1 hybrid mice were 66 and 62%, respectively. X-ray examination revealed bone erosion and spondylitis in the peripheral joints, as well as the formation of new bone tissues in the coccygeal vertebrae and between LIII and LIV vertebrae. The histological study showed lymphocyte and plasma cell infiltration, capillary dilation, congestion, and endochondral ossification of the lumbar vertebrae. This novel model of CII-induced spondylitis in F1 hybrid mice provoked axial and peripheral arthritides inducing chronic inflammation. In this model, the formation of new bone tissue in the stiff spine is characterized by endochondral ossification. The advanced model is an additional and valuable tool for investigation of the autoimmune reactions in spondylitis.

Evaluation of 18F-FAPI-04 Imaging in Assessing the Therapeutic Response of Rheumatoid Arthritis.

PURPOSE: Fibroblast activating protein (FAP) is highly expressed in the synovial tissues of rheumatoid arthritis (RA) patients. The aim of this study was to determine the feasibility of PET imaging with an Al[18F] F-NOTA-labeled FAP inhibitor 04(18F-FAPI-04) for the evaluation of arthritic progression and therapeutic response in experimental arthritis. METHODS: Fibroblast-like synoviocytes (FLSs) were obtained from patients with RA or osteoarthritis (OA), and the relationship between 18F-FAPI-04 uptake and the inflammatory activity of RA FLSs was investigated. Collagen-induce arthritis (CIA) mice models were established and treated with methotrexate (MTX) or etanercept (ETC). Then, PET imaging was performed 24 h following 18F-FAPI-04 injection. The imaging results were compared by assessing macroscopic arthritis scores and histological staining. RESULTS: 18F-FAPI-04 uptake was obvious in RA FLSs that characterizing FAP activation. The higher the uptake of 18F-FAPI-04, the more severity of the inflammatory phenotype in RA FLS. Furthermore, the uptake of 18F-FAPI-04 in inflamed joints could be found even before the deformity of the parental joints could be observed by histological examination. Both MTX and ETC were effective in inhibiting the progression of arthritis in CIA mice was confirmed by macroscopic, histological, and radiographic pathology scores. Importantly, 18F-FAPI-04 uptake declined accordingly in CIA models following MTX and ETC treatment. CONCLUSIONS: These findings suggest that PET imaging of 18F-FAPI-04 can be used to monitor treatment response in RA, and is more sensitive in disease speculation than macroscopic arthritis scoring.

Naru-3 inhibits inflammation, synovial hyperplasia, and neovascularization in collagen-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Naru-3 is a prescribed formulation based on the theory of Mongolian medicine for the treatment of rheumatoid arthritis (RA). Naru-3 consists of three medicinal agents: Aconitum kusnezoffii Reichb (caowu), Terminalia chebula Retz (hezi), and Piper longum L (biba). These medicinal agents are widely distributed in the Mongolian area of China and have been used to treat rheumatism for centuries. BACKGROUND: Mongolian medicine Naru-3 is commonly prescribed to treat RA, but its mechanism of action is unknown. METHODS: A rat collagen-induced arthritis (CIA) model was established to investigate the mechanism of Naru-3. Rats were treated with Naru-3, Etanercept (ETN), and sodium carboxymethylcellulose (CMC) for four weeks. After treatment was terminated, paw thickness, ankle diameter, and arthritis index (AI) were scored. Synovial hyperplasia was evaluated using hematoxylin and eosin (H&E) staining and two-dimensional ultrasonography. Synovitis and neovascularization were assayed using power Doppler imaging (PDI) and contrast-enhanced ultrasonography (CEUS). Levels of vascular endothelial growth factor (VEGF), interleukin (IL)-1, and CD31 in the serum or synovium were detected using ELISA and immunohistochemistry analyses. RESULTS: Naru-3 and ETN alleviated the symptoms of CIA as evidenced by diminished paw thickness, ankle diameter, and AI scores. Mechanistically, Naru-3 inhibited synovial hyperplasia, synovitis, and neovascularization by diminishing systemic and local inflammation, as indicated by the relative expression of CD31, VEGF and IL-1 in the serumor synovium. After four weeks of treatment, no significant neovascularization was observed in the Naru-3 group, but neovascularization and synovitis occurred in the ETN group, as demonstrated by H&E staining, PDI, and CEUS examination. CONCLUSION: Naru-3 inhibited inflammation, synovial hyperplasia, and neovascularization and alleviates RA in our CIA rat model. No symptom recurrence was observed four weeks after drug treatment.

Photoacoustic/ultrasound-guided gene silencing: Multifunctional microbubbles for treating adjuvant-induced arthritis.

AIMS: To effectively deliver small interfering RNA (siRNA) to inflammatory tissues for treating rheumatoid arthritis (RA), we developed the multifunctional microbubbles (MBs) to perform photoacoustic/ultrasound-guided gene silencing. METHODS: Fluorescein amidite (FAM)-labelled tumour necrosis factor-α (TNF-α)-siRNA and cationic MBs were mixed to fabricate FAM-TNF-α-siRNA-cMBs. The cell transfection efficacy of FAM-TNF-α-siRNA-cMBs was evaluated in vitro on RAW264.7 cells. Subsequently, wistar rats with adjuvant-induced arthritis (AIA) were injected intravenously with MBs and simultaneously subjected to low-frequency ultrasound for ultrasound-targeted microbubble destruction (UTMD). Photoacoustic imaging (PAI) was utilized to visualize the distribution of siRNA. And the clinical and pathological changes of AIA rats was estimated. RESULTS: FAM-TNF-α-siRNA-cMBs were evenly distributed within the RAW264.7 cells and significantly reduced TNF-α mRNA levels of the cells. For AIA rats, the entering and collapsing of MBs was visualized by contrast-enhanced ultrasound (CEUS). Photoacoustic imaging showed markedly enhanced signals following injection, indicating localization of the FAM-labelled siRNA. The articular tissues of the AIA rats treated with TNF-α-siRNA-cMBs and UTMD showed decreased TNF-α expression levels. CONCLUSIONS: The theranostic MBs exhibited a TNF-α gene silencing effect under the guidance of CEUS and PAI. The theranostic MBs served as vehicles for delivering siRNA as well as contrast agents for CEUS and PAI.

Pathological Characteristics of Monosodium Iodoacetate-Induced Osteoarthritis in Rats.

BACKGROUND: This study aimed to identify pain-related behavior and pathological characteristics of the knee joint in rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA). METHODS: Knee joint inflammation was induced by intra-articular injection of MIA (4 mg/50 µL, n = 14) in 6-week-old male rats. Knee joint diameter, weight-bearing percentage on the hind limb during walking, the knee bending score, and paw withdrawal to mechanical stimuli were measured to evaluate edema and pain-related behavior for 28 d after MIA injection. Histological changes in the knee joints were evaluated using safranin O fast green staining on days 1, 3, 5, 7, 14, and 28 after OA induction (n = 3, respectively). Changes in bone structure and bone mineral density (BMD) were examined 14 and 28 d after OA (n = 3, respectively) using micro-computed tomography (CT). RESULTS: The knee joint diameter and knee bending scores of the ipsilateral joint significantly increased 1 d after MIA injection, and the increased knee joint diameter and knee bending score persisted for 28 d. Weight-bearing during walking and paw withdrawal threshold (PWT) decreased from 1 and 5 d, respectively, and were maintained up to 28 d after MIA. Cartilage destruction started on day 1, and Mankin scores for bone destruction significantly increased for 14 d, as shown by micro-CT imaging. CONCLUSION: The present study demonstrated that histopathological structural changes in the knee joint due to inflammation started soon after MIA injection, which induced OA pain from inflammation-related acute pain to spontaneous and evoked associated chronic pain.

Size-switchable polymer-based nanomedicines in the advanced therapy of rheumatoid arthritis.

Chronic inflammatory diseases such as rheumatoid arthritis represent a substantial socio-economic impact and have a high prevalence in the modern world. Nano-sized polymer therapeutics have shown suitable characteristics for becoming the next generation of anti-inflammatory nanomedicines. Here, we present biocompatible and stimuli-sensitive N-(2-hydroxypropyl)methacrylamide based polymer conjugates with the anti-inflammatory drug dexamethasone (DEX), which has been tailored for prolonged blood circulation, enhanced inflammatory site accumulation, site-specific drug release and subsequent elimination of the carrier via urine excretion. The hydrodynamic size of novel polymer-DEX nanomedicine was adjusted to prolong its blood circulation whilst maintaining the renal excretability of the polymer carrier after drug release in inflamed tissue. The therapeutic efficacy of the studied polymer nanomedicines was evaluated in a model of dissipated chronic arthritis, i.e. collagen II-induced arthritis, in mice. The pH-sensitive drug attachment enabled enhanced blood circulation with minimal systemic drug release, as well as rapid drug activation in affected joints. Importantly, unlike free DEX, the polymer nanomedicines were able to diminish joint inflammation and arthritis-induced bone damage - even at a reduced dosing regimen - as evaluated by micro computed tomography (micro-CT).

Preclinical evaluation and pilot clinical study of [18F]AlF-NOTA-FAPI-04 for PET imaging of rheumatoid arthritis.

PURPOSE: Fibroblast-like synoviocytes (FLSs) are key effector cells in the inflamed joints of patients with rheumatoid arthritis (RA). Previous studies have suggested that fibroblast activation protein (FAP) is highly expressed in RA-derived FLSs and is a specific marker of activated RA FLSs. In this study, we developed aluminum-[18F]-labeled 1,4,7-triazacyclononane-N,N',N″-triacetic acid-conjugated FAP inhibitor 04 ([18F]AlF-NOTA-FAPI-04) to image RA-FLSs in vitro and arthritic joints in collagen-induced arthritis (CIA) mice and RA patients. METHODS: RA FLSs and NIH3T3 cells transfected with FAP were used to perform in vitro-binding studies. Biodistribution was conducted in normal DBA1 mice. Collagen-induced arthritis (CIA) models with different arthritis scores were subjected to [18F]AlF-NOTA-FAPI-04 and 18F-FDG PET imaging. Histological examinations were performed to evaluate FAP expression and Cy3 dye-labeled FAPI-04(Cy3-FAPI-04) uptake. Blocking studies with excess unlabeled FAPI-04 in CIA mice and NIH3T3 xenografts in immunocompromised mice were used to evaluate the binding specificity of [18F]AlF-NOTA-FAPI-04. Additionally, [18F]AlF-NOTA-FAPI-04 PET imaging was performed on two RA patients. RESULTS: The binding of [18F]AlF-NOTA-FAPI-04 increased significantly in RA FLSs and NIH3T3 cells overexpressing FAP compared to their parental controls (FAP-GFP-NIH3T3 vs. GFP-NIH3T3, 2.40 ± 0.078 vs. 0.297 ± 0.05% AD/105 cells; RA FLSs vs. OA FLSs, 1.54 ± 0.064 vs. 0.343 ± 0.056% AD/105 cells). Compared to 18F-FDG imaging, [18F]AlF-NOTA-FAPI-04 showed high uptake in inflamed joints in the early stage of arthritis, which was positively correlated with the arthritic scores (Pearson r=0.834, P<0.001). In addition, the binding of [18F]AlF-NOTA-FAPI-04 to cells with high FAP expression and the uptake of [18F]AlF-NOTA-FAPI-04 in arthritic joints both could be blocked by excessive unlabeled FAPI-04. Fluorescent staining showed that the intensity of Cy3-FAPI-04 binding to FAP increased accordingly as the expression of FAP protein increased in cells and tissue sections. Furthermore, the uptake of [18F]AlF-NOTA-FAPI-04 in FAP-GFP-NIH3T3 xenografts was significantly higher than that in GFP-NIH3T3 xenograft (35.44 ± 4.27 vs 7.92 ± 1.83% ID/mL). Finally, [18F]AlF-NOTA-FAPI-04 PET/CT imaging in RA patients revealed nonphysiologically high tracer uptake in the synovium of arthritic joints. CONCLUSION: [18F]AlF-NOTA-FAPI-04 is a promising radiotracer for imaging RA FLSs and could potentially complement the current noninvasive diagnostic parameters.

Multifunctional Nanoparticles Co-Loaded with Perfluoropropane, Indocyanine Green, and Methotrexate for Enhanced Multimodal Imaging of Collagen-Induced Arthritis.

Rheumatoid arthritis (RA), a common chronic inflammatory joint disease with features of synovitis and pannus formation, may lead to irreparable joint damage and disability. Methotrexate (MTX) is known as the cornerstone of therapy for RA. However, the therapeutic effects of MTX are unsatisfactory due to its low retention in the inflammatory joints as well as systemic toxic effects. Fortunately, the use of multifunctional nanoparticles for diagnostics and in treatment shows potential for application as a strategy for traceable and targeted RA therapy. This research aims to develop novel nanoparticles that carry with perfluoropropane (PFP), indocyanine green (ICG), and MTX and investigate the corresponding enhancement in multimodal imaging both in vitro and in vivo. A modified double emulsion method was applied for the construction of encapsulated PFP-O2, ICG, and MTX (OIM@NPs), and the essential properties of the developed NPs were determined. The fluorescence and ultrasonic and photoacoustic imaging characteristics were experimentally evaluated both in in vitro and in vivo models. The OIM@NPs are stable and efficient nanoagents. They enable more targeted distribution in the inflammatory joints in RA rats. Moreover, the NPs play an important role as contrast agents for prominent ultrasound and photoacoustic imaging after laser and low-intensity focused ultrasound excitation, providing precision guidance and monitoring for subsequent treatment. This research may provide a novel and efficient strategy to better enable monitoring in inflammatory joints of RA patients and the developed NPs may be a promising nanoplatform for integrating multimodal image monitoring.

Inflammation-Triggered Supramolecular Nanoplatform for Local Dynamic Dependent Imaging-Guided Therapy of Rheumatoid Arthritis.

The aging of population has resulted in a significant increase in the prevalence of rheumatoid arthritis (RA), which is a persistent and recurrent synovial inflammation caused by abnormal immune activation. Herein, the authors designed an inflammation-triggered disassembly (ITD) nanoplatform by a supramolecular assembly method, which controls the decomposition and drug release through changes in cytokine concentrations and redox potentials during the onset of arthritis, and its dual-targeted synergistic effect on collagen-induced arthritis (CIA) rats resulted in higher cell death rate and immunosuppressive rate. Meanwhile, they propose the local dynamic dependent imaging (LDDI) technology to diagnose the disease status, which may produce corresponding changes with the fluctuation of inflammatory activity and improve the accuracy of dual-target therapy by monitoring the synovial changes through in situ photoactivation of the second near infrared light (NIR-II). Very importantly, histological analysis shows that ITD strategy relieved joint destruction and cartilage degeneration and its clinical score is similar to that of the healthy group. Their work provides an effective strategy for the early diagnosis and treatment of acute and chronic inflammation diseases, which can interfere to abnormal immune activation, rather than affecting the normal function of immune system.

TNF-α inhibitors reduce inflammation-induced concentric remodelling, but not diastolic dysfunction in collagen-induced arthritis.

OBJECTIVES: To determine biologic disease-modifying anti-rheumatic drug effects on inflammation-induced cardiac geometry and function changes. METHODS: Male and female Sprague-Dawley rats (n=92) were divided into four groups: control group, collagen-induced arthritis (CIA) group, anti-TNF-α group and anti-IL-6 group. Inflammation was induced by injecting bovine type-II collagen emulsified in incomplete Freund's adjuvant at the base of the tail, in all groups except the control group. Following the onset of arthritis, the anti-TNF-α group received etanercept, and the anti-IL-6 group received tocilizumab, for six weeks. Left ventricular (LV) geometry and function were assessed with echocardiography and circulating inflammatory markers were measured with ELISA. RESULTS: Relative wall thickness in the CIA and anti-IL-6 groups were increased compared to controls (p<0.001 and p=0.02, respectively). TNF-α inhibition protected against inflammation-induced LV concentric remodelling, as relative wall thickness in the anti-TNF-α group was similar to controls (p=0.55). Systolic function variables were not different between the groups. In all groups inoculated with collagen, myocardial relaxation (lateral e') were impaired compared to controls (all p<0.001). LV filling pressures (E/e') were increased in the CIA, anti-TNF-α and anti-IL-6 groups compared to controls (p<0.001, p<0.001 and p=0.05, respectively). Independent of concentric remodelling, circulating CRP concentrations were associated with decreased e' and increased E/e', while TNF-α concentrations were associated with E/A. CONCLUSIONS: TNF-α inhibition protected against inflammation-induced adverse cardiac remodelling, but not diastolic dysfunction. IL-6 receptors blocker effects on inflammation-induced cardiac changes were inconclusive. Systemic inflammation likely impacts LV concentric remodelling and diastolic dysfunction through distinct pathways.

Verification of pain-related neuromodulation mechanisms of icariin in knee osteoarthritis.

Knee osteoarthritis (KOA) is a common disease with no specific treatment. Icariin (ICA) is considered an agent for KOA. This study aimed to confirm the pain-related neuromodulation mechanisms of ICA on KOA. Three experiments were designed: (1) verifying the therapeutic effects of ICA in vivo and in vitro, (2) exploring the potential pain-related neuromodulation pathways involved in ICA treatment by functional magnetic resonance imaging (fMRI) and virus retrograde tracing (VRT) and (3) confirming the pain-related targets by tandem mass tag (TMT)-based quantitative proteomics and bioinformatic analyses. Experiment 1 verified the efficacy of ICA in OA animal and cell models. Experiment 2 found a series of brain regions associated with KOA reversed by ICA treatment, indicating that a pain-related hypothalamic-mediated neuromodulation pathway and an endocannabinoid (EC)-related pathway contribute to ICA mechanisms. Experiment 3 explored and confirmed four pain-related genes involved in KOA and ICA treatment. We confirmed the key role of pain-related neuromodulation mechanisms in ICA treatment associated with its analgesic effect. Our findings contribute to considering ICA as a novel therapy for KOA.

Non-invasive monitoring of arthritis treatment response via targeting of tyrosine-phosphorylated annexin A2 in chondrocytes.

BACKGROUND: The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. METHODS: LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. RESULTS: In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. CONCLUSIONS: We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation.

Different protective efficacies of a novel antigen-specific DNA vaccine encoding chicken type â ¡ collagen via intramuscular, subcutaneous, and intravenous vaccination against experimental rheumatoid arthritis.

Tolerizing DNA vaccines encoding key autoantigens are one of emerging strategies for the treatment of rheumatoid arthritis (RA). Among these vaccines, the most representative is pcDNA-CCOL2A1, an antigen-specific DNA vaccine encoding chicken type Ⅱ collagen (CCⅡ) with significant therapeutic and prophylactic efficacy in collagen-induced arthritis (CIA) rat models. We compared the in situ expression levels of CCOL2A1-mRNA and CCⅡ protein and the protective efficacies against CIA after a single dose (300 µg/kg) of this vaccine via intramuscular (IM), subcutaneous (SC) and intravenous (IV) vaccinations. The IM vaccination routes resulted in good protective efficacies in terms of decreasing CIA incidence and severity and significantly improved radiographic and histopathologic findings and scores of joints. Furthermore, IM, SC, and IV vaccinations markedly decreased serum levels of anti-type Ⅱ collagen (CⅡ) IgG antibodies, but only IM vaccination significantly reduced serum levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody. The vaccine exhibited a continuous CCOL2A1-mRNA expression in the tail and abdominal subcutaneous tissue injection sites, but no CCOL2A1-mRNA signal was observed in muscle. Strikingly, CCⅡ protein expression levels at the three injection sites were comparable with minimal variation. IM administration may be considered the preferred route for RA treatment in clinical practice.

Infliximab prevents systemic bone loss and suppresses tendon inflammation in a collagen-induced arthritis rat model.

Reduced Bone Mineral Density (BMD) and tendon abnormalities, such as tenosynovitis and enthesitis, are prevalent comorbidities in patients with rheumatoid arthritis (RA). The aim of the present study was to investigate the effect of chronic treatment with infliximab on BMD and tendon inflammation in an animal model of inflammatory arthritis. Collagen-Induced Arthritis (CIA) was induced in rats, followed by long-term intraperitoneal administration of infliximab. Two additional groups of animals received methotrexate either as a monotherapy or as a co-treatment to infliximab. BMD was evaluated by Micro-Computed Tomography (Micro-CT) and bone histological examination. Tendon inflammation was assessed histologically and by quantitative ELISA analysis of pro-inflammatory cytokines in tendon tissues. Both methotrexate and infliximab treatment alleviated joint inflammation and reduced paw edema. Infliximab-treated animals exhibited an improved trabecular microarchitecture on micro-CT and histological analysis compared to both non-treated and methotrexate-treated animals. Infliximab almost reversed the pathological changes in tendons induced by CIA. Finally, we observed statistically significant declines in tendon TNF-a and IL-23 levels after infliximab treatment. Our study provides evidence that infliximab prevents arthritis-related osteoporosis and suppresses tendon inflammation in an animal model of inflammatory arthritis, in addition to controlling disease activity. These findings offer perspectives for the management of osteoporosis and enthesitis in RA.

A novel apoptosis probe, cyclic ApoPep-1, for in vivo imaging with multimodal applications in chronic inflammatory arthritis.

Apoptosis plays an essential role in the pathophysiologic processes of rheumatoid arthritis. A molecular probe that allows spatiotemporal observation of apoptosis in vitro, in vivo, and ex vivo concomitantly would be useful to monitoring or predicting pathophysiologic stages. In this study we investigated whether cyclic apoptosis-targeting peptide-1 (CApoPep-1) can be used as an apoptosis imaging probe in inflammatory arthritis. We tested the utility of CApoPep-1 for detecting apoptotic immune cells in vitro and ex vivo using flow cytometry and immunofluorescence. The feasibility of visualizing and quantifying apoptosis using this probe was evaluated in a murine collagen-induced arthritis (CIA) model, especially after treatment. CApoPep-1 peptide may successfully replace Annexin V for in vitro and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for ex vivo in the measurement of apoptotic cells, thus function as a sensitive probe enough to be used clinically. In vivo imaging in CIA mice revealed that CApoPep-1 had 42.9 times higher fluorescence intensity than Annexin V for apoptosis quantification. Furthermore, it may be used as an imaging probe for early detection of apoptotic response in situ after treatment. The CApoPep-1 signal was mostly co-localized with the TUNEL signal (69.6% of TUNEL+ cells) in defined cell populations in joint tissues of CIA mice. These results demonstrate that CApoPep-1 is sufficiently sensitive to be used as an apoptosis imaging probe for multipurpose applications which could detect the same target across in vitro, in vivo, to ex vivo in inflammatory arthritis.

KP-10/Gpr54 attenuates rheumatic arthritis through inactivating NF-κB and MAPK signaling in macrophages.

Rheumatoid arthritis (RA) is an autoimmune disease mainly characterized as chronic inflammation of joint. Both genetic and environmental factors play important roles in RA progression. G protein-coupled receptor 54 (GPR54) and Kisspeptins (KPs), the natural GRP54 ligands encoded by Kiss-1 gene are known to play important roles in immune regulation but the precise role of KP-10/GPR54 in RA remains elusive. Kiss1/Gpr54 expression was determined by immunohistochemistry on protein and real-time PCR on RNA from isolated RA-patient synovial tissue and PBMC. Collagen-induced arthritis (CIA) mouse models were used to investigate the effect of KP-10/Gpr54 on the rheumatic arthritis severity in the mice. The signaling pathway involved in KP-10/GPR54 was assessed by western blot and immunofluorescence.In the present study, we demonstrated that GPR54 upregulation in bone marrow-derived macrophages (BMDM) was associated with the severity of RA. In addition, Gpr54-/- increased the inflammatory cytokines induced by lipopolysaccharide (LPS) in BMDM and diseased severity of CIA (n = 10), while KP-10 reduced the LPS-induced inflammatory cytokines in vitro and ameliorated the CIA symptoms in vivo. Furthermore, we demonstrated that KP-10/GPR54 binds to PP2A-C to suppressed LPS induced NF-κB and MAPK signaling in BMDM. All these findings suggest that KP-10/GPR54 may be a novel therapeutic target for the diagnosis and treatment of RA.

Systems pharmacology-based dissection of mechanisms of Tibetan medicinal compound Ruteng as an effective treatment for collagen-induced arthritis rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Ruteng (CRT) is a prescribed formulation based on the theory of Tibetan medicine for the treatment of yellow-water-disease. It is consisted with 7 medicinal material include Boswellia carterii Birdw (named "Ruxiang" in Chinese); Tinospora sinensis (Lour.) Merr. (named "Kuan-Jin-Teng" in Chinese), Cassia obtusifolia L (named "Jue-Ming-Zi" in Chinese); Abelmoschus manihot (L.) Medic (named "Huang-Kui-Zi" in Chinese); Terminalia chebula Retz. (named "He-Zi" in Chinese); Lamiophlomis rotata (Benth.) Kudo (named "Du-Yi-Wei" in Chinese) and Pyrethrum tatsienense (Bur. et Franch.) Ling (named "Da-Jian-Ju" in Chinese). They are widely distributed in Tibet area of China and have been used to treat rheumatism, jaundice, and skin diseases for centuries. AIM OF THE STUDY: The present study was conducted to investigate the anti-arthritis effect of CRT and to disclose the systems pharmacology-based dissection of mechanisms. MATERIALS AND METHODS: The chemical constituents in CRT were identified using HPLC method, and CRT candidate targets against RA were screened by network pharmacology-based analysis and further experimentally validated based on collagen-induced arthritis (CIA) rat model. Furthermore, therapeutic mechanisms and pathways of CRT were investigated. RESULTS: 391 potential targets (protein) were predicted against 92 active ingredients of 7 medicinal materials in CRT. Enrichment analysis and molecular docking studies also enforced the practiced results. X-ray based physiological imaging showed the attenuated effect of CRT on paw swelling, synovial joints and cartilage with improved inflammation in CIA rats. Moreover, the expression of biomarkers associated with RA such as MMP1, MMP3 and MMP13 and TNF-a, COX2 and iNOS are down-regulated in ankle joints, serum, or liver. CONCLUSION: In conclusion, CRT compound could attenuate RA symptoms and active ingredients of this compound could be considered for drug designing to treat RA.

Collagen-Induced Temporomandibular Joint Arthritis Juvenile Rat Animal Model.

Juvenile idiopathic arthritis can affect the temporomandibular joint (TMJ) can cause growth disturbances of the lower jaw (mandible). The collagen-induced arthritis (CIA) juvenile rat model may be an appropriate model for studying how juvenile arthritis affects this joint during growth. However, studies using this animal model to investigate TMJ arthritis are limited. To validate an animal model for studying TMJ arthritis in growing rats, our study aimed to investigate the changes in mandibular growth and expression of proteins and cytokines in the mandibular condyle of CIA juvenile rat TMJs. A total of 27 male Wistar rats (3 weeks old) were scanned with microcomputed tomography (MicroCT) and divided into three groups (n = 9); CIA was induced in each TMJ in the CIA group, the Saline group received saline injections (sham injections) into their TMJs, and the Healthy group remained untreated (no TMJ injections) as negative controls. After 4 weeks, our results show that mandibular growth was significantly reduced in the CIA group compared with the Saline group (p < 0.01). There was no difference in mandibular growth between the two control groups (Saline and Healthy). Inflamed synovial tissue, cartilage invaginations, and lipid accumulation were observed in the CIA TMJs. Toluidine blue staining revealed decreased proteoglycan production in the CIA cartilage. In addition, immunohistochemistry revealed that type II collagen expression decreased, interleukin-1ß expression increased, and matrix metalloproteinase-13 expression increased in the CIA TMJs in comparison with the two control groups (Saline and Healthy). Immunostaining of tumor necrosis factor-α (TNF-α) was quantified and we showed that TNF-α expression was significantly greater in the CIA cartilage compared with both control groups (p < 0.05), and there was no difference in TNF-α expression between the Saline and Healthy groups. This CIA juvenile rat model of TMJ juvenile arthritis shows that CIA reduced mandibular growth and induced degenerative changes in TMJ condylar cartilage. This new information will help to understand the pathogenesis involved in CIA in juvenile rat TMJs for this animal model to be used in research investigating new therapeutics to treat TMJ juvenile arthritis. Impact statement In this study, the effects of collagen-induced arthritis (CIA) on the temporomandibular joint (TMJ) using a juvenile rat model were investigated. Our results showed that local injection of CIA in the TMJ significantly reduced mandibular growth and caused degenerative changes in condylar cartilage. This information helps to validate this animal model for studying the effect of arthritis in TMJs in growing rats. This model has the potential to be used in future studies to evaluate possible therapies for TMJ arthritis.