[Effect of moxibustion on the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor in ankle synovial tissue of rats with adjuvant arthritis].

OBJECTIVE: To observe the effect of moxibustion on the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in ankle synovial tissue of rats with adjuvant arthritis(AA), so as to explore the mechanism of moxibustion in inhibiting synovial angiogenesis and improving joint symptoms of rheumatoid arthritis. METHODS: Sixty healthy male SD rats were randomly divided into normal group, model group, moxibustion group and medication group, with 15 rats in each group. AA rat model was established by subcutaneous injection of Freund's complete adjuvant into the right hind paw. Rats in the moxibustion group were treated with "Zusanli" (ST36), "Guanyuan" (CV4) and "Ashi" point moxibustion, 20 min each time, once a day, for consecutive 3 weeks. Rats in the medication group were given methotrexate (0.35 mg/kg) intragastric administration, twice a week, for consecutive 3 weeks. Foot plantar volume of rats was measured by toe volume mea-suring instrument. HE staining was used to observe the histopathology of ankle synovium. The protein expressions of HIF-1α and VEGF in ankle synovial tissue were detected by immunohistochemistry and Western blot. RESULTS: Compared with the normal group, the foot plantar volume and the protein expressions of HIF-1α and VEGF in synovial tissue of ankle joint were significantly increased (P<0.01) in the model group, the synovial tissue showed obvious hyperplasia and a large number of neovasculogenesis. Following the interventions, the foot plantar volume and the protein expressions of HIF-1α and VEGF in synovial tissue of ankle joint were significantly decreased (P<0.05, P<0.01) in both moxibustion and medication groups in contrast to the model group, and there was no obvious proliferation of synovial tissue, and only a few neovascularization was observed. Compared with the medication group, the foot plantar volume was decreased (P<0.05) in the moxibustion group. CONCLUSION: Moxibustion can improve joint swelling and inhibit synovial angiogenesis in AA rats, and its mechanism may be related to down-regulating of HIF-1α and VEGF protein expressions.

Acupuncture modulates extracellular ATP levels in peripheral sensory nervous system during analgesia of ankle arthritis in rats.

As an ancient analgesia therapy, acupuncture has been practiced worldwide nowadays. A good understanding of its mechanisms will offer a promise for its rational and wider application. As the first station of pain sensation, peripheral sensory ganglia express pain-related P2X receptors that are involved in the acupuncture analgesia mechanisms transduction pathway. While the role of their endogenous ligand, extracellular ATP (eATP), remains less studied. This work attempted to clarify whether acupuncture modulated eATP levels in the peripheral sensory nerve system during its analgesia process. Male Sprague-Dawley rats underwent acute inflammatory pain by injecting Complete Freund's Adjuvant in the unilateral ankle joint for 2 days. A twenty-minute acupuncture was applied to ipsilateral Zusanli acupoint. Thermal hyperalgesia and tactile allodynia were assessed on bilateral hind paws to evaluate the analgesic effect. eATP of bilateral isolated lumbar 4-5 dorsal root ganglia (DRGs) and sciatic nerves were determined by luminescence assay. Nucleotidases NTPDase-2 and -3 in bilateral ganglia and sciatic nerves were measured by real-time PCR to explore eATP hydrolysis process. Our results revealed that acute inflammation induced bilateral thermal hyperalgesia and ipsilateral tactile allodynia, which were accompanied by increased eATP levels and higher mechano-sensitivity of bilateral DRGs and decreased eATP levels of bilateral sciatic nerves. Acupuncture exerted anti-nociception on bilateral hind paws, reversed the increased eATP and mechanosensitivity of bilateral DRGs, and restored the decreased eATP of bilateral sciatic nerves. NTPDase-2 and -3 in bilateral ganglia and sciatic nerves were inconsistently modulated during this period. These observations indicate that eATP metabolism of peripheral sensory nerve system was simultaneously regulated during acupuncture analgesia, which might open a new frontier for acupuncture research.

Altered sensory innervation and pain hypersensitivity in a model of young painful arthritic joints: short- and long-term effects.

BACKGROUND: Early life experience can cause long-term alterations in the nociceptive processes underlying chronic pain, but the consequences of early life arthritic joint inflammation upon the sensory innervation of the joint is not known. Here, we measure pain sensitivity and sensory innervation in a young, juvenile and adult rodent model of arthritic joints and test the consequences of joint inflammation in young animals upon adult arthritic pain and joint innervation. METHODS: Unilateral ankle joint injections of complete Freund's adjuvant (CFA) (6-20 µl) were performed in young, postnatal day (P)8, adolescent (P21) and adult (P40) rats. A separate cohort of animals were injected at P8, and again at P40. Hindpaw mechanical sensitivity was assessed using von Frey monofilaments (vF) for 10 days. Nerve fibres were counted in sections through the ankle joint immunostained for calcitonin gene-related peptide (CGRP) and neurofilament 200 kDa (NF200). RESULTS: Ankle joint CFA injection increased capsular width at all ages. Significant mechanical pain hypersensitivity and increased number of joint CGRP + ve sensory fibres occurred in adolescent and adult, but not young, rats. Despite the lack of acute reaction, joint inflammation at a young age resulted in significantly increased pain hypersensitivity and CGRP+ fibre counts when the rats were re-inflamed as adults. CONCLUSIONS: Joint inflammation increases the sensory nociceptive innervation and induces acute pain hypersensitivity in juvenile and adult, but not in young rats. However, early life joint inflammation 'primes' the joint such that adult inflammatory pain behaviour and nociceptive nerve endings in the joint are significantly increased. Early life joint inflammation may be an important factor in the generation and maintenance of chronic arthritic pain.

Elevation of Inflammatory Cytokines and Proteins after Intra-Articular Ankle Fracture: A Cross-Sectional Study of 47 Ankle Fracture Patients.

INTRODUCTION: Intra-articular fractures are the leading etiology for posttraumatic osteoarthritis (PTOA) in the ankle. Elevation of proinflammatory cytokines following intra-articular fracture may lead to synovial catabolism and cartilage degradation. We aimed to compare cytokine levels in injured and healthy ankle joints, examine the longer-term cytokine levels in fractured ankles, and investigate the association between cytokine levels in fractured ankles and plasma. MATERIALS AND METHODS: In this cross-sectional study, synovial fluid (SF) and plasma of forty-seven patients with acute intra-articular ankle fractures and eight patients undergoing implant removal were collected prior to surgery. We determined concentrations of sixteen inflammatory cytokines, two cartilage degradation proteins, and four metabolic proteins and compared the levels in acutely injured ankles with those of the healthy contralateral side or during metal removal. Cytokine levels in injured ankles were also compared to serum cytokine levels. Nonparametric Wilcoxon rank-sum and Spearman tests were used for statistical analysis, and a p value below 0.05 was considered significant. RESULTS: Compared to the healthy ankles, the synovial fluid in ankles with acute intra-articular fracture had elevated levels of several proinflammatory cytokines and proteases (IL-1ß, IL-2, IL-6, IL-8, IL-12p70, TNF, IFNγ, MMP-1, MMP-3, and MMP-9) and anti-inflammatory cytokines (IL-1RA, IL-4, IL-10, and IL-13). The levels of cartilage degradation products (ACG, CTX-2) and metabolic mediators (TGF-ß1 and TGF-ß2) were also significantly higher. Synovial concentrations of ACG, IL-12-p70, IFNγ, IL-4, and bFGF correlated with serum levels. While most of the examined synovial cytokines were unchanged after implant removal, IL-4 and IL-6 levels were upregulated. CONCLUSIONS: We show that an acute ankle fracture is followed by an inflammatory reaction and cartilage degeneration. These data contribute to the current understanding of the protein regulation behind the development of PTOA and is a further step towards supplementing the current surgical treatment. This cross-sectional study was "retrospectively registered" on the 31th October 2017 at ClinicalTrials.gov (NCT03769909). The registration was carried out after inclusion of the first patient and prior to finalization of patient recruitment and statistical analyses: https://clinicaltrials.gov/ct2/show/NCT03769909?term=NCT03769909&draw=2&rank=1.

Non-target metabolomic analysis reveals the therapeutic effect of Saposhnikovia divaricata decoction on collagen-induced arthritis rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Saposhnikovia divaricata (SD), a Chinese crude drug, has long been recognized for therapeutic effect to rheumatoid arthritis (RA). At present, the mechanisms of SD treatment in RA have not been fully understood especially on the perspective of metabolomics. AIM OF THE STUDY: To study the pharmacodynamic effects of Saposhnikovia divaricata decoction on CIA rats, and explore the therapeutic mechanism by metabolomics methods. MATERIALS AND METHODS: Wistar rats were randomly divided into normal group, CIA model group, dexamethasone group and SD decoction groups (10 g crude drug/kg, 5 g crude drug/kg and 2.5 g crude drug/kg of SDD). Body weight, arthritis scores, serum cytokine levels and histopathological parameters of rats were assessed. A metabolomics method based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS) was established to collect the metabolic profiles of rats and explore the metabolic changes that occurred after SDD treatment. RESULTS: SDD showed its protective effect on the affected joints, especially in the middle dosage group of SDD. Eighteen and 13 potential biomarkers for the SDD treatment of CIA rats were identified in the plasma and urine, respectively. SDD could regulate the disturbed metabolic pathways including tryptophan metabolism, glycerophospholipid catabolism, primary bile acid biosynthesis and fatty acid metabolism. CONCLUSIONS: In summary, SDD treatment could effectively alleviate symptoms of RA and regulate metabolic disorders in CIA rats.

Synovial fibroblast-derived exosomal microRNA-106b suppresses chondrocyte proliferation and migration in rheumatoid arthritis via down-regulation of PDK4.

Fibroblast-derived exosomes have been reported to transfer microRNAs to recipient cells, where they regulate target gene expression, which is of interest for understanding the basic biology of inflammation, tissue homeostasis, and development of therapeutic approaches. Initial microarray-based analysis carried out in this study identified the rheumatoid arthritis (RA)-related differentially expressed gene pyruvate dehydrogenase kinase 4 (PDK4). Subsequently, the upstream regulatory microRNA-106b (miR-106b) of PDK4 was predicted with bioinformatic analyses. A collagen-induced arthritis (CIA)-induced mouse model was established, and exosomes were isolated from synovial fibroblasts (SFs) and transferred into chondrocytes to identify the role of exosomes in rheumatoid arthritis (RA). We found that PDK4 was poorly expressed in RA cartilage tissues and chondrocytes, while miR-106b was highly expressed in RA SFs and SF-derived exosomes. Notably, PDK4 was confirmed as a target gene of miR-106b. Over-expression of PDK4 promoted the proliferation and migration abilities of chondrocytes and inhibited their apoptosis as well as affected the receptor activator of nuclear factor kappa B ligand (RANKL)/RANK/osteoprotegerin (OPG) system. Meanwhile, miR-106b was delivered from SFs to chondrocytes through exosomes, which suppressed chondrocyte proliferation and migration and accelerated apoptosis as well as affected the RANKL/RANK/OPG system via down-regulation of PDK4. Furthermore, in vivo results validated that miR-106b inhibition could relieve CIA-induced RA. Taken together, SF-derived exosomal miR-106b stimulates RA initiation by targeting PDK4, indicating a physiologically validated potential approach for the prevention and treatment of RA. KEY MESSAGES: PDK4 is decreased in chondrocytes of RA, while miR-106b is increased in SFBs. PDK4 promotes proliferation and migration of chondrocytes. miR-106b could target 3'UTR of PDK4 gene. SFB-exosomal miR-106b inhibits proliferation and migration of chondrocytes. Inhibition of miR-106b attenuates RA progression in a CIA mouse model.

Risk factors of ultrasound-detected tophi in patients with gout.

INTRODUCTION: Tophus is a characteristic manifestation of advanced gout, the clinical significance of which is often underestimated. This study aimed to compare the difference of clinical and ultrasound features between gout patients with and without ultrasound-detected tophus and identify risk factors associated with the presence of ultrasonographic tophus in gout patients. MATERIALS AND METHODS: A total of 85 gout patients were divided into tophaceous (n = 54) and non-tophaceous group (n = 31) according to the presence of ultrasound-detected tophus. All patients underwent ultrasound examination of the bilateral knee, ankle, and first metatarsophalangeal joint (MTP1). Clinical information and ultrasound findings were compared between the groups. A multivariate logistic regression analysis to determine possible risk factors is associated with the number of ultrasound-detected tophaceous joints. RESULTS: Older age, longer gout duration, higher gout flare frequency, lower estimated glomerular filtration rate (eGFR), and higher prevalence of hypertension, hyperlipidemia, and ultrasound manifestations including double contour sign (DCS) and erosion were observed in tophaceous patients from the univariate analysis. Multivariable logistic regression analysis showed that eGFR and disease duration were independently associated with the number of tophaceous joints. Lower eGFR and longer course duration were associated with a higher risk of tophi (B = -0.020, 0.141; P = 0.009, 0.010, respectively). CONCLUSIONS: The main factors that may influence the formation of tophi are disease duration and eGFR.Key Points• Lower eGFR and longer course duration are independent risk factors of tophi formation in gout patients.• The incidence of ultrasound manifestations including double contour sign (DCS) and erosion in patients with tophi were higher than those without tophi.

Bone marrow-derived mesenchymal stem cells-secreted exosomal microRNA-192-5p delays inflammatory response in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease closely correlated to synovial tissue inflammation. Exosomes are known to transfer microRNAs (miRNAs) between cells and have been validated as the vehicles for delivery of therapeutic molecules. AIM AND SCOPE: The current study was set to examine the functional values of bone marrow-derived mesenchymal stem cells (BMSCs)-secreted exosomal miR-192-5p (exo-miR-192-5p) on inflammation in RA. METHODS: Following the screening of differentially expressed genes in RA and miRNA-mRNA target prediction, BMSCs were infected with lentivirus expressing miR-192-5p to obtain miR-192-5p-overexpressed exosomes. To study the effect of exo-miR-192-5p on the expression of ras-related C3 botulinum toxin substrate 2 (RAC2), collagen-induced arthritis (CIA) rat models were established, and the clinical and histopathological changes were evaluated in the rats injected with exosomes. Subsequently, the expression patterns of pro-inflammatory factors were determined by ELISA. RESULTS: miR-192-5p was found to be down-regulated, while RAC2 was up-regulated in RA samples. Bioinformatics prediction revealed that the up-regulated RAC2 in RA may be regulated by miR-192-5p, which was further confirmed by dual luciferase reporter gene assay. The clinical arthritic scores, joint destruction, and inflammatory response were reduced after the injection of exosomes in rats with CIA targeting RAC2, and the treatment efficacy was even better with miR-192-5p-overexpressed exosomes. CONCLUSION: Our study established that the BMSCs-secreted exosomal miR-192-5p can delay the event of the inflammatory response in RA and may represent a possible therapeutic strategy for the treatment of RA.

Risk factors for the formation of double-contour sign and tophi in gout.

BACKGROUND: This study aimed to confirm the diagnostic accuracy of ultrasound (US) on gout and explore the potential risk factors for double-contour sign and tophi formation in gout patients. METHODS: The US analyses were performed on all knee, ankle, and first metatarsophalangeal (MTP 1) joints to reveal the type and location of lesions. While a questionnaire and blood biochemical index were used to explore the potential risk factors for double-contour sign and tophi in gout, the SPSS17.0 software was used for statistical analysis in the present study. RESULTS: Totally, 117 gout patients with 702 joints (38 lesions in knee joint, 93 lesions in ankle joint, and 112 lesions in MTP 1 joint) were enrolled in current analyses. Double-contour sign and joint effusion were the two most outstanding lesion manifestations in knee joints and ankle joints. Tophi and double-contour sign were the two most outstanding lesion manifestations in TMP 1 joints. Moreover, factors including uric acid (UA) level and the highest blood UA were potential risk factors of the double-contour sign, while age and history of US were potential risk factors for tophi. CONCLUSION: US was effective on the joints of gout patients. There was US sensitivity for tophi and double-contour sign in MTP 1 joints. The double-contour sign was a potential specific manifestation in knee joints and ankle joints. Furthermore, UA and highest blood UA level were potential risk factors for double-contour sign, while age and US history were potential risk factors for tophi.

The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

BACKGROUND: The circadian clock plays a crucial role in regulating physiology and is important for maintaining immune homeostasis and responses to inflammatory stimuli. Inflammatory arthritis often shows diurnal variation in disease symptoms and disease markers, and it is now established that cellular clocks regulate joint inflammation. The clock gene Bmal1 is critical for maintenance of 24-h rhythms and plays a key role in regulating immune responses, as well as in aging-related processes. Fibroblast-like synoviocytes (FLS) are circadian rhythmic joint mesenchymal cells which are important for maintenance of joint health and play a crucial role in the development of inflammatory arthritis. The aim of this study was to investigate the importance of the joint mesenchymal cell circadian clock in health and disease. METHODS: Mice were generated which lack Bmal1 in Col6a1-expressing cells, targeting mesenchymal cells in the ankle joints. Joints of these animals were assessed by X-ray imaging, whole-mount staining and histology, and the composition of the synovium was assessed by flow cytometry. Arthritis was induced using collagen antibodies. RESULTS: Bmal1 deletion in joint mesenchymal cells rendered the FLS and articular cartilage cells arrhythmic. Targeted mice exhibited significant changes in the architecture of the joints, including chondroid metaplasia (suggesting a switch of connective tissue stem cells towards a chondroid phenotype), reductions in resident synovial macrophages and changes in the basal pro-inflammatory activity of FLS. Loss of Bmal1 in FLS rendered these resident immune cells more pro-inflammatory in response to challenge, leading to increased paw swelling, localised infiltration of mononuclear cells and enhanced cytokine production in a model of arthritis. CONCLUSIONS: This study demonstrates the importance of Bmal1 in joint mesenchymal cells in regulating FLS and chondrocyte development. Additionally, we have identified a role for this core clock component for restraining local responses to inflammation and highlight a role for the circadian clock in regulating inflammatory arthritis.

Attenuated levels of ghrelin in synovial fluid is related to the disease severity of ankle post-traumatic osteoarthritis.

Post-traumatic osteoarthritis (PTOA) of ankle joints results in pain and reduced joint function. Ghrelin, a 28-amino-acid polypeptide, has been previously identified as the first cognate natural ligand that binds to the growth hormone secretagogue receptor. In the present study, ghrelin has been validated to exert cartilage-protective and anti-inflammatory effects. The current study was aimed at investigating the potential role of the levels of serum and synovial fluid (SF) ghrelin on the severity of disease in patients suffering from ankle PTOA. Ninety-seven patients with ankle osteoarthritis who received an arthroscopical examination and debridement or replacement of the ankle joint were included in the study cohort. Meanwhile, 95 healthy individuals (whose age and sex were matched) who received periodic body checkups were enrolled as healthy controls. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the ghrelin levels in serum and SF. SF was also probed for cartilage degradation enzyme matrix metalloproteinases-3 (MMP-3) and tumor necrosis factor alpha (TNF-α), which is a known pro-inflammatory cytokine. The clinical evaluation was carried out using the American Orthopedic Foot and Ankle Society (AOFAS) ankle-hindfoot rating scale and visual analogue scale (VAS). The radiographic severity was evaluated using the modified Kellgren-Lawrence (K-L) grading system. We scored for the modified Mankin's score to depict histopathological changes due to cartilage lesions. The diagnostic relevance of the ghrelin concentrations in the prediction of the radiographic grading (in comparison with MMP-3 and TNF-α) was evaluated by calculating the area under the curve of the receiver operating characteristic (ROC) curve. The serum abundance of ghrelin was not significantly altered between ankle PTOA patients and healthy controls. SF ghrelin was negatively correlated with radiographic progression determined by modified ankle K-L grades. In addition SF ghrelin concentrations were negatively related to VAS scores, and positively associated with AOFAS ankle-hindfoot rating. Moreover, SF ghrelin was inversely proportional to the expressions of MMP-3 and TNF-α. ROC analysis curve demonstrated that ghrelin serves as a favorable marker for the diagnosis of radiographic severity by modified ankle K-L grade. The ghrelin concentration in SF is negatively proportional to disease progression in patients suffering from ankle PTOA. Local administration of ghrelin may function as a decent adjuvant therapy to delay the progress of ankle PTOA. © 2019 BioFactors, 45(3):463-470, 2019.

Articular chondrocytes isolated from the knee and ankle joints of human tissue donors demonstrate similar redox-regulated MAP kinase and Akt signaling.

OBJECTIVE: To compare key intracellular redox-regulated signaling pathways in chondrocytes derived from knee joint femoral cartilage and ankle joint talar cartilage in order to determine if differences exist that might contribute to the lower prevalence of ankle osteoarthritis (OA). METHODS: Femoral and talar chondrocytes were treated with H2O2 generators (menadione or 2-3-dimethoxy-1,4-napthoquinone (DMNQ), fragments of fibronectin (FN-f)) to stimulate MAP kinase signaling (MAPK), or with IGF-1 to stimulate the Akt signaling pathway. Hyperoxidation of the peroxiredoxins, used as a measure of redox status, and phosphorylation of proteins pertinent to MAPK (p38, ERK, JNK, c-Jun) and Akt (Akt, PRAS40) signaling cascades were detected by immunoblotting. RESULTS: Treatment of femoral and talar chondrocytes with menadione, DMNQ or FN-f led to a time dependent increase in extracellular-regulated kinase (ERK) and p38 phosphorylation. DMNQ and FN-f stimulation enhanced phosphorylation of JNK and its downstream substrate, c-Jun. Menadione treatment did not stimulate JNK activity but hyperoxidized the peroxiredoxins and inhibited IGF-1-induced Akt activation. In all experiments, chondrocytes derived from the femur and talar joints displayed comparable MAP kinase responses after treatment with various catabolic stimuli, as well as similar Akt signaling responses after IGF-1 treatment. CONCLUSIONS: MAP kinase and Akt signaling in response to factors that modulate the intracellular redox status were similar in chondrocytes from knee and ankle joints suggesting that redox signaling differences do not explain differences in OA prevalence. Talar chondrocytes, when isolated from their native matrix, can be used to examine redox-regulated cell signaling events relevant to OA in either joint.

The level of synovial AXL expression determines the outcome of inflammatory arthritis, possibly depending on the upstream role of TGF-ß1.

OBJECTIVE: To investigate the role of AXL, a member of the anti-inflammatory TYRO3, AXL MER (TAM) receptor family, in arthritis. METHODS: KRN serum transfer arthritis was induced in Axl-/- and wild-type mice. Knee and ankle joints were scored macro- and microscopically. Synovial gene and protein expression of Axl was determined in naïve and TGF-ß1-overexpressing joints. AXL expression was determined in M1-like or M2-like macrophages and RA synovium. Human macrophages, fibroblasts and synovial micromasses were stimulated with TGF-ß1 or the AXL inhibitor R428. RESULTS: Ankle joints of Axl-/- mice showed exacerbated arthritis pathology, whereas no effect of Axl gene deletion was observed on gonarthritis pathology. To explain this spatial difference, we examined the synovium of naïve mice. In contrast to the knee, the ankle synovial cells prominently expressed AXL. Moreover, the M2-like macrophage phenotype was the dominant cell type in the naïve ankle joint. Human M2-like macrophages expressed higher levels of AXL and blocking AXL increased their inflammatory response. In the murine ankle synovium, gene expression of Tgfb1 was increased and Tgb1 correlated with Axl. Moreover, TGFB1 and AXL expression also correlated in human RA synovium. In human macrophages and synovial micromasses, TGF-ß1 enhanced AXL expression. Moreover, TGF-ß1 overexpression in naïve murine knee joints induced synovial AXL expression. CONCLUSION: Differences in synovial AXL expression are in accordance with the observation that AXL dampens arthritis in ankle, but not in knee joints. We provide evidence that the local differences in AXL expression could be due to TGF-ß1, and suggest similar pathways operate in RA synovium.

Ketoprofen and MicroRNA-124 Co-loaded poly (lactic-co-glycolic acid) microspheres inhibit progression of Adjuvant-induced arthritis in rats.

Ketoprofen, a non-steroid anti-inflammatory drug, is widely used for relieving the pain and swelling caused by rheumatoid arthritis. However, ketoprofen can't suppress disease progression effectively. In this study, in an effort to improve the therapeutic effect for rheumatoid arthritis (RA), microRNA-124 (miR-124), a promising new therapeutic agent for RA, was co-loaded with ketoprofen into poly (lactic-co-glycolic acid) (PLGA) microspheres and administrated to adjuvant-induced arthritis rats. PLGA microspheres loaded with ketoprofen and miR-124 were prepared by a modified multiple emulsion-solvent evaporation method. In vivo pharmacodynamics experimental results indicated ketoprofen in co-loaded microspheres could significantly reduce inflammation of the joints and miR-124 in the microspheres could reduce bone damage. In addition, ketoprofen and miR-124 co-loaded PLGA microspheres had a remarkable advanced activity over delivery of either miR-124 or ketoprofen in suppressing adjuvant-induced arthritis (AA) in rats. Results of western blot and immunohistochemistry revealed that miR-124 could reduce the level of receptor activator of nuclear factor kappa-B ligand (RANKL). These results suggested co-delivery of ketoprofen and miR-124 could achieve synergistic effects on preventing inflammation and bone damage caused by AA. Ketoprofen and miR-124 co-loaded PLGA microspheres could be a promising combined therapeutic strategy against RA.

Mesenchymal stem cells ameliorate experimental arthritis via expression of interleukin-1 receptor antagonist.

Human bone marrow-derived mesenchymal stem cells (MSCs) have been observed to inhibit arthritis in experimental animal models such as collagen-induced arthritis. However, the exact anti-inflammatory mechanisms remain poorly understood. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine produced by immune and stromal cells. We postulated that MSCs could produce IL-1Ra and attenuate experimental arthritis. In this study, 5x106 MSCs were injected into the peritoneal cavity of IL-1Ra knockout (IL-1RaKO) mice. MSCs reduced the severity of the arthritis by histology and decreased pro-inflammatory cytokine levels in IL-1RaKO mice. The ratio of splenic T helper 17 (Th17) cells to regulatory T cells (Treg) was significantly decreased in MSC-injected IL-1RaKO mice. Purified splenic CD4+ T cells from mice in each of the treatment groups were cultured under Th17 polarizing conditions and analyzed by flow cytometry. Less expansion of the Th17 population was observed in the MSC-treated group. Interestingly, MSCs expressed inducible IL-1Ra against inflammatory environmental stimuli. Human recombinant IL-1Ra could suppress Th17 cells differentiation under Th17 polarizing conditions. These results indicate that IL-1Ra expressed by MSCs can inhibit Th17 polarization and decrease the immune response in IL-1RaKO mice. Therefore, MSC-derived IL-1Ra may inhibit inflammation in IL-1RaKO mice via effects on Th17 differentiation.

Anti-CD11b antibody treatment suppresses the osteoclast generation, inflammatory cell infiltration, and autoantibody production in arthritis-prone FcγRIIB-deficient mice.

BACKGROUND: Previously we established an arthritis-prone FcγRIIB-deficient mouse strain (designated KO1). Anti-mouse CD11b mAb (5C6) has been reported to inhibit the recruitment of peripheral CD11b+ myelomonocytic cells from the blood to the inflammatory site. These cells include neutrophils and monocytes, both of which play important roles in the development of arthritis. Here we treated KO1 mice with 5C6 mAb in order to study its effect on arthritis development. METHODS: To evaluate the disease-preventive effect of 5C6, 4-month-old preclinical KO1 mice were divided into three groups: the first treated with 5C6 for 6 months, the second treated with normal rat IgG for 6 months, as a control, and the third left untreated. Arthritis severity and immunological abnormalities were compared among the groups, along with transcriptional levels of several important arthritis-related factors in ankle joints, spleen, and peripheral blood cells. RESULTS: The 5C6 treatment ameliorated arthritis in KO1 mice, showing decreases in inflammatory cell infiltration and osteoclast formation. Analysis of transcriptional levels in ankle joints revealed that compared with the two control groups, the 5C6-treated group showed downregulated expression of RANK, RANKL, MCP-1, RANTES, TNFα, and IL-6, and at the same time showed significantly up-regulated expression of the decoy receptor for RANKL, i.e. osteoprotegerin. In addition, the disease suppression was associated with the lower serum levels of autoantibodies, and the decreased frequencies of activated B cells and plasma cells. The expression levels of B cell activation/differentiation-related cytokines were suppressed in spleen and peripheral leukocytes of the 5C6-treated mice. Intriguingly, while untreated KO1 mice spontaneously developed marked monocytosis, the 5C6-treated mice showed the significantly down-regulated frequency of monocytes. CONCLUSIONS: The outcome of 5C6 treatment was complex, in which the 5C6-mediated disease-preventive effect is likely due on one hand to the decrease in the recruitment of inflammatory cells and osteoclast precursor monocytes from the periphery into the joints, and on the other hand to the suppression of B cell activation/maturation and of autoantibody production via the suppression of B cell stimulating cytokine production. The lower levels of these cytokines may be the secondary effect of the lower frequency of monocytes, since monocytes/macrophages are the major producers of these cytokines.

Fluid distribution in ankle and midfoot joints: MR findings in asymptomatic volunteers.

PURPOSE: The purpose of this study is to assess the amount of fluid in the joints of the ankle and midfoot on MR imaging in asymptomatic volunteers. MATERIALS AND METHODS: Twenty-one healthy asymptomatic volunteers (42 ankles) were evaluated with MRI imaging. There were 13 men and 8 women. The mean age was 24.7 years (19-42 years). MR imaging was performed on a 3T MR system using proton density weighted images with fat saturation (TR 2969, TE 30 ms, NA 2, slice thickness 2.5 mm). Images were obtained in three orthogonal planes. The images were interpreted by two radiologists in two sessions. The maximum size of the joint effusion was measured in one plane. Descriptive statistics and variation between interpretation sessions were calculated. RESULTS: Fluid in the anterior tibiotalar joint had a mean size of 2.0 mm (0.0-5.5 mm), in the posterior tibiotalar joint 3.1 mm (0.0-6.3 mm), in the talonavicular joint 0.7 mm (0.0-2.9 mm), and in the anterolateral recess 2.0 mm (0.0-4.3 mm). Fluid in the posterior aspect of the posterior subtalar joint had a mean size of 2.6 mm (0.0-9.4 mm), in the anterior aspect of the posterior subtalar joint 1.9 mm (0.0-6.6 mm), at the middle subtalar joint 0.1 mm (0.0-1.7 mm), and at the anterior subtalar joint 1.6 mm (0.0-6.0 mm). Fluid in the tibiofibular joint had a mean height of 8.1 mm (0.0-16.4 mm). CONCLUSION: In asymptomatic volunteers, moderate to large amounts of fluid were common in all joint recesses of ankle and midfoot, and most pronounced in the anterior and posterior tibiotalar joint, anterolateral recess, and posterior subtalar joint. This should not be mistaken for evidence of a pathological condition.

An optimal ultrasonographic diagnostic test for early gout: A prospective controlled study.

Objective To identify the optimal sites for classification of early gout by ultrasonography. Methods Sixty patients with monosodium urate crystal-proven gout (25 with early gout [≤2-year symptom duration], 35 with late gout [>2-year symptom duration], and 36 normouricemic healthy controls) from one centre were prospectively evaluated. Standardized blinded ultrasound examination of 36 joints and the triceps and patellar tendons was performed to identify tophi and the double contour (DC) sign. Results Ultrasonographic sensitivity was lower in early than late gout. Binary logistic regression analysis showed that two ultrasonographic signs (tophi in the first metatarsophalangeal joint [odds ratio, 16.46] and the DC sign in the ankle [odds ratio, 25.18]) significantly contributed to the final model for early gout diagnosis (sensitivity and specificity of 84% and 81%, respectively). The inter-reader reliability kappa value for the DC sign and tophi was 0.712. Conclusions Four-joint investigation (both first metatarsophalangeal joints for tophi and both ankles for the DC sign) is feasible and reliable and could be proposed as a screening test for early ultrasonographic gout classification in daily practice.

Clinically-evident tophi are associated with reduced muscle force in the foot and ankle in people with gout: a cross-sectional study.

BACKGROUND: The foot and ankle represent a common site for tophi in people with gout, yet it is unclear whether the presence of tophi is related to impaired muscle function. This study aimed to determine the association between foot and ankle tophi and muscle force in people with gout. METHODS: Participants with gout were stratified into two groups based on the presence of clinically-evident tophi affecting the foot or ankle on physical examination. Isometric muscle force for plantarflexion, dorsiflexion, inversion and eversion was measured using static dynamometry. Mixed-models regression was used to determine the difference in muscle force between the two groups while adjusting for age, disease duration and foot pain. This model was also used to determine the difference in muscle force between presence and absence of tophi at specific locations within the foot and ankle. In addition, Pearson's correlations were used to determine the association between total foot tophus count and muscle force. RESULTS: Fifty-seven participants were included (22 with foot or ankle tophi and 35 without foot or ankle tophi). Foot and ankle tophi were most often seen at the Achilles tendon. After adjusting for age, disease duration and foot pain, participants with tophi had significantly reduced muscle force during plantarflexion (P < 0.001), dorsiflexion (P = 0.003), inversion (P = 0.003) and eversion (P = 0.001) when compared to participants without tophi. Those with Achilles tophi had significantly reduced force during plantarflexion (P < 0.001), inversion (P = 0.008) and eversion (P = 0.001). No significant differences in muscle force were observed between the presence and absence of tophi at other foot or ankle locations. There were also no significant correlations between total foot tophus count and muscle force (all P > 0.05). CONCLUSION: In people with gout, clinically-evident foot or ankle tophi are associated with muscle force deficits during foot plantarflexion, dorsiflexion, inversion and eversion, which persist despite adjusting for age, disease duration and foot pain. Tophi at the Achilles tendon, which associate with force deficits, may contribute to reduced muscular activation and consequent disuse muscle atrophy.

Differences in type II collagen turnover of osteoarthritic human knee and ankle joints.

PURPOSE: We analysed hyaline cartilage of human knee and ankle joints for collagen and proteoglycan turnover in order to find differences in the metabolism and biochemical content of the extracellular matrix that could explain the higher prevalence of osteoarthritis (OA) in the knee joint, compared to the ankle joint. METHODS: Cartilage tissue from ankle and knee joints of OA patients were assessed for total collagen and proteoglycan content. For turnover, the aggrecan 846-epitope (CS 846), the type II collagen C-propeptide (CP2) and the collagenase-generated intrahelical cleavage neoepitope (C2C) were quantified. RESULTS: Molecular analyses showed that type II collagen turnover (CP2 and C2C) was significantly elevated in the ankle, whereas aggrecan turnover (CS 846), total proteoglycan and total collagen were comparable between both joints. Analysis of the inter-relationships in the components of cartilage matrix turnover showed a significant positive correlation of C2C vs CP2. CONCLUSIONS: The data suggest an increased type II collagen turnover in ankle vs knee OA cartilage but a comparable aggrecan turnover and comparable contents of type II collagen and proteoglycan. These findings point towards a focused attempt in advanced OA cartilage to structurally repair the collagen network that was more pronounced in the ankle joint and may explain in part the higher prevalence of OA in the knee as compared to the ankle joint.