Inflamed Ankle Research Articles

Evaluation of the Anti-Inflammatory/Immunomodulatory Effect of Teucrium montanum L. Extract in Collagen-Induced Arthritis in Rats.

The anti-inflammatory/immunomodulatory effects of Teucrium montanum L. (TM), a plant distributed in the Mediterranean region, have been insufficiently examined. The effects of the TM ethanol extract were tested in a rat collagen-induced arthritis (CIA) model of rheumatoid arthritis. LC-MS was used for the phytochemical analysis of the TM extract. Dark Agouti rats were immunized with bovine type II collagen (CII) in incomplete Freund's adjuvant for CIA, and treated with 100 or 200 mg/kg of TM extract daily via oral administration. Clinical and histopathological evaluations and a flow cytometric analysis of the phenotypic and functional characteristics of splenocytes and draining lymph node cells were performed. The cytokines in the paw tissue culture supernatants and anti-CII antibodies in serum were determined by ELISA. The TM extract, with the dominant components verbascoside and luteolin 7-O-rutinoside, reduced the arthritic score and ankle joint inflammation in CIA rats, promoted the antioxidant profile in serum, and lowered pro-inflammatory TNF-α, IL-6 and IL-1β production. It suppressed the activation status of CD11b+ cells by lowering CD86, MHCII and TLR-4 expression, and promoted the Th17/T regulatory cell (Tregs) balance towards Tregs. A lower frequency of B cells was accompanied by a lower level of anti-CII antibodies in treated rats. These findings imply the favorable effect of TM extract on the clinical presentation of CIA, suggesting its anti-inflammatory/immunomodulatory action and potential therapeutic effect.

Biomimetic Integrated Nanozyme for Flare and Recurrence of Gouty Arthritis

Flare and multiple recurrences pose significant challenges in gouty arthritis. Traditional treatments provide temporary relief from inflammation but fail to promptly alleviate patient pain or effectively prevent subsequent recurrences. It should also be noted that both anti-inflammation and metabolism of uric acid are necessary for gouty arthritis, calling for therapeutic systems to achieve these two goals simultaneously. In this study, we propose a biomimetic integrated nanozyme, HMPB-Pt@MM, comprising platinum nanozyme and hollow Prussian blue. It demonstrates anti-inflammatory properties by eliminating reactive oxygen species and reducing infiltration of inflammatory macrophages. Additionally, it rapidly targets inflamed ankles through the camouflage of macrophage membranes. Furthermore, HMPB-Pt@MM exhibits urate oxidase-like capabilities, continuously metabolizing locally elevated uric acid concentrations, ultimately inhibiting multiple recurrences of gouty arthritis. In summary, HMPB-Pt@MM integrates ROS clearance with uric acid metabolism, offering a promising platform for the treatment of gouty arthritis.

Macrophage mannose receptor CD206 targeting of fluoride-18 labeled mannosylated dextran: A validation study in mice

PurposeAluminum fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated mannosylated dextran derivative (Al[18F]F-NOTA-D10CM) is a new tracer for PET imaging. We report here on in vitro and in vivo validation of the tracer’s ability to target the macrophage mannose receptor CD206.MethodsFirst, the uptake of intravenously (i.v.) administered Al[18F]F-NOTA-D10CM was compared between wild-type (WT) and CD206−/− knockout (KO) mice. C57BL/6N mice were injected with complete Freund’s adjuvant (CFA) in the left hind leg and the uptake of Al[18F]F-NOTA-D10CM after i.v. or intradermal (i.d.) injection was studied at 5 and 14 days after CFA induction of inflammation. Healthy C57BL/6N mice were studied as controls. Mice underwent PET/CT on consecutive days with [18F]FDG, i.v. Al[18F]F-NOTA-D10CM, and i.d. Al[18F]F-NOTA-D10CM. After the last imaging, Al[18F]F-NOTA-D10CM was i.v. injected for an ex vivo biodistribution study and autoradiography of inflamed tissues. Blood plasma samples were analyzed using high-performance liquid chromatography. To evaluate the specificity of Al[18F]F-NOTA-D10CM binding, an in vitro competitive displacement study was performed on inflamed tissue sections using autoradiography. CD206 expression was assessed by immunohistochemical staining.ResultsCompared with WT mice, the uptake of Al[18F]F-NOTA-D10CM was significantly lower in several CD206−/− KO mice tissues, including liver (SUV 8.21 ± 2.51 vs. 1.06 ± 0.16, P < 0.001) and bone marrow (SUV 1.63 ± 0.37 vs. 0.22 ± 0.05, P < 0.0001). The uptake of i.v. injected Al[18F]F-NOTA-D10CM was significantly higher in inflamed ankle joint (SUV 0.48 ± 0.13 vs. 0.18 ± 0.05, P < 0.0001) and inflamed foot pad skin (SUV 0.41 ± 0.10 vs. 0.04 ± 0.01, P < 0.0001) than in the corresponding tissues in healthy mice. The i.d.-injected Al[18F]F-NOTA-D10CM revealed differences between CFA-induced lymph node activation and lymph nodes in healthy mice. Ex vivo γ-counting, autoradiography, and immunohistochemistry supported the results, and a decrease of ~ 80% in the binding of Al[18F]F-NOTA-D10CM in the displacement study with excess NOTA-D10CM confirmed that tracer binding was specific. At 60 min after i.v. injection, an average 96.70% of plasma radioactivity was derived from intact Al[18F]F-NOTA-D10CM, indicating good in vivo stability. The uptake of Al[18F]F-NOTA-D10CM into inflamed tissues was positively associated with the area percentage of CD206-positive staining.ConclusionThe uptake of mannosylated dextran derivative Al[18F]F-NOTA-D10CM correlated with CD206 expression and the tracer appears promising for inflammation imaging.Graphical abstract

Mechanism investigation of Duhuo Jisheng pill against rheumatoid arthritis based on a strategy for the integration of network pharmacology, molecular docking and in vivo experimental verification

Context Duhuo Jisheng pill (DHJS) is a classic traditional Chinese medicine (TCM) formula for rheumatoid arthritis (RA). The effective components and therapeutic mechanisms of DHJS for treating RA are still unclear. Objective To explore the potential mechanism of DHJS against RA by means of network pharmacology and experimental verification. Materials and methods A network pharmacology and molecular docking analysis based on phytochemistry was used to elucidate the mechanism of DHJS against RA. The targets of DHJS anti-RA active ingredient were obtained by searching TCMSP, ETCM and TCMSID. The RA model induced by collagen was established in Wistar rats. The rats in the DHJS group were administered doses of 0.5, 1.0 and 2.0 g/kg for a period of 10 d. The expression of targets was measured with Western blot. Results Network pharmacology analysis showed that the anti-RA effect of DHJS was mediated by targets involved in immunity, inflammation and apoptosis, as well as PI3K-Akt and NF-κB signalling pathways. Of 2.0 g/kg DHJS significantly alleviated the ankle inflammation (IL-6: 62.73 ± 8.39 pg/mL, IL-1β: 50.49 ± 11.47 pg/mL, TNF-α: 16.88 ± 3.05 pg/mL, IL-17A: 12.55 ± 1.87 pg/mL, IL-10: 16.24 ± 3.00 pg/mL), comparing with the model group (IL-6: 92.02 ± 13.25 pg/mL, IL-1β: 71.85 ± 4.12 pg/mL, TNF-α: 25.64 ± 3.69 pg/mL, IL-17A: 22.14 ± 4.56 pg/mL, IL-10: 9.51 ± 3.03 pg/mL) (p < 0.05). Moreover, the protein expression of p-PI3K, p-AKT and p-p65 significantly decreased after DHJS administration. Conclusions DHJS could alleviate the collagen-induced arthritis (CIA) by the PI3K/AKT/NF-κB signalling pathway.

Electroacupuncture improves gout arthritis pain via attenuating ROS-mediated NLRP3 inflammasome overactivation

BackgroundGout results from disturbed uric acid metabolism, which causes urate crystal deposition in joints and surrounding tissues. Gout pain management is largely limited to colchicine and nonsteroidal anti-inflammatory drugs. Constant usage of these medications leads to severe side effects. We previously showed electroacupuncture (EA) is effective for relieving pain in animal model of gout arthritis. Here we continued to study the mechanisms underlying how EA alleviates gout pain.MethodsMonosodium urate was injected into ankle joint to establish gout arthritis model in mice. EA or sham EA was applied at ST36 and BL60 acupoints of model animals. Biochemical assays, immunostaining, live cell Ca2+ imaging and behavioral assays were applied.ResultsModel mice displayed obvious mechanical allodynia, accompanied with gait impairments. EA attenuated mechanical hypersensitivities and improved gait impairments. EA reduced the overexpression of NLRP3 inflammasome signaling molecules in ankle joints of model animals. EA-induced anti-allodynia, as well as inhibition on NLRP3 inflammasome, were mimicked by antagonizing but abolished by activating NLRP3 inflammasome via pharmacological methods. EA attenuated oxidative stress, an upstream signaling of NLRP3 inflammasome in ankle joints of model mice. Exogenously increasing oxidative stress abolished EA’s inhibitory effect on NLRP3 inflammasome and further reversed EA’s anti-allodynic effect. EA reduced neutrophil infiltrations in ankle joint synovium, a major mechanism contributing to oxidative stress in gout. Pharmacological blocking NLRP3 inflammasome or EA reduced TRPV1 channel overexpression in dorsal root ganglion (DRG) neurons. Ca2+ imaging confirmed that EA could reduce functional enhancement in TRPV1 channel in DRG neurons during gout.ConclusionsOur results demonstrate that EA reduces gout pain possibly through suppressing ROS-mediated NLRP3 inflammasome activation in inflamed ankle joints and TRPV1 upregulation in sensory neurons, supporting EA as a treatment option for gout pain.

IL-18 receptor-α signalling pathway contributes to autoantibody-induced arthritis via neutrophil recruitment and mast cell activation.

The interleukin (IL)-18 signalling pathway is involved in animal models of collagen-induced arthritis, but the role of this pathway in autoantibody-induced arthritis is poorly understood. An autoantibody-induced arthritis model, K/BxN serum transfer arthritis, reflects the effector phase of arthritis and is important in innate immunity including neutrophils and mast cells. This study aimed to investigate the role of the IL-18 signalling pathway in autoantibody-induced arthritis using IL-18 receptor (IL-18R) α-deficient mice. K/BxN serum transfer arthritis was induced in IL-18Rα-/- and wild-type B6 (controls) mice. The severity of arthritis was graded, and histological and immunohistochemical examinations were performed on paraffin-embedded ankle sections. Total Ribonucleic acid (RNA) isolated from mouse ankle joints was analysed by real-time reverse transcriptase-polymerase chain reaction. IL-18 Rα-/- mice had significantly lower arthritis clinical scores, neutrophil infiltration, and numbers of activated, degranulated mast cells in the arthritic synovium than in controls. IL-1β, which is indispensable for the progression of arthritis, was significantly downregulated in inflamed ankle tissue in IL-18 Rα-/- mice. IL-18/IL-18Rα signalling contributes to the development of autoantibody-induced arthritis by enhancing synovial tissue expression of IL-1β and inducing neutrophil recruitment and mast cell activation. Therefore, inhibition of the IL-18Rα signalling pathway might be a new therapeutic strategy for rheumatoid arthritis.

Total Ankle Replacement Outcome in Patients With Inflammatory Versus Noninflammatory Arthritis: A Systematic Review and Meta-analysis.

The decision regarding total ankle replacement (TAR) is challenging in patients with inflammatory arthritis (IA) regarding more inferior bone quality, large bone cysts, and increased risk of infections. This systematic review and meta-analysis aimed to compare the functional outcome, revision rate, complication rate, and survival rate of TAR between IA (including rheumatoid arthritis [RA]) and noninflammatory arthritis (NIA) (primary and posttraumatic). After reviewing the full texts, 30 articles fulfilled all inclusion criteria from 1985 until 2021, comparing TAR results. The eligible studies included 5508 patients, of whom 1565 patients had IA and 3943 patients had NIA. At the time of surgery, the average age was 58 years in the IA group and 63 in the NIA group. The average follow-up was 67.2 months in the IA group and 67 months in the NIA group. The outcome measures were the American Orthopaedic Foot and Ankle Society (AOFAS) score and the rate of complications, revisions, and survival. The mean final AOFAS score was 82 (95% confidence interval [CI]: 78-86) in the IA group and 83 (95% CI: 78-88) in the NIA group, with no significant difference. There was no significant difference in the mean preoperative to postoperative AOFAS score change between the IA and the NIA. The complication rate was 16% (95% CI: 9%-27%) in the IA group and 15% (95% CI: 8%-27%) in the NIA group with no significant difference. The revision rate was 12% (95% CI: 10%-15%) in the IA group and 13% (95% CI: 10%-18%) in the NIA group, which was significant (P = .04). There was no significant difference in the survival rate between IA and NIA. Total ankle replacement is a safe procedure in inflammatory ankle arthritis, specifically in RA patients with relatively minor and major complications close to other reasons for ankle replacement. Level IV: prognostic.

P36 Crohn’s-associated inflammatory arthritis treated successfully with upadacitinib

Introduction/BackgroundEnteropathic arthritis is inflammatory arthritis which is associated with inflammatory bowel disease such as Crohn’s disease and ulcerative colitis. The optimal treatment for patients is one which is effective for both arthritis and bowel disease. First line treatments include DMARDs such as methotrexate and sulfasalazine. Second line options include anti-TNF biologics such as infliximab and adalimumab, and the anti-IL-12/23 biologic ustekinumab. There is currently little guidance on choice of treatment if these options fail. We present a case of Crohn’s-associated inflammatory arthritis which did not respond to two anti-TNFs but was successfully treated with the JAK inhibitor upadacinitib.Description/MethodA 30-year-old male was referred to gastroenterology with a 4 month history of diarrhoea and weight loss. His faecal calprotectin was raised at 982µg/g. He had a history of seronegative inflammatory arthritis diagnosed 5 years previously. He had been treated initially with methotrexate and sulfasalazine, which were discontinued due to side effects and inefficacy, and his arthritis was subsequently well-controlled on etanercept for the last year.Colonoscopy showed patchy colitis at the rectum which was continuous in the transverse and ascending colon and caecum. The appearances and mucosal biopsy were consistent with moderately active Crohn’s colitis. In view of this, etanercept was switched to adalimumab, which was administered using the accelerated loading regimen used in IBD.Five weeks after switching the patient developed a flare of arthritis with knee and ankle inflammation and CRP 30. He was given oral steroids and reviewed in a combined rheumatology/gastroenterology clinic. The frequency of adalimumab was increased to 40mg weekly and subcutaneous methotrexate 10mg weekly was introduced.Despite this, the patient’s arthritis remained active, although with some improvement in his bowel symptoms and a fall in faecal calprotectin to 117µg/g. Adalimumab was switched to certolizumab and methotrexate increased to 15mg weekly with intra-muscular steroids. On certolizumab he developed progressively more active arthritis with CRP 83, bilateral ankle inflammation and large knee effusions requiring aspiration and steroid injection.After 8 weeks of certolizumab, there was no improvement and the decision was made to switch to upadacitinib 15mg daily. Within 4 weeks of treatment with upadacitinib he noticed a significant improvement in his arthritis and after 12 weeks was in clinical remission with normal CRP and faecal calprotectin. Methotrexate was later discontinued due to nausea. Over 12 months later the patient remains in remission for both bowel disease and arthritis on upadacitinib monotherapy.Discussion/ResultsIn enteropathic arthritis, joint symptoms may precede or follow bowel disease. Etanercept, a recombinant TNF-receptor fusion protein, is not an effective treatment for IBD. Indeed, there are case reports and observational data to suggest that etanercept may be associated with an increased risk of developing IBD in some patients. In our case the patient developed Crohn’s disease while taking etanercept and the switch to adalimumab precipitated a flare of his arthritis which was not controlled by treatment intensification. The failure of two anti-TNF biologics, adalimumab and certolizumab, may point to the activation of alternative cytokine inflammatory pathways as the driver of the patient’s disease.Our patient was managed in a combined rheumatology/gastroenterology clinic which enabled an inter-specialty approach to managing both aspects of his disease. After the failure of certolizumub we made the decision to trial upadacitinib, a selective JAK1 inhibitor, owing to our experience of its effectiveness in rheumatoid and psoriatic arthritis, and its promising results in Phase 3 trials in Crohn’s disease. We considered ustekinumab but opted against it as in our experience it may be suboptimal in patients in whom arthritis is the most troublesome symptom. Upadacitinib worked quickly and effectively to control our patient’s arthritis alongside his bowel disease and was well tolerated.There are currently limited treatments which are effective for both IBD and arthritis, and there is little guidance on treatment strategies in enteropathic arthritis if anti-TNF and ustekinumab are unsuccessful. Tofacitinib, a JAK1/3 inhibitor, is licensed for rheumatoid and psoriatic arthritis and ulcerative colitis, but is not effective for Crohn’s disease. Upadacitinib is undergoing Phase 3 trials in Crohn’s disease and our case highlights its potential role in the treatment of patients with Crohn’s disease and inflammatory arthritis in whom anti-TNF therapy is unsuccessful.Key learning points/Conclusion1. Etanercept is not an effective treatment for inflammatory bowel disease and may necessitate a change to another anti-TNF biologic such as infliximab or adalimumab.2. Upadacitinib, a selective JAK1 inhibitor, was successfully used in our case to treat both arthritis and bowel disease. Although this was just one case, the results of Phase 3 trials suggest a promising role for upadacitinib in Crohn’s disease and it is already used in the treatment of rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis.3. Patients with enteropathic arthritis may be best managed in a specialist combined gastroenterology and rheumatology clinic where a holistic approach to the management of both aspects of their disease can be undertaken.

Mechanism of total flavonoids from Ampelopsis grossedentata against gouty arthritis based on multi-level interactive network and in vivo experimental validation

The present study investigated the mechanism of total flavonoids from Ampelopsis grossedentata(AGTF) against gouty arthritis(GA) by network pharmacology and experimental validation. The main active ingredients and targets of AGTF, as well as disease targets, were screened out using relevant databases and literature data. The &quot;protein-protein interaction&quot;(PPI) network and &quot;drug-ingredient-target-pathway&quot; network were constructed, and the potential targets and mechanism of AGTF against GA were predicted. The hyperuricemia(HUA) combined with GA model was induced in rats. The gait behaviors of rats were scored, and ankle swelling degree was observed. The uric acid(UA) level and xanthine oxidase(XOD) activity in the rat serum were detected, and the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) were measured. The protein expression of toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), and nuclear factor-kappa B(NF-κB) in the synovial tissues of the rat ankle joint was determined by immunohistochemistry. Ten active ingredients of AGTF and 73 candidate targets of AGTF against GA were screened out by network pharmacology. Eighty-six signaling pathways were enriched, including TNF signaling pathway, NF-κB signaling pathway, TLR signaling pathway, Nod-like receptor signaling pathway, and purine metabolism signaling pathway, which were closely related to AGTF against GA. Animal experimental results showed that AGTF could effectively improve the abnormal gait behaviors of GA rats, relieve ankle inflammation, and reduce ankle joint swelling. In addition, AGTF could significantly reduce UA level, inhibit XOD activity, decrease TNF-α, IL-6, and IL-1β content, and down-regulate the expression of TLR4, MyD88, and NF-κB in ankle synovial tissues(P&lt;0.05, P&lt;0.01). The results of network pharmacology and experimental validation are consistent, indicating that AGTF exerts its therapeutic effect on GA by regulating UA metabolism, improving abnormal UA level, reducing the release of inflammatory factors, and regulating immunity and the TLR4/MyD88/NF-κB inflammatory pathway.

PPAR-γ alleviates the inflammatory response in TNF-α-induced fibroblast-like synoviocytes by binding to p53 in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by synovial inflammation, synoviocyte expansion and damage to cartilage and bone. We recently reported that peroxisome proliferator-activated receptor (PPAR)-γ inhibited the proliferation and activation of fibroblast-like synoviocytes (FLS), and was downregulated in RA synovial. In this study we investigated the role of PPAR-γ in RA and the underlying mechanisms. Adjuvant-induced arthritis (AIA) was induced in rats; from D15, AIA rats were orally administered pioglitazone (30 mg·kg-1·d-1) or rosiglitazone (4 mg·kg-1·d-1) for 14 days. Collagen-induced arthritis (CIA) was induced in wild-type and Ppar-γ+/- mice. We showed that the expression of PPAR-γ was significantly reduced, whereas that of TNF-α was markedly increased in human RA FLS. In CIA mice, knockdown of PPAR-γ expression (Ppar-γ+/-) aggravated the ankle inflammation. Similarly, T0070907 (a PPAR-γ antagonist) or si-PPAR-γ promoted the activation and inflammation of TNF-α-induced FLS in vitro. On the contrary, administration of PPAR-γ agonist pioglitazone or rosiglitazone, or injection of ad-Ppar-γ into the ankle of AIA rat in vivo induced overexpression of PPAR-γ, reduced the paw swelling and inflammation, and downregulated activation and inflammation of FLS in RA. Interesting, injection of ad-Ppar-γ into the ankle also reversed the ankle inflammation in Ppar-γ+/- CIA mice. We conducted RNA-sequencing and KEGG pathway analysis, and revealed that PPAR-γ overexpression was closely related to p53 signaling pathway in TNF-α-induced FLS. Co-IP study confirmed that p53 protein was bound to PPAR-γ in RA FLS. Taken together, PPAR-γ alleviates the inflammatory response of TNF-α-induced FLS by binding p53 in RA.

Systematic analysis of inflammation and pain pathways in a mouse model of gout.

Gout is a prevalent and painful inflammatory arthritis, and its global burden continues to rise. Intense pain induced by gout attacks is a major complication of gout. However, systematic studies of gout inflammation and pain are lacking. Using a monosodium urate (MSU) crystal-induced gout model, we performed genome-wide transcriptome analysis of the inflamed ankle joint, dorsal root ganglion (DRG), and spinal cord of gouty mice. Our results revealed important transcriptional changes, including highly elevated inflammation and broad activation of immune pathways in both the joint and the nervous system, in gouty mice. Integrated analysis showed that there was a remarkable overlap between our RNAseq and human genome-wide association study (GWAS) of gout; for example, the risk gene, stanniocalcin-1 (STC1) showed significant upregulation in all three tissues. Interestingly, when compared to the transcriptomes of human osteoarthritis (OA) and rheumatoid arthritis (RA) joint tissues, we identified significant upregulation of cAMP/cyclic nucleotide-mediated signaling shared between gouty mice and human OA with high knee pain, which may provide excellent drug targets to relieve gout pain. Furthermore, we investigated the common and distinct transcriptomic features of gouty, inflammatory pain, and neuropathic pain mouse models in their DRG and spinal cord tissues. Moreover, we discovered distinct sets of genes with significant differential alternative splicing or differential transcript usage in each tissue, which were largely not detected by conventional differential gene expression analysis approaches. Based on these results, our study provided a more accurate and comprehensive depiction of transcriptomic alterations related to gout inflammation and pain.

A novel synergistic device for joint inflammation – efficacy on ankle sprain cases

Joint inflammation results from soft-tissue injuries and cartilage damage. PRICE is the standard treatment approach for acute soft tissue injuries like ankle sprain. Electrical stimulation, application of orthotic braces, etc. is also effective for this. In a synergistic device all these components are combined and applied simultaneously. This device was developed for joint inflammation and tested for grade I & II acute ankle joint sprain. To test a synergistic – semirigid device, combining PRICE & electrical stimulation for acute ankle sprains of grades I & II for pain, range of motion and swelling is a case series was the objective. Device was developed using novel concept of synergistic applications of PRICE with electrical stimulation. The joint contour of ankle and specific biomechanical bony surface landmarks were considered. Ethical approval was taken from NTCC committee, AIPT. Recordings were taken from eight patients of acute ankle sprain with – in two days of injury, after getting ethical approval. Elevation to the ankle was provided by keeping the part over the pillow and data was recorded with the help of: 1.VAS scale for pain; 2. Measuring tape; 3. Goniometry. t-test was used to find out the significant difference pre and post the application of device. There was a significant reduction in pain (p = 0.006), edema (p = 0.011), dorsi-flexion (p = 0.015), and plantar flexion (p =0.008). The synegistic device has been effective for acute ankle inflammation - grade I & II ankle sprains in 5 sessions; sufficient for the return of function.

The real-time detection of acupuncture-induced extracellular ATP mobilization in acupoints and exploration of its role in acupuncture analgesia.

Our and in vitro studies had confirmed that mechanosensitive ATP release and accumulation in acupoints was elicited by acupuncture (AP), which might be a pivotal step for triggering AP analgesia. But to date, the dynamics of extracellular ATP (eATP) in the interstitial space during AP process was poorly known, mainly due to the low temporal resolution of the current detection approach. This study attempted to capture rapid eATP signals in vivo in the process of needling, and further explored the role of this eATP mobilization in initiating AP analgesic effect. Ipsilateral 20-min needling was applied on Zusanli acupoint (ST36) of complete Freund's adjuvant (CFA)-induced ankle arthritis rats. Pain thresholds were assessed in injured-side hindpaws. eATP in the interstitial space was microdialyzed and real-time quantified by luciferin-luciferase assay at 1-min interval with the aid of the microfluid chip. We revealed in behavioral tests that modulation of eATP levels in ST36 influenced AP analgesic effect on ankle arthritis. A transient eATP accumulation was induced by needling that started to mobilize at 4min, climbed to the peak of 11.21nM within 3.25min and gradually recovered. Such AP-induced eATP mobilization was significantly impacted by ankle inflammation, needling depth, needle manipulation, and the presence of local ecto-nucleotidases. This work reveals that needling elicits a transient eATP mobilization in acupoints, which contributes to initiating AP analgesia. This study will help us better understand the peripheral mechanism of AP analgesia and guide clinicians to optimize the needle manipulations to improve AP efficacy.

Anti-arthritic and anti-inflammatory effects of Baccharis conferta Kunth in a kaolin/carrageenan-induced monoarthritis model

Ethnopharmacological relevancePopularly known as “escoba” (broom) or “escobilla china” (Chinese brush), Baccharis conferta Kunth (Asteraceae), is a plant widely used in Mexican folk medicine for alleviating muscular and rheumatic pain. A recent study described that dichloromethane extract as well as fractions and isolated compounds, possess anti-inflammatory activity in TPA-induced acute edema. Aim of the studyBased on the popular medicinal uses of B. conferta as well as previous studies on its anti-inflammatory activity, the aim of this research was to evaluate the anti-arthritic and anti-inflammatory effects of dichloromethane extract, fractions, and compounds from B. conferta in a monoarthritis model induced with kaolin/carrageenan (K/C). Materials and methodsAerial parts of B. conferta were collected, dried, and macerated with dichloromethane. The dichloromethane extract (BcD) was separated by open column chromatography to obtain the BcD2 fraction where the diterpene kingidiol (KIN) was isolated and from the BcD3 fraction the flavonoid cirsimaritin (CIR), which are the most active compounds in the TPA model. In addition, the flavonoids acacetin, pectolinaringenin and 6-methoxykaempferide were identified and isolated from the BcD2 fraction. The content of the main compounds was estimated in BcD, BcD2 and BcD3. The anti-arthritic and anti-inflammatory effects of B. conferta were investigated by evaluating ankle joint inflammation, hyperalgesia using the hot plate test, and pro- and anti-inflammatory cytokine levels in the synovial capsule as well as histological changes in ankle joint tissue in a monoarthritis model induced with K/C in Balb/c mice. ResultsOral administration of BcD2 fraction (25 mg/kg) and KIN (10 mg/kg) reduced the ankle thickness induced by K/C and decreased the levels of TNF-α, IL-1β, IL-6 and IL-17, while BcD2 increased IL-10. In addition, BcD2 and KIN showed significant edema attenuation of the synovial membrane and decreased inflammatory infiltration and cartilage erosion compared to the VEH group. Finally, BcD (50 mg/kg), KIN (10 mg/kg) and CIR (5 mg/kg) decreased hyperalgesia. ConclusionsB. conferta constitutes a therapeutic or preventive candidate for osteoarthritis, because of decreased articular inflammation and pain accompanied with the modulation of cytokine concentrations, which confirms the anti-arthritic and anti-inflammatory activities of B. conferta and support its popular use.

β-arrestin-2 alleviates rheumatoid arthritis injury by suppressing NLRP3 inflammasome activation and NF- κB pathway in macrophages

ABSTRACT Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder that inflicts damage to the joints of the hands and wrist. The aim of this study was to investigate the protective effect of β-Arrestin-2 (βArr2) on RA in vivo and in vitro. The βArr2 adenovirus (βArr2-Ad) or the control (Con-Ad) was injected into the ankle joint cavity of collagen-induced arthritis (CIA) mice. According to the results, an improvement was shown in the symptoms and pathological injury of RA after an upregulation of βArr2. Correspondingly, the inflammatory response was attenuated, as evidenced by the decreased serum pro-inflammatory cytokines levels and NF-κB pathway-related proteins. Nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome activation was inhibited in CIA mice treated with βArr2-Ad injection, as reflected by the diminished IL-18 level and declined protein levels of inflammasome components in the ankle joint. Likewise, the anti-inflammatory effect of macrophages was also validated by in vitro experiments. In summary, βArr2 effectively ameliorates ankle inflammation in CIA mice via NF-κB/NLRP3 inflammasome, providing theoretical and clinical basis for RA therapy.

P17 A case of treatment resistant severe destructive arthritis in a young boy

Case report - IntroductionMajor advances in medical and therapy management of inflammatory arthritis has been associated with marked improvement in outcomes. We present a very unusual case of a young boy with destructive and incapacitating arthritis of knees, feet and ankles refractory to such management. He is now wheelchair dependent. We have evidence of reversal of bone loss after treatment. However, synovitis and joint destruction has left few surgical options with above knee amputation considered to have the best long-term functional outcome.Case report - Case descriptionAge five, he had persistent pain and swelling of the right knee after a minor fall. In the emergency department a sclerotic, lucent area was highlighted on X-ray. Neoplasia was ruled out and the changes were felt to be a result of erosive arthropathy.By the time of rheumatology referral, symptoms progressed to significant bilateral knee effusions with left ankle inflammation. All three joints underwent steroid injections which had a good response, but unfortunately this was short-lived and they were repeated. A synovial biopsy was sent to exclude other pathologies and loose bone fragments were removed from both knees. Biopsy confirmed chronic inflammation and methotrexate was commenced. Unfortunately, there was minimal response.Over the next 5 years he trialled etanercept, infliximab, tocilizumab, pamidronate, azathioprine and abatacept. His destructive arthritis of the knees, ankles and feet continued to progress with relentless bone loss and associated osteochondritis dissecans (OCD).Opinions from five other tertiary rheumatology units worldwide, three radiology departments and four orthopaedic units were sought. Unable to control the synovitis and in the presence of complete loss of the lateral femoral condyle we elected to refocus our attention to bone integrity and preparation for definitive surgery. This included the use of denosumab. Serial radiology showed reversal of some bone loss over 2 years but surgical teams could not identify intervention to optimise weight-bearing capacity. Mobility deteriorated to leave our patient wheelchair dependent outside, using crutches or his knees to move around inside. Further surgical consideration has indicated the best long-term will be associated with above knee amputations.Case report - DiscussionOur team are aware of one less severe case in a 13-year-old male in Oxford that stabilised after 5 years, allowing definitive treatment with bilateral knee replacement followed by excellent recovery with full mobility. None of the international centres we contacted reported a case as severe as ours. From discussions with our paediatric and adult orthopaedic colleagues, the only comparable patients they have seen this level of bony knee destruction in has been uncontrolled haemophilia patients. However, the damage is not seen at such a young age which affects the surgical options due to ongoing growth in childhood and adolescence. This caused us to question; why was our patient so severely affected? Would a different treatment course have been feasible? And would this have changed the outcome?Despite trial of many immunomodulatory agents, both those routinely used for arthritis and those used in treatment resistance, the persistent inflammation and destruction progressed. As a result he developed persistent and problematic OCD. OCD is considered a relatively common diagnosis that can occur in adults and children of unclear aetiology. A small subchondral bone fragment becomes loose in the joint space as a result of disrupted blood supply. OCD seems to be more common in males and is often managed conservatively. In children, the knee is the most commonly affected joint and inflammation is hypothesised as a possible cause which we will elaborate on in our presentation. For cases requiring surgical intervention this is normally localised drilling, fixation or bone grafts; however, most cases only affect one site, with one loose fragment which was very different from our patient.Case report - Key learning pointsFrom this case we have been reminded that each patient follows a different clinical course and response to medications is difficult to predict. We have gained experience managing difficult inflammatory arthritis-related OCD working closely with our orthopaedic colleagues. Although we managed to control the destructive inflammatory process this did not reverse the damage to the patient’s joints and we have been left with no choice but to progress to surgical treatment options.By sharing this case we hope to learn from any similar experiences or challenges faced by other clinical teams to help inform our practice for any other cases in the future.

Treatment of collagen-induced arthritis rat model by using Notch signalling inhibitor.

BackgroundThe Notch signalling pathway has been reported to play a key role in rheumatoid arthritis (RA) development. Thus, inhibition of the activation of this signalling pathway may be a promising approach to the treatment of RA. In this study, the Notch signalling inhibitor LY411575, which can inhibit both Notch1 and Notch3, was used for the treatment of collagen-induced arthritis (CIA) rats.MethodsWistar rats were immunised with bovine type II collagen (CII) to establish rats CIA model. The inhibitory effects of LY411575 on Notch1 intracellular domain (N1ICD) and Notch3 intracellular domain (N3ICD) protein was verified by western blot (WB) in vitro. CIA rats were treated with different doses of LY411575 for 15 and 28 days, respectively. Methotrexate and sodium carboxymethyl cellulose (CMC-Na) were used as positive and negative (vehicle) control respectively. Destruction of the rat ankle joint and the bone loss on the periarticular side were evaluated by micro-computed tomography (Micro-CT). In addition, destruction of the ankle articular cartilage and the osteoclast numbers were determined by histology. Expression of N1ICD and N3ICD in the ankle joint was detected by immunohistochemistry.ResultsLY411575 could significantly inhibit the expression of N1ICD and N3ICD in vitro. Micro-CT test showed that the ankle joint destruction significantly improved after treatment with LY411575 (5 ​mg/kg and 10 ​mg/kg, respectively). The bone quality in the LY411575 (5 ​mg/kg and 10 ​mg/kg, respectively) groups were improved compared with the vehicle group. Histological analysis showed that LY411575 (5 ​mg/kg and 10 ​mg/kg, respectively) treatment reduced the severity of ankle joint inflammation in CIA rats (including ankle joint destruction, pannus formation, and cartilage damage) and reduced the expression of N1ICD and N3ICD in CIA rats ankle joints significantly.ConclusionThe inhibitor of Notch signalling LY411575 is an effective treatment for CIA.The translational potential of this articleOur study provides new evidence to support the potential clinical application of Notch signalling pathway inhibitor LY411575 as a drug candidate for the treatment of RA.

IL-12p40/IL-23p40 Blockade With Ustekinumab Decreases the Synovial Inflammatory Infiltrate Through Modulation of Multiple Signaling Pathways Including MAPK-ERK and Wnt.

Background: Psoriatic arthritis (PsA) is a chronic inflammatory joint disease within the spondyloarthritis spectrum. IL-12p40/IL-23p40 blockade reduces PsA disease activity, but its impact on synovial inflammation remains unclear.Objectives: To investigate the cellular and molecular pathways affected by IL-12p40/IL-23p40 blockade with ustekinumab in the synovium of PsA patients.Methods: Eleven PsA patients with at least one inflamed knee or ankle joint were included in a 24-week single-center open-label study and received ustekinumab 45 mg/sc according to standard care at week 0, 4, and 16. Besides clinical outcomes, synovial tissue (ST) samples were obtained by needle arthroscopy from an inflamed knee or ankle joint at baseline, week 12 and 24 and analyzed by immunohistochemistry, RNA-sequencing and real-time quantitative polymerase chain reaction (qPCR).Results: We obtained paired baseline and week 12, and paired baseline, week 12 and 24 ST samples from nine and six patients, respectively. Eight patients completed 24 weeks of clinical follow-up. At 12 weeks 6/11 patients met ACR20, 2/11 met ACR50 and 1/11 met ACR70 improvement criteria, at 24 weeks this was 3/8, 2/8 and 1/8 patients, respectively. Clinical and serological markers improved significantly. No serious adverse events occurred. We observed numerical decreases of all infiltrating cell subtypes at week 12, reaching statistical significance for CD68+ sublining macrophages. For some cell types this was even more pronounced at week 24, but clearly synovial inflammation was incompletely resolved. IL-17A and F, TNF, IL-6, IL-8, and IL-12p40 were not significantly downregulated in qPCR analysis of W12 total biopsies, only MMP3 and IL-23p19 were significantly decreased. RNA-seq analysis revealed 178 significantly differentially expressed genes between baseline and 12 weeks (FDR 0.1). Gene Ontology and KEGG terms enrichment analyses identified overrepresentation of biological processes as response to reactive oxygen species, chemotaxis, migration and angiogenesis as well as MAPK-ERK and PI3K-Akt signaling pathways among the downregulated genes and of Wnt signaling pathway among the upregulated genes. Furthermore, ACR20 responders and non-responders differed strikingly in gene expression profiles in a post-hoc exploratory analysis.Conclusions: Ustekinumab suppresses PsA synovial inflammation through modulation of multiple signal transduction pathways, including MAPK-ERK, Wnt and potentially PI3K-Akt signaling rather than by directly impacting the IL-17 pathway.

ST36 Acupuncture Alleviates the Inflammation of Adjuvant-Induced Arthritic Rats by Targeting Monocyte/Macrophage Modulation.

Background Rheumatoid arthritis (RA) is a chronic systemic chronic autoimmune disease characterized by the aggregation of immune cells and secretion of cytokines in the joint synovium, causing hyperblastosis and even bone destruction. Acupuncture has been proven effective in RA treatment. This study aimed to investigate the anti-inflammatory action of acupuncture, specifically, in relation to immune cell interactions and key mediators. Methods Rats with adjuvant-induced arthritics (AIA) were treated with manual acupuncture (MA) at Zusanli (ST36). Joint edema and paw withdrawal latency were monitored to observe the effects on inflammation. The levels of 24 cytokines, chemokines, and growth factors in ankle joints during the treatment (on days 1, 7, 15, and 21) were detected by multiplex immunoassay. A bioinformatics analysis based on a directed weighted mathematical model was used to construct cell communication network diagrams and identify the key cells through calculation. The monocyte/macrophage polarization in inflamed joints was investigated by detecting M1- and M2-phenotypic populations and their related cytokines. Results ST36 MA alleviated paw edema and upregulated the nociceptive threshold of AIA rats. Several innate and adaptive immune cytokines were dynamically regulated by MA, and MA-treated rats showed a significant improvement in symptoms compared with AIA rats by day 21. The immune cell-cell communication networks were intensified with the development of RA but were significantly reduced after treatment with MA. MA was found to specifically regulate monocytes/macrophages in inflamed ankle joints ST36 MA also inhibited M1-phenotype macrophages accompanied by decreased levels of IL-1β. Conclusions ST36 MA showed anti-inflammatory and analgesic effects as well as inhibition of immune cell communication networks in inflamed joints of AIA rats. Inhibiting the polarization of macrophages to the M1-phenotype in inflamed joints may be one of the key mechanisms of MA anti-inflammatory action. This research highlighted a systematic research paradigm for investigating mechanisms of acupuncture action.

Chemical characterization and anti-arthritic appraisal of Monotheca buxifolia methanolic extract in Complete Freund's Adjuvant-induced arthritis in Wistar rats.

The current study was designed to evaluate the anti-oxidant and anti-arthritic potential of a traditionally used herb, Monotheca buxifolia. The M. buxifolia methanolic extract (MBME) was prepared from the aerial parts of the plant followed by chemical characterization with GC-MS. The anti-oxidant potential of the MBME was demonstrated by DPPH scavenging activity. The effects of MBME on protein denaturation and membrane stabilization were determined by inhibition of egg albumin denaturation and RBC membrane stabilization assays, respectively. The in vivo anti-arthritic potential of the MBME at 50, 100, and 150mg/kg/day was evaluated in Complete Freund's Adjuvant-induced polyarthritis in Wistar rats treated for 21days. Phytochemicals, such as linolenic acid methyl ester, n-hexadecanoic acid, vitamin E, α-amyrin, and β-amyrin were detected in the GC-MS analysis. The plant extract exhibited a 55.20 ± 0.69% scavenging of free radicals at 100µg/ml concentration. It significantly (p < 0.05) stabilized human RBC membrane (65.06 ± 0.22%) and inhibited protein denaturation (70.53 ± 0.34%) at 100mg/ml concentration. The diclofenac sodium (DS) and MBME at 150,100, and 50mg/kg reduced the paw edema, restored the body weight, and altered blood parameters including CRP. The MBME significantly reduced the MDA and increased the SOD, CAT, and GSH levels in liver tissue homogenate in treated rats. The serum concentration of TNF-α and PGE2 was remarkably (p < 0.01-< 0.0001) restored by the DS and MBME dose dependently. The histopathological study showed that MBME 150mg/kg commendably restored the ankle joint inflammation, bone erosion, and cartilage damage in polyarthritic rats. It was concluded that the anti-oxidant, anti-inflammatory and anti-arthritic effects of MBME might be attributed to phenols, flavonoids, triterpenoids, vitamin E, phytol, and other fatty acids. This study showed the anti-arthritic potential of Monotheca buxifolia and thus validates its traditional claim.