Ankle Joints Of Mice Research Articles

PFKFB3 decreases α-ketoglutarate production while partial PFKFB3 knockdown in macrophages ameliorates arthritis in tumor necrosis factor-transgenic mice.

Aberrant 6-phosphofructo-2kinase/fructose-2,6-bisphoshatase 3 (PFKFB3) expression is tightly correlated with multiple steps of tumorigenesis; however, the pathological significance of PFKFB3 in macrophages in patients with rheumatoid arthritis (RA) remains obscure. In this study, we examined whether PFKFB3 modulates macrophage activation and promotes RA development. Peripheral blood mononuclear cells (PBMCs) from patients with RA, THP-1 cells, and bone marrow-derived macrophages from conditional PFKFB3-knockout mice were used to investigate the mechanism underlying PFKFB3-induced macrophage regulation of RA. We demonstrated that patients with RA have higher PFKFB3 levels than healthy volunteers. PFKFB3 silencing suppressed M1 macrophage polarization and downregulated IL-1β, CD80, IFIT1, CCL8, and CXCL10 in macrophages of patients with RA. PFKFB3 overexpression markedly upregulated IRF5, HIF1α, IL-1β, CD80, IFI27, IFI44, IFIT1, IFIT3, CCL2, CCL8, CXCL10, CXCL11, and MMP13 in phorbol 12-myristate 13-acetate-induced THP-1 cells, although these changes were partially reversed by PFK15, an inhibitor of PFKFB3 enzyme activity. Co-immunoprecipitation assays revealed that PFKFB3 interacted with GLUD1 and decreased glutamate dehydrogenase (GDH) activity and α-ketoglutarate production. PFKFB3, TNFα, IL-6, IFNγ, CXCL9, CXCL10, CXCL11, MMP13, and MMP19 were downregulated in bone marrow-derived macrophages of conditional PFKFB3-knockout mice relative to those of wild-type mice. Partial PFKFB3 knockdown in macrophages ameliorated the clinical signs of arthritis and bone destruction, inhibited proinflammatory factor expression, and promoted GDH activity and α-ketoglutarate production in tumor necrosis factor-transgenic mice. Single-cell sequencing revealed that macrophages were the most abundant cells in the ankles of arthritic mice, and partial PFKFB3 knockdown promoted M2-like polarization and was correlated with TREM2, SPP1, APOE, and C1Q expression. PFKFB3 is upregulated in macrophages in patients with RA. PFKFB3 aggravates arthritis by modulating macrophage activity, which may be related to decreased α-ketoglutarate production.

Voluntary exercise in mice triggers an anti-osteogenic and pro-tenogenic response in the ankle joint without affecting long bones

Biomechanical stimulation is proposed to occupy a central place in joint homeostasis, but the precise contribution of exercise remains elusive. We aimed to characterize in vivo the impact of mechanical stimulation on the cell-controlled regulation of ossification within the ankles of healthy mice undergoing mild physical activity. DBA/1 male mice were subjected to voluntary running exercise for two weeks, and compared to mice housed in standard conditions (n = 20 per group). Free access to activity wheels resulted in a running exercise of 5.5 ± 0.8 km/day at 14.5 ± 0.5 m/min. Serum levels of alkaline phosphatase, IL-6, IL-8/Kc, IL-17a, and TNF-α were measured. No change in systemic inflammation was detected. The bone architecture of the femur and the calcaneus was unchanged, as revealed by μCT and histology of the enthesis of the Achilles tendon. mRNAs were extracted from femurs, tibias, and ankle joints before RT-qPCR analysis. The expression of the mechanosensitive genes Sclerostin (Sost) and Periostin (Postn) was not impacted by the exercise in long bones. Oppositely, Sost and Postn levels were modulated by exercise in joints, and osteogenic markers (Col10a1, Runx2, Osx, and Dmp1) were downregulated in the exercise group. In addition, the tenogenic markers Scx, Mkx, and Tnmd were upregulated by exercise. Thus, voluntary exercise affected the phenotype of joint cells without impacting long bones. As gene expression of Bmp2, Bmp4, and Id1 was also reduced in these cells, an off-regulation of BMP signaling could be partly responsible for their mechanosensitive response. Running exercise seemed to preserve the tendon from its progressive ossification, as seen in numerous enthesopathies. This study paves the way to future experiments for investigating the effects of mechanical stimulation in various mouse models.

Columbianadin ameliorates rheumatoid arthritis by attenuating synoviocyte hyperplasia through targeted vimentin to inhibit the VAV2/Rac-1 signaling pathway

IntroductionRheumatoid arthritis (RA) is an autoimmune disease pathologically characterized by synovial inflammation. The abnormal activation of synoviocytes seems to accompany the progression of RA. The role and exact molecular mechanism in RA of columbianadin (CBN) which is a natural coumarin is still unclear. ObjectivesThe present research aimed to investigate the effect of vimentin on the abnormal growth characteristics of RA synoviocytes and the targeted regulatory role of CBN. MethodsCell migration and invasion were detected using the wound healing and transwell method. Mechanistically, the direct molecular targets of CBN were screened and identified by activity-based protein profiling. The expression of relevant proteins and mRNA in cells and mouse synovium was detected by western blotting and qRT-PCR. Changes in the degree of paw swelling and body weight of mice were recorded. H&E staining, toluidine blue staining, and micro-CT were used to visualize the degree of pathological damage in the ankle joints of mice. Small interfering RNA and plasmid overexpression of vimentin were used to observe their effects on MH7A cell proliferation, migration, apoptosis, and downstream molecular signaling. ResultsThe TNF-α-induced proliferation and migration of MH7A cells could be significantly repressed by CBN (25,50 μM), and the expression of apoptosis and autophagy-associated proteins could be modulated. Furthermore, CBN could directly bind to vimentin and inhibit its expression and function in synoviocytes, thereby ameliorating foot and paw swelling and joint damage in CIA mice. Silencing and overexpression of vimentin might be involved in developing RA synovial hyperplasia and invasive cartilage by activating VAV2 phosphorylation-mediated expression of Rac-1, which affects abnormal growth characteristics, such as synoviocyte invasion and migration. ConclusionCBN-targeted vimentin restrains the overactivation of RA synoviocytes thereby delaying the pathological process in CIA mice, which provides valuable targets and insights for understanding the pathological mechanisms of RA synovial hyperplasia.

Effects of chronic ankle instability after grade I ankle sprain on the post-traumatic osteoarthritis

BackgroundUntreated acute ankle sprains often result in chronic ankle instability (CAI) and can ultimately lead to the development of post-traumatic osteoarthritis (PTOA). At present, a typical animal model of ankle instability in mice is established by transecting the ligaments around the ankle joint. This study aimed to establish a grade I acute ankle sprain animal model by rapid stretching of peri-ankle joint ligaments. Furthermore, we tried to explore the pathophysiological mechanism of ankle osteoarthritis.MethodsIn all, 18 male C57BL/6 J mice (7 weeks) were randomly divided into three groups: calcaneofibular ligament (CFL) laxity group, deltoid ligament (DL) laxity group, and SHAM group. One week after the surgical procedure, all mice were trained to run in the mouse rotation fatigue machine daily. The mice were tested on the balance beam before surgery and three days, 4 weeks, 8 weeks, and 12 weeks after surgery. Footprint analyses were performed on each mouse before surgery and 12 weeks after surgery. Micro-CT scanning was then performed to evaluate the degeneration of ankle joints and histological staining was performed to analyze and evaluate PTOA caused by ankle joint instability.ResultsAfter surgery, the mice in the CFL and DL laxity groups took longer to cross the balance beam and slipped more often than those in the SHAM group (p < 0.05). The step length and width in the CFL and DL laxity groups were significantly shorter and smaller than those in the SHAM group 12 weeks after surgery (p < 0.05). There was a significant increase in the bone volume fraction (BV/TV) in the CFL and DL laxity groups compared with the SHAM group (p < 0.05). Histological staining results suggested obvious signs of PTOA in the CFL and DL laxity groups.ConclusionsBased on CFL and DL laxity in a mouse ankle instability model, this study suggests that grade I ankle sprain can contribute to chronic ankle instability, impair motor coordination and balance, and eventually lead to PTOA of ankle with significant degeneration of its adjacent joints.

Research on preventive effect of Akkermansia muciniphilaAKKPROBIO on acute gouty arthritis in mice.

In mice with acute gouty arthritis, this study intends to examine the mechanism of action of Akkermansia muciniphila AKK PROBIO. We developed a mouse model of acute gouty arthritis using sodium urate. The efficiency and mechanism of AKK PROBIO in preventing acute gouty arthritis in mice were then determined by examining the degree of foot swelling, pain threshold, blood biochemical indicators, histological alterations, and messenger RNA (mRNA) expression changes. The results of the animal experiment showed that AKK PROBIO can lessen mouse foot edema severity and increase pain threshold. AKK PROBIO can enhance the enzyme activity of superoxide dismutase (SOD) and the level of glutathione (GSH) in the ankle joint tissues of mice with acute arthritis while decreasing the enzyme activity of myeloperoxidase (MPO) and the level of malondialdehyde (MDA). Interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels are all reduced by AKK PROBIO in the blood of mice with acute arthritis. Results from histopathology showed that AKK PROBIO reduced tissue damage in the mouse ankle and foot joints. In the tissues of the ankle joints of mice with acute arthritis, the results of the quantitative polymerase chain reaction (qPCR) and Western blot experiments suggested that AKK PROBIO may inhibit the mRNA and protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) in the tissues. AKK PROBIO can also regulate gut microbiota, inhibit harmful bacteria, and enhance valeric acid in the intestine, isobutyric acid, and isovaleric acid. Therefore, it is evident that AKK PROBIO prevents acute gouty arthritis better than glucosamine sulfate. It is a strain that has probiotic potential.

Analgesic Effect of SKI306X on Chronic Postischemic Pain and Spinal Nerve Ligation-Induced Neuropathic Pain in Mice.

Neuropathic pain (NP) results from lesions or diseases affecting the peripheral or central somatosensory system. However, there are currently no drugs that are particularly effective in treating this condition. SKI306X is a blend of purified extracts of three oriental herbs (Clematis mandshurica, Trichosanthes kirilowii, and Prunella vulgaris) commonly used to treat osteoarthritis for their chondroprotective effects. Chronic postischemic pain (CPIP) and spinal nerve ligation (SNL) models were created by binding the upper left ankle of mice with an O-ring for 3 h and ligating the L5 spinal nerve, respectively. Mice with allodynia were injected intraperitoneally with 0.9% normal saline (NS group) or different doses (25, 50, or 100 mg/kg) of SKI306X (SKI groups). We assessed allodynia using von Frey filaments before injection and 30, 60, 90, 120, 180, and 240 min and 24 h after injection to confirm the antiallodynic effect of SKI306X. We also measured glial fibrillary acidic protein (GFAP) levels in the spinal cord and dorsal root ganglia to confirm the change of SKI306X administration. Both models exhibited significant mechanical allodynia. The intraperitoneal injection of SKI306X significantly increased the paw withdrawal threshold in a dose-dependent manner, as the paw withdrawal threshold was significantly increased after SKI306X administration compared with at baseline or after NS administration. GFAP levels in the SKI group decreased significantly (p < 0.05). Intraperitoneal administration of SKI306X dose-dependently attenuated mechanical allodynia and decreased GFAP levels, suggesting that GFAP is involved in the antiallodynic effect of SKI306X in mice with CPIP and SNL-induced NP.

Preventive effects of Chinese Xinjiang naturally fermented yogurt separated from Lactobacillus rhamnosusAFY02 on acute gouty arthritis in mice.

Lactobacillus rhamnosus AFY02 (LR-AFY02) is a newly discovered strain isolated and identified from naturally fermented yogurt in Xinjiang, China. This research aims to investigate the mechanism of action of LR-AFY02 in mice with acute gouty arthritis. We examined the degree of foot swelling, pain threshold, blood biochemical indicators, histopathological changes, and mRNA expression. LR-AFY02 can decrease the severity of mouse foot edema and raise the pain threshold. LR-AFY02 can increase the enzyme activity of superoxide dismutase (SOD) and the level of glutathione (GSH) while lowering the enzyme activity of myeloperoxidase (MPO) and the level of malondialdehyde (MDA) in mice with acute arthritis. Interleukin-6 (IL-6), IL-10, interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels in the blood of mice with acute arthritis are also decreased by LR-AFY02. Histopathological findings demonstrated that LR-AFY02 reduced tissue damage in the mouse foot and ankle joints. LR-AFY02 may suppress the mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), IL-6, interferon gamma (IFN-γ), and TNF-α in the tissues of the ankle joints of mice with acute arthritis. Additionally, LR-AFY02 has the ability to increase the expression of catalase (CAT), manganese superoxide dismutase (Mn-SOD), and copper/zinc superoxide dismutase (Cu/Zn-SOD). As a result, it is clear that L. rhamnosus AFY02 is more effective than glucosamine sulfate at preventing acute gouty arthritis.

Honokiol alleviates monosodium urate-induced gouty pain by inhibiting voltage-gated proton channels in mice.

To investigate whether honokiol (HNK) acted as an analgesic in connection with inhibiting the voltage-gated proton channel (Hv1). The model of gouty arthritis was induced by injecting monosodium urate (MSU) crystals into the hind ankle joint of mice. HNK was given by intragastric administration. Ankle swelling degree and mechanical allodynia were evaluated using ankle joint circumference measurement and von Frey filaments, respectively. Hv1 current, tail current, and action potential in dorsal root ganglion (DRG) neurons were recorded with patch-clamp techniques. HNK (10, 20, 40mg/kg) alleviated inflammatory response and mechanical allodynia in a dose-dependent manner. In normal DRG neurons, 50µM Zn2+ or 2-GBI significantly inhibited the Hv1 current and the current density of Hv1 increased with increasing pH gradient. The amplitude of Hv1 current significantly increased on the 3rd after MSU treatment, and HNK dose-dependently reversed the upregulation of Hv1 current. Compared with MSU group, 40mg/kg HNK shifted the activation curve to the direction of more positive voltage and increased reversal potential to the normal level. In addition, 40mg/kg HNK reversed the down-regulation of tail current deactivation time constant (τtail) but did not alter the neuronal excitability of DRG neurons in gouty mice. HNK may be a potential analgesic by inhibiting Hv1 current.

Hypertonic saline- and detergent-accelerated EDTA-based decalcification better preserves mRNA of bones

Ethylenediaminetetraacetic acid (EDTA), a classically used chelating agent of decalcification, maintains good morphological details, but its slow decalcification limits its wider applications. Many procedures have been reported to accelerate EDTA-based decalcification, involving temperature, concentration, sonication, agitation, vacuum, microwave, or combination. However, these procedures, concentrating on purely tissue-outside physical factors to increase the chemical diffusion, do not enable EDTA to exert its full capacity due to tissue intrinsic chemical resistances around the diffusion passage. The resistances, such as tissue inner lipids and electric charges, impede the penetration of EDTA. We hypothesized that delipidation and shielding electric charges would accelerate EDTA-based penetration and the subsequent decalcification. The hypothesis was verified by the observation of speedy penetration of EDTA with additives of detergents and hypertonic saline, testing on tissue-mimicking gels of collagen and adult mouse bones. Using a 26% EDTA mixture with the additives at 45°C, a conventional 7-day decalcification of adult mouse ankle joints could be completed within 24 h while the tissue morphological structure, antigenicity, enzymes, and DNA were well preserved, and mRNA better retained compared to using 15% EDTA at room temperature. The addition of hypertonic saline and detergents to EDTA decalcification is a simple, rapid, and inexpensive method that doesn't disrupt the current histological workflow. This method is equally or even more effective than the currently most used decalcification methods in preserving the morphological details of tissues. It can be highly beneficial for the related community.

CD8 T cell-derived perforin regulates macrophage-mediated inflammation in a murine model of gout.

Gout is characterized by hyperuricemia and recurrent inflammatory episodes caused by intra-articular crystal deposition of monosodium urate (MSU). There is a clear relationship between gout and metabolic syndrome. Recent evidence indicates that perforin plays a role in regulating glucose homeostasis and provides protection in diet-induced non-alcoholic steatohepatitis models. However, the impact of perforin on immune inflammation in gout remains unclear. We induced acute gout models in both wild-type (WT) mice and Prf1null mice by administering intra-articular injections of MSU crystals. We compared the ankle joint swelling and the histological score between the two groups. Furthermore, we investigated underlying mechanisms through in vitro co-culture experiments involving CD8 T cells and macrophages. In this study, Prf1null mice showed significantly more pronounced ankle swelling with increased inflammatory cell infiltrations compared with WT mice 24h after local MSU injection. Moreover, MSU-induced Prf1null mice exhibited increased accumulation of CD8 T cells but not NK cells. Perforin-deficient CD8 T cells displayed reduced cytotoxicity towards bone marrow-derived M0 and M1 macrophages and promoted TNF-α secretion from macrophage. Perforin from CD8 T cells limits joint inflammation in mice with acute gout by downregulating macrophage-mediated inflammation. Key Points • Perforin deficiency increased swelling in the ankle joints of mice upon MSU injection. • Perforin deficiency is associated with increased immune cell recruitment and severe joint damage in gout. • Perforin regulated CD8 T cell accumulation in gout and promoted CD8 T cell cytotoxicity towards M0 and M1 macrophages. • CD8 T cell-derived perforin regulated pro-inflammatory cytokine secretion of macrophage.

Novel mechanistic study of HDAC6 regulation of rheumatoid arthritis via CMA: exploring potential therapeutic targets.

Rheumatoid arthritis (RA) is a systemic autoimmune disease. Its pathogenesis has not yet been clarified, so it is urgent to explore therapeutic targets. Here, we clarified the role of HDAC6 in the mechanism of action of RA through mediating chaperone-mediated autophagy (CMA) to provide a clinical treatment of RA. We used rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and collagen-induced arthritis mice (CIA mice) as models of RA and pharmacological inhibitors as well as genetic interference with adeno-associated viruses to reduce the expression of HDAC6. We explored the influence of CAY10603 on RA-FLS proliferation and inflammation, as well as the expression of proteins related to the CMA signaling pathway. CIA model was constructed using DBA/1J mice. Arthritis symptoms in CIA mice were evaluated, and the expression and localization of CMA-related proteins in mouse ankle joints were examined. CAY10603 inhibited proliferation as well as the level of the molecular chaperone autophagy in RA-FLS. HDAC6 shRNA significantly reduced the clinical signs of arthritis in CIA mice, as did the expression of HDAC6 in the serum and ankle synovial tissues of CIA mice. Finally, it significantly inhibited the level of Hsc70 and LAMP-2A, which are involved in the CMA signaling pathway, in ankle joint tissues. Downregulation of HDAC6 may inhibit CMA and thereby ameliorate RA.

Identification of citrullinated α1-antitrypsin (A1AT) in saliva in a mouse model of rheumatoid arthritis

ObjectivesRheumatoid arthritis (RA) is an autoimmune disease characterized by progressive joint destruction. Early diagnosis and treatment, before joint deformation or destruction occurs, are crucial. Identifying novel biomarkers for RA in saliva could potentially enable early detection of the disease, prior to its onset. MethodsWe conducted a comprehensive proteomic analysis of salivary proteins in a mouse model of RA. Proteins were identified using western blotting and enzyme-linked immunosorbent assay in the serum, saliva, and ankle joints of DBA/1JJmsSlc mice, a model of RA. Ankle joints and submandibular glands were stained with hematoxylin and eosin and immunostained, and the results were compared with those of control mice. ResultsCitrullinated alpha-1 antitrypsin (A1AT, 46 kDa) was commonly detected in the saliva, serum, and ankle joints of mice with severe RA and was confirmed through proteomic analysis. Western blotting showed a band corresponding to 46 kDa in the serum, saliva, and ankle joints. Immunostaining of the ankle joints with the A1AT antibody showed a strong positive signal in the synovium. ConclusionsIn DBA/1JJmsSlc mice, cyclic citrullinated peptide antibodies and A1AT may be involved in citrullination and contribute to the development and severity of RA, making them valuable treatment targets requiring further study.

Exploring the efficacy and mechanism of Glycyrrhizae Radix et Rhizoma in improving collagen-induced arthritis in mice

Ethnopharmacological relevanceRheumatoid arthritis (RA), a chronic auto-immune disease, will cause serious joint damage and disability. Glycyrrhizae Radix et Rhizoma (GRR) is commonly included in many anti-RA formulas used in the clinical practice in China. Aim of the studyTo elucidate the alleviation of GRR and its active compounds on RA and the possible engaged mechanism. Materials and methodsThe clinical score, paw swelling degree and pain threshold were detected in the collagen-induced arthritis (CIA) in DBA/1 mice. The ankle joints of mice were observed by using X-Ray, hematoxylin-eosin (H&E), masson's trichrome (Masson), and safranin O and fast green (Safranin O) staining. The potential targets of GRR were predicted by network pharmacology and further verified by using enzyme-linked immunosorbent assay (ELISA) and western-blot. Real-time polymerase chain reaction (Real-time PCR) and wound healing assay were conducted in synovial MH7A cells. The interaction between active compounds and potential targets predicted by molecular docking was confirmed by using cellular thermal shift assay (CETSA). ResultsGRR (615 mg/kg) obviously alleviated CIA in mice. Network pharmacology implied that GRR might affect angiogenesis and inflammation, among which vascular endothelial growth factor-A (VEGF-A), tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), IL-6 and phosphorylated protein kinase B (AKT) might be the key targets involved in this process. GRR decreased AKT phosphorylation and reduced the elevated amounts of TNFα, VEGF-A, IL-1β and IL-6. Next, in vitro results demonstrated that glycyrrhetinic acid (GA) and isoliquiritigenin (ISL) were two active compounds that inhibited TNFα-induced synovial cell angiogenesis and inflammation. Moreover, GA and ISL actually improved RA in CIA mice. The results of molecular docking and CETSA displayed that ISL and GA might interact with TNF receptor-1 (TNFR1), toll-like receptor-4 (TLR4) and VEGF receptor-2 (VEGFR2), thereby contributing to their inhibition on angiogenesis and inflammation. ConclusionGRR and two active compounds, including ISL and GA, alleviated RA via inhibiting angiogenesis and inflammation.

The role of acid-sensing ion channels in monosodium urate-induced gouty pain in mice.

Acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons play an important role in inflammatory pain. The objective of this study is to observe the regulatory role of ASICs in monosodium urate (MSU) crystal-induced gout pain and explore the basis for ASICs in DRG neurons as a target for gout pain treatment. The gout arthritis model was induced by injecting MSU crystals into the ankle joint of mice. The circumference of the ankle joint was used to evaluate the degree of swelling; the von Frey filaments were used to determine the withdrawal threshold of the paw. ASIC currents and action potentials (APs) were recorded by patch clamp technique in DRG neurons. The results displayed that injecting MSU crystals caused ankle edema and mechanical hyperalgesia of the paw, which was relieved after amiloride treatment. The ASIC currents in DRG neurons were increased to a peak on the second day after injecting MSU crystals, which were decreased after amiloride treatment. MSU treatment increased the current density of ASICs in different diameter DRG cells. MSU treatment does not change the characteristics of AP. The results suggest that ASICs in DRG neurons participate in MSU crystal-induced gout pain.

Xanthohumol relieves arthritis pain in mice by suppressing mitochondrial-mediated inflammation.

Chronic pain is the most common symptom for people who suffer from rheumatoid arthritis and it affects approximately 1% of the global population. Neuroinflammation in the spinal cord induces chronic arthritis pain. In this study, a collagen-induced arthritis (CIA) mice model was established through intradermally injection of type II collagen in complete Freund's adjuvant solution. Following CIA inducement, the paws and ankles of mice were found to swell, mechanical pain and spontaneous pain were induced, and their motor coordination was impaired. The spinal inflammatory reaction was triggered, which presented as severe infiltration of inflammatory cells, and the expression levels of GFAP, IL-1β, NLRP3, and cleaved caspase-1 increased. Oxidative stress in the spinal cord of CIA mice was manifested as reduced Nrf2 and NDUFB11 expression and SOD activity, and increased levels of DHODH and Cyto-C. At the same time, spinal AMPK activity was decreased. In order to explore the potential therapeutic options for arthritic pain, Xanthohumol (Xn) was intraperitoneally injected into mice for three consecutive days. Xn treatment was found to reduce the number of spontaneous flinches, in addition to elevating mechanical pain thresholds and increasing latency time. At the same time, Xn treatment in the spinal cord reduced NLRP3 inflammasome-mediated inflammation, increased the Nrf2-mediated antioxidant response, and decreased mitochondrial ROS level. In addition, Xn was found to bind with AMPK via two electrovalent bonds and increased AMPK phosphorylation at Thr174. In summary, the findings indicate that Xn treatment activates AMPK, increases Nrf2-mediated antioxidant response, reduces Drp1-mediated mitochondrial dysfunction, suppresses neuroinflammation, and can serve to relieve arthritis 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.

Triptolide alleviates collagen-induced arthritis in mice by modulating Treg/Th17 imbalance through the JAK/PTEN-STAT3 pathway.

This study aimed to investigate the effects of triptolide (TP) on collagen-induced arthritis (CIA) mice and the related mechanisms. CIA mice were administered TP for 35 days. Mouse ankle joints and serum antibodies and cytokines were examined to assess the therapeutic effects of TP. The ratios of Treg, Th1, and Th17 cells were measured by flow cytometry and RT-qPCR. Reverse docking was used to characterize the binding modes of TP against target proteins. The expression of the STAT3 pathway in CIA mice was evaluated by western blotting and immunofluorescence staining. Mouse spleen lymphocytes were extracted and the expression of the STAT3 pathway after IL-6 stimulation was analyzed. TP could significantly alleviate joint swelling, reduce bone destruction, and downregulate serum inflammation levels. TP improved the imbalance of Treg/Th17 cells in CIA mice. TP could form stable complexes with target proteins. TP significantly inhibited the activation of the JAK/PTEN-STAT3 pathway in mice. Moreover, TP regulated the activation of the JAK1/2-STAT3 signaling pathway in mouse spleen lymphocytes under inflammatory stimulation. TP can inhibit inflammation and alleviate bone destruction in CIA mice. The underlying mechanism is related to the regulation of the imbalance of Treg/Th17 cells through the JAK/PTEN-STAT3 pathway.

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

A scanning focus nuclear microscope with multi-pinhole collimation

Microscopic nuclear imaging down to spatial resolutions of a few hundred microns can already be achieved using low-energy gamma emitters (e.g. 125I, ∼30 keV) and a basic single micro-pinhole gamma camera. This has been applied to in vivo mouse thyroid imaging, for example. For clinically used radionuclides such as 99mTc, this approach fails due to penetration of the higher-energy gamma photons through the pinhole edges. To overcome these resolution degradation effects, we propose a new imaging approach: scanning focus nuclear microscopy (SFNM). We assess SFNM using Monte Carlo simulations for clinically used isotopes. SFNM is based on the use of a 2D scanning stage with a focused multi-pinhole collimator containing 42 pinholes with narrow pinhole aperture opening angles to reduce photon penetration. All projections of different positions are used to iteratively reconstruct a three-dimensional image from which synthetic planar images are generated. SFNM imaging was tested using a digital Derenzo resolution phantom and a mouse ankle joint phantom containing 99mTc (140 keV). The planar images were compared with those obtained using a single-pinhole collimator, either with matched pinhole diameter or with matched sensitivity. The simulation results showed an achievable 99mTc image resolution of 0.04 mm and detailed 99mTc bone images of a mouse ankle with SFNM. SFNM has strong advantages over single-pinhole imaging in terms of spatial resolution.

Modulation of Neutrophil Function by Recombinant Human IgG1 Fc Hexamer in the Endogenous K/BxN Mouse Model of Rheumatoid Arthritis

Introduction: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1β in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. Methods: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1β. Results: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1β was measured in mice treated with rFc-µTP-L309C. Conclusion: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1β production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.