TET1-mediated epigenetic regulation of tumor necrosis factor-α in trigeminal ganglia contributes to chronic temporomandibular joint pain.

Chronic temporomandibular joint (TMJ) pain profoundly affects patients' quality of life. Trigeminal tumor necrosis factor-α (TNFα) plays a pivotal role in mediating TMJ pain in mice, yet the underlying epigenetic mechanisms remain enigmatic. To unravel these epigenetic intricacies, we employed a multifaceted approach. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and chromatin immunoprecipitation (ChIP) followed by qPCR were employed to investigate the demethylation of TNFα gene (Tnfa) and its regulation by ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in a chronic TMJ pain mouse model. The global levels of 5-hydroxymethylcytosine (5hmc) and percentage of 5hmc at the Tnfa promoter region were measured in the trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) following complete Freund's adjuvant (CFA) or saline treatment. TET1 knockdown and pain behavioral testing were conducted to ascertain the role of TET1-mediated epigenetic regulation of TNFα in the pathogenesis of chronic TMJ pain. Our finding revealed an increase in 5hmc at the Tnfa promoter region in both TG and Sp5C of CFA-treated mice. TET1 was upregulated in the mouse TG, and the ChIP result showed TET1 direct binding to the Tnfa promoter, with higher efficiency in the CFA-treated group. Immunofluorescence revealed the predominant expression of TET1 in trigeminal neurons. TET1 knockdown in the TG significantly reversed CFA-induced TNFα upregulation and alleviated chronic TMJ pain. In conclusion, our study implicates TET1 as a vital epigenetic regulator contributing to chronic inflammatory TMJ pain via trigeminal TNFα signaling. Targeting TET1 holds promise for epigenetic interventions in TMJ pain management.

BMP-induced non-canonical signaling is upregulated during autophagy-mediated regeneration in inflamed mesothelial cells.

Previously, we showed that after Freund's adjuvant-induced peritonitis, rat mesothelial cells regain their epithelial phenotype through mesenchymal-epithelial transition (MET) accompanied by autophagy. Since bone morphogenetic proteins (BMPs) are well-known MET-inducers, we were interested in the potential expression of BMPs and BMP-induced pathways. Although mesothelial cells expressed lower amounts of BMP7, its level in the peritoneal cavity and mesothelial synthesis of BMP4 were significantly increased during inflammation. BMPR1A and BMPR2 were also significantly expressed. Expression of transforming growth factor beta-activated kinase (TAK1) and c-Jun NH2-terminal kinases (JNK1-JNK2) were more intense than that of phosphorylated Mothers Against Decapentaplegic homolog 1/5 (p-SMAD1/5), confirming that the non-canonical pathway of BMPs prevailed in our model. JNK signaling through B-cell lymphoma-2 (Bcl-2) can contribute to Beclin-1 activation. We demonstrated that TAK1-JNK-Bcl-2 signaling was upregulated simultaneously with the autophagy-mediated regeneration. A further goal of our study was to prove the regenerative role of autophagy after inflammation. We used a specific inhibitor, bafilomycin A1 (BafA1), and found that BafA1 treatment decreased the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3B) and resulted in morphological signs of cell death in inflamed mesothelial cells indicating that if autophagy is arrested, regeneration turns into cell death and consequently, mesothelial cells die.

Anti-arthritic effects of geranium essential oil loaded chitosan nanoparticles in Freund's complete adjuvant induced arthritic rats through down-regulation of inflammatory cytokines.

Geranium essential oil (GEO) has been widely used in aromatherapy and traditional medicines. Nanoencapsulation, a novel technique has emerged to overcome the environmental degradation and less oral bioavailability of essential oils. This work was undertaken to encapsulate geranium essential oil in chitosan nanoparticles (GEO-CNPs) by ionic gelation technique and to explore anti-arthritic and anti-inflammatory potential in FCA-induced arthritic model in rats. The GEO was characterized by gas chromatography flame ionization detector (GCFID) and the nanosuspension was characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-rays diffraction (XRD). The Wistar albino rats (n = 32) were separated into four groups; Group 1 and 2 were considered as normal and arthritic controls. Group 3 was positive control that received oral celecoxib for 21 days while Group 4 was treated with oral GEO-CNPs after the induction of arthritis. Hind paw ankle joints diameters were weekly measured throughout the study and significant decrease (5.5 ± 0.5 mm) was observed in GEO-CNPs treatment group in comparison to arthritic group (9.17 ± 0.52 mm). Blood samples were drawn at end for evaluation of hematological, biochemical and inflammatory biomarkers. A significant upregulation of red blood cells and hemoglobin while downregulation of white blood cells, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP) and rheumatoid factor (RF) was observed. Ankles were transected for the histopathological and radiographic examination after animals were sacrificed which confirmed the alleviation of necrosis along cellular infiltration. It was concluded that GEO-CNPs were found to possess excellent therapeutic potential and promising candidates to reduce FCA-induced arthritis.

Cassia absus-mediated upregulation of IL-4, IL-10 and downregulation of IL-1ß, IL-6, TNF- α, NF-κB, IFN-γ in CFA-induced arthritis model.

Traditional use of Cassia absus as an anti-inflammatory in conjunctivitis and bronchitis is well reported. Owing to its anti-inflammatory potential, the current study appraised in vivo anti-arthritic activity of n-hexane and aqueous extracts of Cassia absus seeds (200 mg/kg) using Complete Freund's Adjuvant (CFA) rat model of arthritis. Changes in paw size (mm), joint diameter (mm), and pain response (sec) were recorded at the baseline and then after CFA induction at the interval of 4 days till the 28th day. Blood samples of anesthetized rats were collected for the estimation of hematological, oxidative, and inflammatory biomarkers. Results showed percent inhibition in paw edema (45.09% and 60.79%) with both n-hexane and aqueous extracts, respectively. Significant reduction in paw size and ankle joint diameter (P < 0.01) was seen in extracts treated rats. Erythrocyte Sedimentation rate, C-Reactive Protein, White Blood Cell levels significantly lowered, and Hemoglobin, Platelets and Red Blood Cell count significantly increased post-treatments. Superoxide Dismutase, Catalase, and Glutathione were significantly improved (P < 0.0001) in treated groups as compared to CFA induced arthritic control. Real-time polymerase chain reaction investigation showed significant downregulation (P < 0.05) of Interleukin-1ß, Tumor Necrosis Factor-α, Interleukin-6, Cycloxygenase-2, Nuclear Factor-κB, Prostaglandin E Synthase 2, Interferon Gamma and upregulation of Interleukin-4, Interleukin-10 in both n-hexane and aqueous extract-treated groups. It is thereby concluded that Cassia absus can significantly attenuate CFA-induced arthritis by modulation of oxidative and inflammatory biomarkers.

Bioactive fraction of Tragia involucrata Linn leaves attenuates inflammation in Freund's complete adjuvant-induced arthritis in Wistar albino rats via inhibiting NF-κB.

Tragia involucrata Linn. (T. involucrata) belongs to the family of Euphorbiaceae found in the subtropical regions. Traditionally, the plant parts are used to treat inflammation, wounds and skin infection by people of the Western Ghats, India. Few studies on the acute anti-inflammatory activity of T. involucrata extracts were reported earlier. The present study aims to identify the bioactive fraction of T. involucrata and to evaluate its mechanism in Complete Freund's Adjuvant-induced arthritic rat model. The leaf extract was highly effective among the methanolic leaf and root extracts. The hexane (HF) and a methanolic fraction (MF) of the leaf extract of T involucrata were further identified as a bioactive fraction evaluated through protein denaturation assay. The HF and MF were further studied for their anti-inflammatory potential in a chronic inflammatory model, and their mechanism of action was explored further. Arthritis was induced by administering 0.1 ml of CFA intradermally. The treatment was started the next day with HF (100 and 250 mg/kg/day) and MF (100 and 250 mg/kg/day), while the HF and MF alone group served as the drug control, Indomethacin-treated group served as the positive control. On the 25th day, the animals were euthanized, and their body weight, paw thickness, arthritic score, spleen and thymus weight, haematological parameters, biochemical parameters, radiographs and histopathology were analyzed. Results showed that the MF-treated animals maintained dry weight, reduced paw thickness, arthritic scores, and haematological and biological parameters compared to the HF-treated and CFA-induced arthritic rats. Both radiological and histopathological analyses of the joints revealed that the MF-treated groups restored bone architecture without any erosion and normal tissue architecture with nil signs of active inflammation. Western blot analysis revealed that MF has effectively inhibited the protein expression levels of MMP-3, MMP-9, and NF-κB in the synovial tissues compared to that of CFA-induced arthritic rats. Besides, HPLC analysis revealed the presence of flavonoids, including gallic acid, rutin and Quercetin, in the MF of T. involucrata, which had shown to have potent anti-inflammatory potential. Thus, it can be emphasized that T. involucrata could be a potential therapeutic candidate for treating inflammatory diseases, which needs further experimental studies to confirm its safety and efficacy.

Anti-Arthritic Effect of Edaravone Against Complete Freund Adjuvant Induced Arthritis via Osteoclast Differentiation and HIF-1α-VEGF-ANG-1 Axis.

Background: Bone dysfunction is a crucial problem that occurs during rheumatoid arthritis (RA) disease. Osteoclast plays a significant role in bone resorption and osteoclast differentiation and its enhancement of bone destruction. Edaravone remarkably exhibited free radical scavenging and anti-inflammatory effects. The objective of the current investigation is to comfort the inhibitory effect of Edaravone (ED) against complete Freund adjuvant (CFA) rat model via inhibition of angiogenesis and inflammation. Methods: Subcutaneous injection of CFA (1%) was used to induce arthritis; the rats were divided into different groups and received the oral administration of ED. Paw edema, body weight, and arthritis score were regularly estimated. Biochemical parameters were estimated, respectively. We also estimate the level of hypoxia-inducible factor-1α (HIF-1α), angiopoietin 1 (ANG-1), and vascular endothelial growth factor (VEGF). We also checked into how ED affected the differentiation of osteoclasts utilising a co-culture system with monocytes and synovial fibroblasts in arthritis rats. Results: ED treatment significantly (P<0.001) suppressed the arthritis score and paw edema and improved the body weight. ED treatment significantly (P<0.001) altered the antioxidant parameters and pro-inflammatory cytokines: inflammatory mediator nuclear kappa B factor (NF-κB), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2), respectively. Furthermore, ED treatment significantly (P<0.001) suppressed the level of ANG-1, HIF-1α, and VEGF, respectively. The results suggest that ED suppressed osteoclast differentiation and also decreased the level of cytokines and osteopontin (OPN), receptor activator for nuclear factor-κ B Ligand (RANKL) and macrophage colony stimulating factor (M-CSF) in the co-culture supernatant of monocytes and synovial fibroblasts. Conclusion: Edaravone could mitigate CFA via inhibiting angiogenesis and inflammatory reactions, which may be linked with the HIF-1α-VEGF-ANG-1 axis and also enhance the bone destruction of murine arthritis via suppression of osteoclast differentiation and inflammatory reaction.

[Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome].

OBJECTIVE: To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. METHODS: Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1ß, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). RESULTS: Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1ß, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1ß, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. CONCLUSION: Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1ß, IL-6 and CRP.

Jobelyn® extends the life span and improves motor function in Drosophila melanogaster exposed to lipopolysaccharide via augmentation of antioxidant status.

Jobelyn® (JB), a dietary supplement, derived from polyphenol-rich leaf sheath of Sorghum bicolor, has been reported to attenuate sensorimotor deficits and oxidative stress evoked by complete Freund-adjuvant in mice. This present study evaluated its effects on the life span, motor function and changes in oxidative stress parameters as well as acetylcholinesterase activity in Drosophila melanogaster exposed to lipopolysaccharide (LPS). The flies (50 per vial), in 5 replicates were fed with LPS (250 µg/kg diet) alone or in combination with JB (0.25-1.0 mg/kg diet) daily for 7 days. The mortality rate and motor function were evaluated on day 7. The flies were afterwards processed for determination of oxidative stress parameters and acetylcholinesterase activity. The effects of JB (0.25-1.0 mg/g diet) on the longevity of Drosophila was also investigated wherein the flies were monitored daily for mortality throughout their lifespan. The flies exposed to LPS (250 µg/kg diet) had reduced life span and elevated oxidative stress when compared with control. However, JB (0.25 and 1.0 mg/kg diet) improved the motor function and also reduced the mortality rate of the flies exposed to LPS. It also restored the cellular antioxidant status and reduced acetylcholinesterase activity, accumulation of hydrogen peroxide as well as nitric oxide in Drosophila fed with LPS. JB also extended the longevity of the flies relative to control. The findings that JB improves motor function and extended the lifespan of Drosophila flies by boosting the antioxidant status and cholinergic function, suggest it might be helpful in delaying the onset of neuropsychiatric illnesses associated with the aging processes.

Co-treatment of Nimbolide augmented the anti-arthritic effects of methotrexate while protecting against organ toxicities.

Prolonged exposure to the pharmacological doses of disease-modifying anti-rheumatic drugs (DMARDs) often results in major organ toxicities resulting in poor patient compliance. Methotrexate (MTX) is one of the commonly prescribed DMARDs for the treatment of arthritis, which results in vital organ dysfunction. To retain the anti-arthritic activity of MTX with the reduction in toxicities, combination therapies are warranted. Nimbolide (NMB) is a potent anticancer, anti-inflammatory and anti-fibrotic agent whose potential has been demonstrated in various pre-clinical models. Monoarthritis was developed with Complete Freund's Adjuvant in the knees of Wistar rats and treatment was given with either NMB (3 mg/kg/day) or MTX (2 mg/kg/week) alone or combination therapy (NMB + MTX). The anti-arthritic effects were evaluated by arthritic scoring, radiological imaging, synovial tissue proteins analysis, and histopathological staining. While hepato-renal toxicity was assessed in serum by evaluating the kidney and liver functional parameters, in tissues by oxidative-nitrosative stress markers, and pro-inflammatory cytokines levels. Histopathological analysis was performed to study the extent of tissue damage. Molecular studies like immunoblotting and immunohistochemistry were performed to understand the effect of combination therapy. We thereby report that monotherapy with either NMB or MTX exhibited significant anti-arthritic effects, while combination therapy resulted in augmented anti-arthritic effects with significant reduction in hepato-renal toxicity produced by MTX probably through anti-inflammatory and anti-oxidant effects. Therefore, our proposed combination of NMB and MTX may serve as a potential strategy for the effective management of arthritis.

Appraisal of Anti-Arthritic and Anti-Inflammatory Potential of Folkloric Medicinal Plant Peganum harmala.

BACKGROUND & OBJECTIVE: Peganum harmala has been traditionally used to manage rheumatoid arthritis (RA) and other inflammatory conditions. However, its use against RA has not been scientifically evaluated. The current study was designed to assess the anti-arthritic and anti-inflammatory activities of the methanolic extract of P. harmala leaves by in vitro and in vivo methods. METHODS: The in vitro assays were carried out to determine the effect of plant extract on inhibition of egg albumin denaturation and human red blood cell membrane (HRBC) stabilization. Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity was performed to determine the antioxidant potential. In vivo anti-arthritic activity was performed by determining the curative effect against Complete Freund's adjuvant (0.1 ml). The plant extract was administered to rats orally at 200, 400 and 600 mg/kg/day for 21 days. RESULTS: The values of IC50 of plant extract in protein denaturation, stabilization of HRBC and DPPH assays were 77.54 mg/ml, 23.90 mg/ml and 58.09 µg/ml, respectively. Moreover, the plant extract significantly attenuated the poly-arthritis and weight loss, anemia and paw edema. The plant extract restored the level of C-reactive protein, rheumatoid factor, alanine transaminase, aspartate transaminase and alkaline phosphatase in poly-arthritic rats. Moreover, the plant extract restored the immune organs' weight in treated rats. Treatment with P. harmala also significantly subdued the oxidative stress by reinstating superoxide dismutase, reduced glutathione, catalase and malondialdehyde in poly-arthritic rats. The plant extract notably restored the prostaglandin-E2 and tumor necrosis factor (TNF)-α in the serum of poly-arthritic rats. CONCLUSION: It was concluded that P. harmala extract had potential antioxidant, anti-inflammatory and antiarthritic activities, which primarily might be attributed to alkaloids, flavonoids and phenols.

Referral and adjuvant treatment patterns after nephrectomy in high-risk locoregional renal cell carcinoma.

BACKGROUND: It is unclear whether patients with renal cell carcinoma (RCC) are routinely assessed for recurrence risk post-nephrectomy and whether patients at high recurrence risk are seen by providers who can evaluate candidacy for adjuvant systemic therapy (AST) and clinical trials. MATERIALS AND METHODS: We identified all patients with locoregional RCC who underwent nephrectomy via an institutional database within Duke University Health System between 1 April 2015 and 31 December 2019. Medical records were reviewed to identify patient characteristics, post-nephrectomy referrals, treatment, and follow-up. Patients with tumor stage ≥3 and grade ≥2, regional lymph node metastasis, or both, were classified as high recurrence risk. RESULTS: Of 618 patients with locoregional RCC who underwent nephrectomy, 136 (22%) had high recurrence risk. Of those, 25 patients with high-risk disease (18%) were referred to medical oncology for discussion of AST; 23 (92%) of these referrals took place in 2018-2019. One patient received adjuvant sunitinib and two patients participated in adjuvant immunotherapy trials. The decision not to receive AST was primarily made by the oncologist in 10 (46%), the patient in 8 (36%), and unrecorded in 4 (18%) of 22 cases, for multiple reasons. Individual surgeons referred high-risk patients for discussion of AST with varying frequency, ranging from 0% to 100% in 2019. CONCLUSIONS: Despite increasing number of patients with locoregional RCC at high recurrence risk referred to medical oncologists after nephrectomy, few patients received AST, including participation in clinical trials. With increasing AST options and ongoing clinical trials in this space, these findings highlight the need for continued efforts at identifying effective AST and referring patients most likely to benefit to medical oncologists. ClinicalTrials.gov, NCT04309617.

Neuromedin B receptor stimulation of Cav3.2 T-type Ca2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity.

Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.

14-3-3ζ: A suppressor of inflammatory arthritis.

Inflammatory arthritis (IA) is a common disease that affects millions of individuals worldwide. Proinflammatory events during IA pathogenesis are well studied; however, loss of protective immunity remains underexplored. Earlier, we reported that 14-3-3zeta (ζ) has a role in T-cell polarization and interleukin (IL)-17A signal transduction. Here, we demonstrate that 14-3-3ζ knockout (KO) rats develop early-onset severe arthritis in two independent models of IA, pristane-induced arthritis and collagen-induced arthritis. Arthritic 14-3-3ζ KO animals showed an increase in bone loss and immune cell infiltration in synovial joints. Induction of arthritis coincided with the loss of anti-14-3-3ζ antibodies; however, rescue experiments to supplement the 14-3-3ζ antibody by passive immunization did not suppress arthritis. Instead, 14-3-3ζ immunization during the presymptomatic phase resulted in significant suppression of arthritis in both wild-type and 14-3-3ζ KO animals. Mechanistically, 14-3-3ζ KO rats exhibited elevated inflammatory gene signatures at the messenger RNA and protein levels, particularly for IL-1ß. Furthermore, the immunization with recombinant 14-3-3ζ protein suppressed IL-1ß levels, significantly increased anti-14-3-3ζ antibody levels and collagen production, and preserved bone quality. The 14-3-3ζ protein increased collagen expression in primary rat mesenchymal cells. Together, our findings indicate that 14-3-3ζ causes immune suppression and extracellular remodeling, which lead to a previously unrecognized IA-suppressive function.

Blockade of Spinal EphA4 Reduces Chronic Inflammatory Pain in Mice.

Background: Erythropoietin-producing hepatocellular (Ephs) receptor and their ligands, ephrins, orchestrate the induction of cell proliferation and migration, axonal guidance, synaptic genesis and synaptic plasticity in the central nervous system. Previous studies demonstrated that EphBs/ephrinBs participate in the pathophysiology of neuropathic pain, inflammatory pain and bone cancer pain, but the role of EphA4 in the regulation of pain in the spinal cord is unknown. Therefore, we explored the role of EphA4 receptor in regulating chronic inflammatory pain.Methods: We established a mouse model of chronic inflammatory pain through plantar injection of complete freund's adjuvant (CFA) and assessed EphA4 expression in spinal cord by western blotting. EphA4 receptor was blocked by intrathecal injection of EphA4-Fc, an EphA4 antagonist, and pain behaviors were measured by assessing thermal hyperalgesia and mechanical allodynia. Finally, immunohistochemistry was performed to analyze the changes in the expression of Fos protein in spinal cord after blocking EphA4 receptor.Results: Plantar injection of CFA produced persistent thermal hyperalgesia and mechanical allodynia, which was accompanied by significant increases in spinal EphA4 and Fos expression. Blocking spinal EphA4 receptor suppressed CFA-induced pain behaviors and reduced the expression of Fos protein in spinal cord.Conclusions: Our study demonstrated that EphA4 receptor is involved in the generation and maintenance of CFA-induced chronic inflammatory pain and that blocking the spinal EphA4 receptor could relieve persistent pain behaviors in mice.

Polymyxin B prevents the development of adjuvant arthritis via modulation of TLR/Cox-2 signaling pathway.

AIMS: Several microbial toll-like receptor (TLR) ligands, bacterial DNA and bacterial cell wall fragments have been identified in the synovium of rheumatoid arthritis (RA) patients, proving bacterial involvement in the pathogenesis of RA. The current study aimed to verify that low dose polymyxin B could prevent the development of chronic inflammatory arthritis. METHODS: Twelve days post adjuvant injection, Sprague-Dawley rats were treated twice weekly with methotrexate (0.5 mg/kg) or daily with polymyxin B (1 mg/kg) or with combination of both for 1 or 2 weeks. Arthritis progression was assessed by hind paw swelling, serum levels of tumor growth factor-1ß (TGF-1ß), tumor necrosis factor-alpha (TNF-α), high sensitivity C-reactive protein (HS-CRP) and nuclear factor kappa B (NF-κB) were measured using ELISA. Cyclooxygenase-1 (Cox-1) and Cox-2 activities, as well as mRNA expression of TLR-2 and TLR-4 were determined. Histopathological examination of the ankle joint was performed as well as immunohistochemistry for anti-TLR-4. Histopathological assessment of toxic effects on the kidney was performed. KEY FINDINGS: Adjuvant arthritis led to a significant swelling of the hind paw and alteration in all serum parameters, TLR-2 and TLR-4 expression, as well as Cox-2 activity. These alterations were associated with histopathological changes of the joints. Polymyxin B reduced significantly all biomarkers of inflammation, showing better effect of the combination in most of the studied parameters, with minimal signs of nephrotoxicity. SIGNIFICANCE: In conclusion, results showed that polymyxin B possesses significant anti-arthritic activity which may be attributed to inhibition of the TLR-4, NF-κB and Cox-2 signaling pathway.

Polystichum braunii extracts inhibit Complete Freund's adjuvant-induced arthritis via upregulation of I-κB, IL-4, and IL-10, downregulation of COX-2, PGE2, IL-1ß, IL-6, NF-κB, and TNF-α, and subsiding oxidative stress.

Polystichum braunii (Spenn.) Fée is a traditional remedy for rheumatoid arthritis, a chronic inflammatory disorder of polygenetic origin. The current project was intended to demonstrate the role of inflammatory and oxidative stress biomarkers in the anti-arthritic activity of the P. braunii extracts. Methanolic and aqueous extracts of the plant roots were prepared by triple maceration. The phytochemical evaluation of the plant extracts was carried out by high-performance liquid chromatography (HPLC). The plant extracts at 150, 300, and 600 mg/kg/day and piroxicam (10 mg/kg/day) were orally administered to Wistar rats for 21 days that were previously immunized with Complete Freund's adjuvant (150 µl on right hind paw) except normal and arthritic control rats. Both plant extracts mitigated the paw oedema, restored the immune organ and body weights, and ameliorated the level of blood parameters such as haemoglobin, red blood cells, platelets, white blood cells, alkaline phosphatase (ALP), C-reactive proteins, and rheumatoid factor. The evaluation of gene expression using quantitative-real-time polymerase chain reaction (qRT-PCR) revealed the substantial downregulation of tumor necrosis factor (TNF)-α, Interleukin (IL)-6, IL-1ß, cyclo-oxygenase (COX)-2, nuclear factor (NF)-κB, and upregulation of IL-4, IL-10 and I-κB in polyarthritic rats treated with the plant extracts. Methanolic plant extract exhibited the maximum effect on upregulation of IL-4 (79 ± 3%), IL-10 (62.66 ± 4.93%), and I-κB (73.66 ± 3.05%) at 600 mg/kg/day. Treatment with the plant extracts also reduced the level of prostaglandin E2 and TNF-α in the serum of arthritic rats' dose dependently. It was also found that the plant extracts and piroxicam increased (p < 0.05) the activities of superoxide dismutase and catalase in the liver tissue while reduced the level of malondialdehyde in arthritic rats. Histological examination of ankle joints revealed that the plant extracts decreased the pannus formation, inflammation, and synovial hyperplasia in arthritic animals. HPLC analysis depicted that the plant extracts had contained kaempferol, quercetin, gallic acid, and other phenolic acids. It can be elucidated from the results that the extracts of P. braunii roots exhibited anti-arthritic activity in Wistar rats through modulation of inflammatory cytokines and boosting the antioxidant defense mechanism.

Phytol, a Chlorophyll Component, Produces Antihyperalgesic, Anti-inflammatory, and Antiarthritic Effects: Possible NFκB Pathway Involvement and Reduced Levels of the Proinflammatory Cytokines TNF-α and IL-6.

Phytol is a diterpene constituent of chlorophyll and has been shown to have several pharmacological properties, particularly in relation to the management of painful inflammatory diseases. Arthritis is one of the most common of these inflammatory diseases, mainly affecting the synovial membrane, cartilage, and bone in joints. Proinflammatory cytokines, such as TNF-α and IL-6, and the NFκB signaling pathway play a pivotal role in arthritis. However, as the mechanisms of action of phytol and its ability to reduce the levels of these cytokines are poorly understood, we decided to investigate its pharmacological effects using a mouse model of complete Freund's adjuvant (CFA)-induced arthritis. Our results showed that phytol was able to inhibit joint swelling and hyperalgesia throughout the whole treatment period. Moreover, phytol reduced myeloperoxidase (MPO) activity and proinflammatory cytokine release in synovial fluid and decreased IL-6 production as well as the COX-2 immunocontent in the spinal cord. It also downregulated the p38MAPK and NFκB signaling pathways. Therefore, our findings demonstrated that phytol can be an innovative antiarthritic agent due to its capacity to attenuate inflammatory reactions in joints and the spinal cord, mainly through the modulation of mediators that are key to the establishment of arthritic pain.

Sex Differences Revealed in a Mouse CFA Inflammation Model with Macrophage Targeted Nanotheranostics.

Monocyte derived macrophages (MDMs) infiltrate sites of infection or injury and upregulate cyclooxygenase-2 (COX-2), an enzyme that stimulates prostaglandin-E2 (PgE2). Nanotheranostics combine therapeutic and diagnostic agents into a single nanosystem. In previous studies, we demonstrated that a nanotheranostic strategy, based on theranostic nanoemulsions (NE) loaded with a COX-2 inhibitor (celecoxib, CXB) and equipped with near-infrared fluorescent (NIRF) reporters, can specifically target circulating monocytes and MDMs. The anti-inflammatory and anti-nociceptive effects of such cell-specific COX-2 inhibition lasted several days following Complete Freund's Adjuvant (CFA) or nerve injury in male mice. The overall goal of this study was to investigate the extended (up to 40 days) impact of MDM-targeted COX-2 inhibition and any sex-based differences in treatment response; both of which remain unknown. Our study also evaluates the feasibility and efficacy of a preclinical nanotheranostic strategy for mechanistic investigation of the impact of such sex differences on clinical outcomes. Methods: CFA was administered into the right hind paws of male and female mice. All mice received a single intravenous dose of NIRF labeled CXB loaded NE twelve hours prior to CFA injection. In vivo whole body NIRF imaging and mechanical hypersensitivity assays were performed sequentially and ex vivo NIRF imaging and immunohistopathology of foot pad tissues were performed at the end point of 40 days. Results: Targeted COX-2 inhibition of MDMs in male and female mice successfully improved mechanical hypersensitivity after CFA injury. However, we observed distinct sex-specific differences in the intensity or longevity of the nociceptive responses. In males, a single dose of CXB-NE administered via tail vein injection produced significant improved mechanical hypersensitivity for 32 days as compared to the drug free NE (DF-NE) (untreated) control group. In females, CXB-NE produced similar, though less prominent and shorter-lived effects, lasting up to 11 days. NIRF imaging confirmed that CXB-NE can be detected up to day 40 in the CFA injected foot pad tissues of both sexes. There were distinct signal distribution trends between males and females, suggesting differences in macrophage infiltration dynamics between the sexes. This may also relate to differences in macrophage turnover rate between the sexes, a possibility that requires further investigation in this model. Conclusions: For the first time, this study provides unique insight into MDM dynamics and the early as well as longer-term targeted effects and efficacy of a clinically translatable nanotheranostic agent on MDM mediated inflammation. Our data supports the potential of nanotheranostics as presented in elucidating the kinetics, dynamics and sex-based differences in the adaptive or innate immune responses to inflammatory triggers. Taken together, our study findings lead us closer to true personalized, sex-specific pain nanomedicine for a wide range of inflammatory diseases.

Andrographolide ameliorates oxidative stress, inflammation and histological outcome in complete Freund's adjuvant-induced arthritis.

OBJECTIVES: As one of the main active ingredients of Chinese herbal medicine Andrographis paniculate, andrographolide is used in domestic clinical treatment for respiratory infections and inflammation. This study was designed to investigate the effects of andrographolide as an antioxidant on the level of oxidative stress, neutrophil accumulation and infiltration in joints and synovial tissue of arthritis rats induced by complete freund's adjuvant. METHODS: A rat model of rheumatoid arthritis was induced by subcutaneous injection of complete Freund's adjuvant in the footpad. The model was established 14 days after induction. The treatment was performed from 14th day to 35th day with different doses of andrographolide (25, 50, 100 mg/kg) and positive control methotrexate (3 mg/kg). The effects of andrographolide on oxidative stress, neutrophil accumulation and infiltration were measured by the paw swelling, arthritis score, the hot plate test, biochemical analysis, and histology. RESULTS: The medium and high-dose andrographolide (50, 100 mg/kg) group declined the levels of tumor necrosis factor-α, interleukin-6 and CXC chemokine ligand2, articular elastase and myeloperoxidase, and increased the levels of antioxidant enzymes superoxide dismutase, catalase, and glutathione. The activity of malondialdehyde and nitrite/nitrate in andrographolide (50, 100 mg/kg) group was weakened than the model group. The degree of swelling and arthritis score of andrographolide group was lower than the model group. The results of hot plate test showed that high dose of andrographolide significantly improved the anti-injury ability of rats; Radiological and histological results showed that the joint osteoporosis, inflammatory cell infiltration, synovial hyperplasia and other phenomena in the andrographolide group were significantly improved. CONCLUSIONS: Andrographolide acts as a protective agent for the treatment of complete freund's adjuvant induced rheumatoid arthritis by inhibiting lipid peroxidation and nitrite/nitrate levels in a dose-dependent manner, enhancing antioxidant enzyme activity, reducing levels of chemokines and inflammatory factors, preventing neutrophil accumulation and infiltration.

Protective effects of a polyphenol-enriched fraction of the fruit peel of Annona crassiflora Mart. on acute and persistent inflammatory pain.

Different parts of Annona crassiflora Mart., a native species from Brazilian savanna, were traditionally used for the treatment of a wide variety of ailments including arthritis. Thus, this study aimed to investigate the possible antinociceptive and anti-inflammatory properties of a polyphenol-enriched fraction of the fruit peel of A. crassiflora, named here as EtOAc, in mice. Pro-inflammatory cytokines and nitric oxide (NO) production were evaluated in LPS-activated macrophages. Then, EtOAc fraction was administered by oral route in male C57BL/6/J mice, and the animals were submitted to glutamate-induced nociception and complete Freund's adjuvant (CFA)-induced monoarthritis tests to assess nociception (mechanical, spontaneous and cold pain) and inflammation (edema and neutrophil infiltration), and to the open-field and rotarod tests for motor performance analysis. EtOAc fraction inhibited the production of IL-6 and NO in the LPS-induced macrophages, and reduced spontaneous nociception induced by glutamate, without altering the animals' locomotor activity. In addition, the polyphenol-enriched fraction was able to revert the early and late hyperalgesia induced by CFA, as well as edema at the acute phase. Reduction of myeloperoxidase activity and inflammatory cell infiltration was observed in the paw tissue of mice injected with CFA and treated with EtOAc fraction. Together, our results support the antinociceptive and anti-inflammatory effects of the polyphenol-enriched fraction of A. crassiflora fruit peel and suggest that these effects are triggered, at least in part, by suppressing pro-inflammatory cytokines and neutrophils infiltration.