Tridecylcyclohexane in incomplete Freund's adjuvant is a critical component in inducing experimental autoimmune diseases.

Incomplete Freund's adjuvant (IFA) has been used for many years to induce autoimmune diseases in animal models, including experimental autoimmune encephalitis and collagen-induced arthritis. However, it remains unclear why it is necessary to emulsify autoantigen and heat-killed Mycobacterium tuberculosis (HKMtb) with IFA to induce experimental autoimmune diseases. Here, we found that immunization with self-antigen and HKMtb was insufficient to induce autoimmune diseases in mice. Furthermore, IFA or one of its components, mineral oil, but not mannide monooleate, was required for the development of experimental autoimmune disease. Immunization with autoantigen and HKMtb emulsified in mineral oil facilitated innate immune activation and promoted the differentiation of pathogenic CD4+ T cells, followed by their accumulation in neuronal tissues. Several water-soluble hydrocarbon compounds were identified in mineral oil. Of these, immunization with HKMtb and autoantigen emulsified with the same amount of hexadecane or tridecylcyclohexane as mineral oil induced the development of experimental autoimmune encephalitis. In contrast, immunization with HKMtb and autoantigen emulsified with tridecylcyclohexane, but not hexadecane, at doses equivalent to those found in mineral oil, resulted in neuronal dysfunction. These data indicate that tridecylcyclohexane in mineral oil is a critical component in the induction of experimental autoimmune disease.

miR-147-3p in pathogenic CD4 T cells controls chemokine receptor expression for the development of experimental autoimmune diseases.

Incomplete Freund's adjuvant (IFA) has long been used to trigger autoimmune diseases in animal models, such as experimental autoimmune encephalitis and collagen-induced arthritis. However, the molecular mechanisms that control CD4 T cell effector functions and lead to the development of autoimmune diseases are not well understood. A self-antigen and heat-killed Mycobacterium tuberculosis emulsified in IFA augmented the activation of CD4 T cells, leading to the differentiation of pathogenic CD4 T cells in the draining lymph nodes. In contrast, IFA emulsification did not elicit Foxp3+ regulatory T cell expansion. We found that pathogenic Th1 cells expressed miR-147-3p, which targets multiple genes to affect T cell function. Finally, miR-147-3p expressed in CXCR6+SLAMF6- Th1 cells was required for the onset of neurological symptoms through the control of CXCR3 expression. Our findings demonstrate that miR-147-3p expressed in pathogenic CD4 T cells regulates the migratory potential in peripheral tissues and impacts the development of autoimmune diseases.

Andrographolide inhibits the activation of spinal microglia and ameliorates mechanical allodynia.

Andrographolide (Andro), a labdane diterpene, possesses anti-inflammatory properties and has been used to treat numerous inflammatory diseases. Novel findings revealed that Andro might be vital in regulating pain. However, the contribution of Andro to chronic inflammatory pain has yet to be determined, and its underlying mechanism of action remains unknown. In this study, we observed that Andro attenuated mechanical allodynia in inflammatory pain mice induced by injecting complete Freund's adjuvant (CFA) into the right hind paws. This analgesic effect of Andro is mainly dependent on its inhibition of microglial overactivation and the release of proinflammatory cytokines (TNF and IL-1ß) in lumbar spinal cords of inflammatory pain model mice. More importantly, our data in vivo and in vitro revealed a negative role for Andro in regulating the TLR4/NF-κB signaling pathway, which might contribute to the inhibition of spinal microglial activation and proinflammatory cytokines production, and the improvement of paw withdrawal thresholds in a mouse model of chronic inflammatory pain evoked by CFA. We further found the potential interaction of Andro with TLR4/myeloid differentiation factor 2 heterodimer using molecular modeling, implying that TLR4 might be a potential target for Andro to exert an analgesic effect. Taken together, our findings demonstrated that the modulation of spinal microglial activation by Andro might be substantially conducive to managing chronic pain triggered by neuroinflammation.

In Silico and In Vivo Evaluation of Anti-Arthritic Effects of Bakuchiol from Psoralea corylifolia Seeds in Experimental Rat Model.

Rheumatoid arthritis is an autoimmune disease mainly affecting the joints categorized by inflammation, swelling of the synovium and decrease in the joint movement. Bakuchiol, a meroterpene class of natural product present in Psoralea corylifolia known to possess anti-inflammatory effects by a variety of mechanisms. However, its effects in rheumatoid arthritis still remain unclear. In the present investigation, we studied the anti-arthritic effects of bakuchiol via in silico and in vivo experiments. It also showed antioxidant effects measured using DPPH assay where it showed free radical scavenging activity with IC50 value 468.26 µg/ml. Molecular Docking studies carried out on COX-1 (PDB ID: 3 N8Z), COX-2 (PDB ID: 4PH9) and TNF-α (PDB ID: 7JRA), proteins involved in inflammation in arthritis. Bakuchiol showed the maximum binding affinity for TNF-α with binding affinity score is -7.29 kcal/moland less affinity was observed for COX-1 and 2. In vivo antiarthritic effects were studied in arthritic female wistar rats model prepared by intradermal injection of freund's complete adjuvant. Bakuchiol was administered orally at dose of 10,20 and 40 mg/kg for 21 days. Our treatment showed that bakuchiol at 20 and 40 mg/kg exhibited significant anti-inflammatory effects (p<0.001) showed by significant decrease in paw volume, paw diameter, spleen and thymus weight and increase in pain threshold and body weight in arthritic rat model. A significant decrease in hematological parameters such as total leukocyte count (TLC), platelet count, CRP and rheumatoid arthritis factor (RF) and increase in red blood cells count, ESR and hemoglobin further demonstrated that bakuchiol treatment suppresses the progression of adjuvant induced arthritis (AIA) in arthritic rat model. Histological analysis further revealed that bakuchiol ameliorates the pathological manifestations of AIA and reverse the abnormality induced by AIA in rats shown by protection against bone necrosis involved with low influx of inflammatory cells. Therefore, in silico and in vivo results revealed that bakuchiol has the potential to be developed as potent antiarthritic agent.

Fumaric acid per se and in combination with methotrexate arrests inflammation via moderating inflammatory and oxidative stress biomarkers in arthritic rats.

Objective: Fumaric acid is a dicarboxylic acid that belongs to the phenolic class enriched in fruits and vegetables that are traditionally used for the treatment of various ailments. The research was planned to find out the anti-inflammatory and anti-arthritic activities of fumaric acid using in-vitro and in-vivo assays. Moreover, safety study was also done.Materials and methods: The 0.1 ml complete Freund's adjuvant was injected in left hind paw in all Wistar rats except normal rats at day 1 to induced arthritis. The treatment with fumaric acid at 10, 20, 40, and fumaric acid 40 mg/kg together with methotrexate (MTX) was administered to immunized rats at 8th day via oral gavage and continued till 28th day though, MTX was administered as standard control.Results: The fumaric acid notably (p < 0.0001) lessened the paw edema and arthritic scoring, reinstated body and immune organ weight, and oxidation status in treated rats. Fumaric acid notably restored altered C-reactive protein, rheumatoid factor, liver function tests, ESR, WBCs, RBCs and Hb levels in treated rats. The fumaric acid in combination noticeably (p < 0.01-0.0001) suppressed the expression of TNF- α, IL-6, IL-1ß, NF-kß, and COX-2, and over expressed IL-4, and IL-10 in contrast to other treated groups. Fumaric acid had presented a dose-dependent antioxidant, anti-inflammatory and anti-arthritic activities while notable activity exhibited by fumaric acid in combination with MTX. The fumaric acid exhibited non-significant clinical signs of toxicity and mortality in acute toxicity study. The LD50 was more than 2000 mg/kg.Conclusion: Fumaric acid in combination can be used as disease-modifying anti-rheumatic drug but it will need extensive pre-clinical and clinical studies.

Assessing the Impact of Influenza Vaccination Timing on Experimental Arthritis: Effects on Disease Progression and Inflammatory Biomarkers.

Numerous studies have indicated a link between vaccines and the exacerbation of autoimmune diseases including rheumatoid arthritis (RA). However, there is no consensus in clinical practice regarding the optimal timing of immunization. Therefore, this study aimed to investigate the impact of the 3Fluart influenza vaccine on the complete Freund's adjuvant (CFA)-induced chronic arthritis rat model and to identify new biomarkers with clinical utility. CFA was injected into the plantar surface of one hind paw and the root of the tail on day 0, and the tail root injection was repeated on day 1. Flu vaccination was performed on day 1 or 7. Paw volume was measured by plethysmometry, mechanonociceptive threshold by dynamic plantar aesthesiometry, neutrophil myeloperoxidase (MPO) activity, and vascular leakage using in vivo optical imaging throughout the 21-day experiment. Inflammatory markers were determined by Western blot and histopathology. CFA-induced swelling, an increase in MPO activity, plasma extravasation in the tibiotarsal joint. Mechanical hyperalgesia of the hind paw was observed 3 days after the injection, which gradually decreased. Co-administration of the flu vaccine on day 7 but not on day 1 resulted in significantly increased heme oxygenase 1 (HO-1) expression. The influenza vaccination appears to have a limited impact on the progression and severity of the inflammatory response and associated pain. Nevertheless, delayed vaccination could alter the disease activity, as indicated by the findings from assessments of edema and inflammatory biomarkers. HO-1 may serve as a potential marker for the severity of inflammation, particularly in the case of delayed vaccination. However, further investigation is needed to fully understand the regulation and role of HO-1, a task that falls outside the scope of the current study.

Assessment of antiarthritic potential of Asparagus dumosus using formaldehyde and CFA-induced arthritic models in rats via modulation of oxidative stress biomarkers and mRNA expression of IL-1b, IL-6, RANKL, OPG, TNF-α and COX-2.

Medicinal plants play a pivotal role in the prevention of chronic non-communicable diseases including arthritis. Despite the traditional use of Asparagus dumosus in arthritis, it has not been studied yet for its effectiveness in arthritis. This study was aimed to explore the antiarthritic potential of A. dumosus in formaldehyde and complete Freund's adjuvant (CFA)-induced arthritic rats. Body weight, arthritic index, hepatic oxidative stress, hematological, biochemical and inflammatory markers were assessed using ELISA, whilst qRT-PCR studies were carried out for the mRNA expression of IL-1b, IL-6, RANKL, OPG, TNF-α and COX-2 genes. GCMS and HPLC analysis were performed to identify the secondary metabolites of A. dumosus. From day 8 to 28 post-administration of formaldehyde and CFA, oral administration of A. dumosus (600, 300 and 150 mg/kg) showed a noteworthy improvement (p < 0.001) in the body weights, immune organ weights, serum levels of rheumatoid (RA) factor, C-reactive protein, TNF-α and IL-6 levels in arthritic rats similar to the effect of piroxicam and methotrexate. Subsequently, the administration of A. dumosus to formaldehyde and CFA-challenged rats, caused a marked decrease (p < 0.001) in the mRNA expression of IL-1b, IL-6, OPG, RANKL, TNF-α and COX-2 genes in treated rats. Likewise, when assessed for antioxidant potential, A. dumosus produced a pronounced (p < 0.001) reduction in malondialdehyde (MDA) levels and hydrogen peroxide (H2O2) production, whilst a dose-dependent (p < 0.001) increase in catalase (CAT) and superoxide dismutase (SOD) activities was recorded. GCMS profiling of A. dumosus presented benzaldehyde, 3-hydroxy-4-methoxy-, 1-decanol and undecane as plant compositions, whereas HPLC fingerprinting displayed quercetin, benzaldehyde, 3-hydroxy-4-methoxy-, gallic acid and cinnamic acid as plants constituents. These results depict that A. dumosus possesses anti-arthritic effect mediated possibly through attenuation of arthritic indices, chronic inflammatory and oxidative stress biomarkers along with down-regulation in the mRNA expression of arthritic candid genes.

Molecular insights of Eucalyptol (1,8-Cineole) as an anti-arthritic agent: in vivo and in silico analysis of IL-17, IL-10, NF-κB, 5-LOX and COX-2.

The monoterpene oxide, Eucalyptol (1,8-Cineole), a primary component of eucalyptus oil, has been evaluated pharmacologically for anti-inflammatory and analgesic activity. Current research aimed to evaluate Eucalyptol's anti-arthritic potential in a Complete Freund's adjuvant induced arthritis that resembles human rheumatoid arthritis. Polyarthritis developed after 0.1 mL CFA injection into the left hind footpad in rats. Oral administration of Eucalyptol at various doses (100, 200 and 400 mg/kg) significantly reduced paw edema, body weight loss, 5-LOX, PGE2 and Anti-CCP levels. Real-time PCR investigation showed significant downregulation of COX-2, TNF-α, NF-κB, IL-17, IL-6, IL-1ß and upregulation of IL-4 and IL-10 in Eucalyptol treated groups. Hemoglobin and RBCs counts significantly increased post-treatment with Eucalyptol while ESR, CRP, WBCs and platelets count significantly decreased. Eucalyptol significantly increased Superoxide Dismutase, Catalase and Glutathione levels compared to CFA-induced arthritic control however, MDA significantly decreased post-treatment. Further, radiographic and histopathological examination of the ankle joints of rodents administered Eucalyptol revealed an improvement in the structure of the joints. Piroxicam was taken as standard. Furthermore, molecular docking findings supported the anti-arthritic efficacy of Eucalyptol exhibited high binding interaction against IL-17, TNF-α, IL-4, IL-10, iNOS NF-κB, 5-LOX, and COX-2. Eucalyptol has reduced the severity of CFA induced arthritis by promoting anti-inflammatory cytokines for example IL-4, IL-10 and by inhibiting pro-inflammatory cytokines such as 5-LOX, COX-2, IL-17, NF-κB, TNF-α, IL-6 and IL-1ß. Therefore, Eucalyptol might be as a potential therapeutic agent because of its pronounced anti-oxidant and anti-arthritic activity.

Artemisia herba-alba: antioxidant capacity and efficacy in preventing chronic arthritis in vivo.

Arthritis is a debilitating condition impacting the quality of life for millions worldwide, characterized by pain and inflammation. Understanding the mechanisms of arthritis and developing effective treatments are crucial. This study investigated the hydroethanolic extract of Artemisia herba-alba for its protective potential against arthritis hallmarks, oxidative stress, and lipid peroxidation in vitro. It also assessed its in vivo anti-arthritic activity. The phytochemical analysis identified various compounds within the extract, with high concentrations of polyphenols and flavonoids. These compounds are associated with numerous health benefits, making A. herba-alba a potential source of valuable phytochemicals. A. herba-alba demonstrated a notable effect in body weight loss, paw edema, and arthritic severity. Histopathological examination revealed structural improvements in bone and muscle tissues, emphasizing its therapeutic potential in managing chronic arthritis. Furthermore, while these findings are promising, further studies are necessary to delve deeper into the mechanisms underlying the observed hematological changes and to gain a more comprehensive understanding of the in vivo results. This research sets the stage for continued exploration, ultimately aiming to unlock the full potential of A. herba-alba in addressing chronic arthritis and enhancing the lives of those affected by this condition.

Anti-rheumatoid arthritis potential of Halodule pinifolia: development, characterization and in vivo evaluation of H. pinifolia-based oral suspension and lipid nano-emulsion.

For treating chronic diseases like rheumatoid arthritis, herbal medicines are preferred due to their evident therapeutic effects and lesser side effects as compared to the long-term used conventional drugs. In this study, the anti-rheumatoid arthritis effect of an unexplored marine grass Halodule pinifolia (HP), and a combination of it with Glycyrrhiza glabra (liquorice; LQ), prepared as a conventional suspension (C1) and a lipid nano-emulsion (C1-N) was evaluated in Freund's complete adjuvant (FCA)- and collagen-induced arthritis (CIA) models. Formulations C1 and C1-N contained standardized extract HP (100 mg/kg) as major active ingredient and liquorice LQ (50 mg/kg) as both active ingredient (anti-inflammatory and anti-ulcer) and sweetening agent. Oral administration of HP and C1 to FCA-induced Sprague-Dawley rats significantly reduced the paw oedema, spleen index, controlled the haematological parameters, cytokine levels (IL-1ß, IL-6, TNF-α estimated by ELISA), mRNA expression of cytokines and osteoclast markers (RANK, TRAP and cathepsin K measured by RTPCR). Histopathology and radiological scanning demonstrated lesser joint deterioration in sample-treated rats, as evident phenotypically. The downregulation of CD51 and MMP-3 (western blot) corroborated the anti-arthritic effect of HP and C1. HP showed better results among all. Further, under the CIA model, both C1 and C1-N were found to be potentially active as evidenced by their effect on rat paw oedema, spleen index, haematological parameters, rheumatoid factor, cytokines, osteoclast markers, histology and X-rays. The results proved the anti-arthritic effect of HP and the formulations, particularly the lipid nano-emulsion that showed improved stability as well as activity.

Launaea fragilis extract attenuated arthritis in rats through modulation of IL-1ß, TNF-α, IL-6, NF-κB, COX-2, IL-4, and IL-10.

Launaea fragilis (Asso) Pau is a Cholistan desert medicinal plant. Launaea species are used as traditional remedies against various inflammatory conditions. The current research was designed to evaluate the anti-nociceptive, anti-inflammatory, and anti-arthritic potential of ethanolic extract of L. fragilis (Et-LF). The plant extract was prepared by triple maceration. GC-MS screening explored the presence of various bioactive phytoconstituents including n-tetracosanol-1, 1-heptacosanol, and n-hexadecanoic acid. DPPH assay demonstrated the antioxidant potential of Et-LF. Safety profile data indicated that Et-LF was safe up to the oral dose of 5000 mg/kg in female rats. Anti-nociceptive activity of Et-LF was assessed in hot plate method and acetic acid-induced writhing model and the results suggested that Et-LF had significant analgesic effects in both animal models. Carrageenan, histamine, and serotonin-induced edema models were used to estimate the anti-inflammatory effects of Et-LF and were found to prevent paw edema development dose dependently. The anti-arthritic effect of Et-LF was estimated in CFA-induced arthritic rat model. Treatment with Et-LF 125, 250, 500 and flurbiprofen (FP) 10 mg/kg/day significantly attenuated the paw edema, reversed the reduced body weight, and restored the altered hematological parameters in arthritic rats. Gene expression studies revealed the significant downregulation of IL-1ß, TNF-α, IL-6, NF­κB, and COX-2, and upregulation of IL-4 and IL-10 in arthritic rats treated with various doses of plant extract. Histological evaluation of ankle joints showed that Et-LF mitigated pannus formation, infiltration of inflammatory cells, and fibrous connective tissue formation in the diseased rats. Thereof, it may be concluded that the recent study demonstrated the anti-nociceptive, anti-inflammatory, and anti-arthritic effects ascribed to the signifying presence of phytoconstituents in L. fragilis.

Novel antiarthritic mechanisms of Azelaic acid against CFA-induced arthritis in rats by modulating pro- and anti-inflammatory cytokines network.

An immunologic system attacking the body's own tissues is a hallmark of autoimmune disorders, which encompass a wide range of unique conditions. Numerous essential biologic functions, including the regulation of the immune system, inflammation, cell division, and tissue repair, are carried out by cytokines. Natural compounds are an effective treatment for autoimmune illnesses by modulation of inflammatory cytokines and infiltration of leukocytes into the inflamed tissue. Here, anti-arthritic study was carried out using oral administration of Azelaic acid (AzA) for 28 days with doses (20, 40, and 80 mg/kg) in Complete Freund's Adjuvant (CFA) induced arthritis model. AzA ameliorated the adjuvant-induced arthritis by decreasing arthritic score, paw volume, improved body-weight alterations and serum levels of PGE2, 5-LOX and anti-ccp. AzA showed significant down regulation of NF-κB, COX-II, TNF-α, IL-17, IL-1ß, IL-6, and up regulation of IL4 and IL10. Hemoglobin and RBCs count remarkably increased and ESR, CRP, platelets, WBCs levels markedly reduced in post treatment. In addition, the weakened SOD (superoxide dismutase), Catalase (CAT), Glutathione (GSH) activity and the increased levels of malondialdehyde (MDA) were all reversed by AzA treatment. And showed improved radiographical and histologic alterations in the structure of the joints. Molecular docking studies targeting COX-II, iNOS, TNF-α, 5-LOX, IL4, IL10, IL-6, and IL-17 establish a correlation between theoretical and experimental results. Results showed that AzA inhibit pro-inflammatory cytokines (COX-II, TNF-α, 5-LOX, IL-17, NF-κB, IL-1ß, and IL-6) and increase anti-inflammatory cytokines, which supported the anti-arthritic and immunomodulatory potential of AzA.

Salidroside suppresses cell growth and inflammatory response of fibroblast-like synoviocytes via inhibition of phosphoinositol-3 kinase/threonine kinase signaling in rheumatoid arthritis.

BACKGROUND: Salidroside (Sal) is a natural product commonly isolated from Rhodiola rosea L., which has been found to have numerous pharmacological activities (e.g., ameliorating apoptosis and inflammation, and acting as an antioxidant) in various diseases, but its concrete function in rheumatoid arthritis (RA) has not been revealed yet. Here, we aimed to explore the specific role and underlying mechanisms of Sal in RA-fibroblast-like synoviocytes (RA-FLSs). METHODS: Cell counting kit 8 (CCK-8) was used to assess the viability of normal-FLSs and RA-FLSs. Cell apoptosis in RA-FLSs was evaluated by flow cytometry. Western blotting was prepared to examine the levels of apoptosis- and signaling-related proteins. Wound-healing and Transwell assays were conducted to examine RA-FLSs migration and invasion. Enzyme-linked immunosorbent assay (ELISA) was used to assess the effect of Sal on tumor necrosis factor-alpha (TNF-α)-induced inflammation in RA-FLSs. RA animal model was established through complete Freund's adjuvant (CFA) induction, and the histopathological changes in synovial tissues of the rat model were analyzed by H&E staining. RESULTS: RA-FLSs were treated with 200 µM Sal for 24 h, and cell viability was significantly suppressed. Sal promoted RA-FLSs apoptosis. The migratory and invasive abilities of RA-FLSs were markedly inhibited by Sal. Sal incubation reduced the levels of inflammatory cytokines interleukin­8 (IL-8), IL-1ß, and IL­6 in RA-FLSs under the stimulation of TNF­α. Subsequently, Sal downregulated phosphorylated phosphatidylinositol­3 kinase (p-PI3K) and protein kinase (p-AKT) expression in RA-FLSs. After the treatment with pathway activator 740Y­P (20 µM) in RA-FLSs, the promotive effect of Sal on cell apoptosis was reversed, and inhibitory effects of it on cell viability, migration, invasion, and inflammatory response were abolished. Sal inhibited RA development in the CFA-induced rat model. CONCLUSION: Sal suppressed cell growth and inflammation in RA-FLSs by inactivating PI3K/AKT-signaling pathways.

Study of PT1 Peptide Analgesic and Anti-Inflammatory Activity on a Local Inflammation Model in Mice CD-1.

PT1 peptide isolated from the venom of spider Geolycosa sp. is a modulator of P2X3 receptors that play a role in the development of inflammation and the transmission of pain impulses. The anti-inflammatory and analgesic efficacy of the PT1 peptide was studied in a model of complete Freund's adjuvant-induced paw inflammation in CD-1 mice. The analgesic activity of PT1 peptide was maximum after intramuscular injection at a dose of 0.01 mg/kg, which surpassed the analgesic effect of diclofenac at a dose of 1 mg/kg. The anti-inflammatory activity was maximum after intramuscular injection at a dose of 0.0001 mg/kg; a decrease in paw thickness was observed as soon as 2 h after the administration of the PT1 peptide against the background of inflammation development. All tested doses of PT1 peptide showed high anti-inflammatory activity 4 and 24 h after administration. PT1 peptide at a dose of 0.01 mg/kg when injected intramuscularly simultaneously produced high anti-inflammatory and analgesic effects compared to other doses of the peptide. Increasing the dose of PT1 peptide led to a gradual decrease in its analgesic and anti-inflammatory activity; increasing the dose of intramuscular injection to 0.1 and 1 mg/kg is inappropriate.

Anti-inflammatory and analgesic effect of Forsythiaside B on complete Freund's adjuvant-induced inflammatory pain in mice.

Chronic pain is frequently reported in clinical practice. Therefore, it is important to identify effective therapy to relieve pain. In this work, we selected Forsythoside B (FB), a phenylethanoid glycoside isolated from Forsythia suspensa (Thunb.) Vahl, to evaluate its effect in modulating inflammatory pain induced by complete Freund's adjuvant (CFA) and the involved mechanisms. We discovered that FB could attenuate inflammatory pain triggered by CFA injection and exert anti-anxiety effects. In detail, proinflammatory cytokines, consisting of IL-6 and TNF-α, were decreased after FB administration in the CFA-injected mice. Furthermore, the FB application ameliorated the activation of ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), the microglia and astrocytes markers respectively. Therefore, our findings indicate that FB could be a promising treatment for chronic inflammatory pain.

Fibroblast-derived PI16 sustains inflammatory pain via regulation of CD206+ myeloid cells.

Originally identified in fibroblasts, Protease Inhibitor (PI)16 was recently shown to be crucial for the development of neuropathic pain via effects on blood-nerve barrier permeability and leukocyte infiltration, though its impact on inflammatory pain has not been established. Using the complete Freund's Adjuvant inflammatory pain model, we show that Pi16-/- mice are protected against sustained inflammatory pain. Accordingly, intrathecal delivery of a PI16 neutralizing antibody in wild-type mice prevented sustained CFA pain. In contrast to neuropathic pain models, we did not observe any changes in blood-nerve barrier permeability due to PI16 deletion. Instead, Pi16-/- mice display reduced macrophage density in the CFA-injected hindpaw. Furthermore, there was a significant bias toward CD206hi (anti-inflammatory) macrophages in the hindpaw and associated dorsal root ganglia. Following CFA, intrathecal depletion of CD206+ macrophages using mannosylated clodronate liposomes promoted sustained pain in Pi16-/- mice. Similarly, an IL-10 neutralizing antibody also promoted sustained CFA pain in the Pi16-/ when administered intrathecally. Collectively, our results point to fibroblast-derived PI16 mediating substantial differences in macrophage phenotype in the pain neuroaxis under conditions of inflammation. The co-expression of PI16 alongside fibroblast markers in human DRG raise the likelihood that a similar mechanism operates in human inflammatory pain states. Collectively, our findings may have implications for targeting fibroblast-immune cell crosstalk for the treatment of chronic pain.

Transcrocetin Meglumine Salt Inhibits Spinal Glial Cell-Mediated Proinflammatory Cytokines and Attenuates Complete Freund's Adjuvant-Induced Inflammatory Pain.

INTRODUCTION: Inflammatory pain is a significant global clinical challenge that involves both unpleasant sensory and emotional experiences. The treatment of pain is imminent, and we are committed to seeking new analgesics for pain relief. Transcrocetin meglumine salt (TCMS), a saffron metabolite derived from the crocin apocarotenoids, has exhibited the ability to cross the blood-brain barrier and exert neuroprotective effects. In this study, we aimed to investigate whether TCMS could ameliorate complete Freund's adjuvant (CFA)-induced inflammatory pain in mice and elucidate its underlying mechanisms. METHODS: Here, we established an inflammatory pain model in mice by injecting CFA into the left hind paw. Three days later, we administered intraperitoneal injections of TCMS (10 mg/kg) or saline to the animals. We examined mechanical allodynia, thermal hypersensitivity, and anxiety behavior. Furthermore, the activation of glial cells and proinflammatory cytokines in the spinal cord were detected. RESULTS: Our results showed that TCMS significantly reversed the mechanical allodynia and thermal hypersensitivity in the CFA-injected mice. Furthermore, TCMS administration effectively inhibited the activation of microglia and astrocytes in the spinal cord induced by CFA. Additionally, TCMS suppressed the production and release of spinal proinflammatory cytokines, including TNF-α, IL-1ß, and IL-6, in CFA-injected mice. CONCLUSION: Taken together, our findings demonstrate that TCMS holds promise as an innovative analgesic due to its ability to ameliorate inflammatory reactions.

Inoculation of Freund's adjuvant in European eel (Anguilla anguilla) revealed key KEGG pathways and DEGs of host anti-Edwardsiella anguillarum infection.

Freund's complete (FCA) and incomplete adjuvants (FIA), generally applied in subunit fishery vaccine, have not been explored on the molecular mechanism of the nonspecific immune enhancement. In this study, we examined the RNA-seq in the spleen of European eel (Anguilla anguilla) inoculated with FCA and FIA (FCIA group) to elucidate the key KEGG pathways and differential expressed genes (DEGs) in the process of Edwardsiella anguillarum infection and A. anguilla anti-E. anguillarum infection using genome-wide transcriptome. After eels were challenged by E. anguillarum at 28 d post the first inoculation (dpi), compared to the control uninfected eels (Con group), the control infected eels (Con_inf group) showed severe pathological changes in the liver, kidney and spleen, although infected eels post the inoculation of FCIA (FCIA_inf group) also formed slight bleeding. Compared to the FCIA_inf group, there was more than 10 times colony forming unit (cfu) in the Con_inf group per 100 µg spleen, kidney or blood, and the relative percent survival (RPS) of eels was 44.4% in FCIA_inf vs Con_inf. Compared to the Con group, the SOD activity in the FCIA group increased significantly in the liver and spleen. Using high-throughput transcriptomics, DEGs were identified and 29 genes were verified using fluorescence real-time polymerase chain reaction (qRT-PCR). The result of DEGs clustering showed 9 samples in 3 groups of Con, FCIA and FCIA_inf were similar, contrast to distinct differences of 3 samples in the Con_inf group. We found 3795 up and 3548 down regulated DEGs in the compare of FCIA_inf vs Con_inf, of which 5 enriched KEGG pathways of "Lysosome", "Autophagy", "Apoptosis", "C-type lectin receptor signaling" and "Insulin signaling" were ascertained, and 26 of 30 top GO terms in the compare were significantly enriched. Finally, protein-protein interactions between the DEGs of the 5 KEGG pathways and other DEGs were explored using Cytoscape 3.9.1. The compare of FCIA_inf vs Con_inf showed 110 DEGs from the 5 pathways and 718 DEGs from other pathways formed total of 9747° in a network, of which 9 hub DEGs play vital roles in anti-infection or apoptosis. Together, the interaction networks revealed that 9 DEGs involved in the 5 pathways underlies the key process of A. anguilla anti-E. anguillarum infection or host cell apoptosis.

Efficacy of radial shock wave therapy on rat models of adjuvant arthritis.

BACKGROUND: Extracorporeal shock wave therapy (ESWT) is an effective treatment for musculoskeletal pain, tendinopathy, and fasciitis with an anti-inflammatory effect. ESWT can be categorized into two groups: radial pressure wave (RPW) and focused shock wave (FSW). Although there have been several studies on the inflammation and pain-improvement mechanisms of FSW, there are few studies on the pain-improvement mechanisms of RPW. This study aimed to elucidate the efficacy of RPW in a rat model of adjuvant arthritis. METHODS: Ninety-six rats were randomly categorized into three groups: RPW, control, and sham as follows: (I) RPW group, which received RPW application after complete Freund's adjuvant (CFA) injection; (II) Control group, which received only CFA injection; and (III) Sham group, which received only saline injection. All rats were evaluated at 0, 4, 7, 14, 28, and 56 days post-RPW application based on foot circumference, von Frey test, and immunohistochemistry of nerve fibers for calcitonin gene-related peptide (CGRP) and protein gene product (PGP) 9.5 in plantar skins. RESULTS: There were no significant differences in foot circumference between the RPW and control groups at any time point. The RPW group showed significant improvements in the von Frey test results on days 7 and 14. The total CGRP-immunoreactive (ir) and PGP9.5-ir nerve fiber lengths in the RPW group decreased on day 0; however, both were increased in the control group. The CGRP-ir and PGP9.5-ir nerve fibers in the RPW group were significantly shorter than those in the control group until day 14 after RPW. CONCLUSIONS: RPW improved the mechanical hypersensitivity between days 7 and 14 after application. Like FSW, RPW also induced the degeneration of sensory nerve fibers in the skin in the early period after irradiation, and reinnervation occurred between 14 and 28 days. Thus, our results demonstrate one of the pain relief mechanisms after RPW application.

Assessment of anti-arthritic activity of lipid matrix encased berberine in rheumatic animal model.

The purpose of this study was to evaluate the drug delivery and therapeutic potential of berberine (Br) loaded nanoformulation in rheumatoid arthritis (RA)-induced animal model. The Br-loaded NLCs (nanostructured lipid carriers) were prepared employing melt-emulsification process, and optimised through Box-Behnken design. The prepared NLCs were assessed for in-vitro and in-vivo evaluations. The optimised NLCs exhibited a mean diameter of 180.2 ± 0.31 nm with 88.32 ± 2.43% entrapment efficiency. An enhanced anti-arthritic activity with reduced arthritic scores to 0.66 ± 0.51, reduction in ankle diameter to 5.80 ± 0.27 mm, decline in paw withdrawal timing, and improvements in walking behaviour were observed in the Br-NLCs treated group. The radiographic images revealed a reduction in bone and cartilage deformation. The Br-NLCs showed promising results in the management of RA disease, can be developed as an efficient delivery system at commercial levels, and may be explored for clinical application after suitable experiments in the future.