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.

Macrophage Activation in Synovitis and Osteoarthritis of Temporomandibular Joint and Its Relationship with the Progression of Synovitis and Bone Remodeling.

This study investigates the regulatory mechanisms of synovial macrophages and their polarization in the progression of temporomandibular joint osteoarthritis (TMJOA). Macrophage depletion models were established by intra-articular injection of clodronate liposomes and unloaded liposomes. TMJOA was induced by intra-articular injection of 50 µL Complete Freund's Adjuvant and the surgery of disc perforation. The contralateral joint was used as the control group. The expression of F4/80, CD86, and CD206 in the synovium was detected by immunofluorescence staining analysis. Hematoxylin and eosin staining and TMJOA synovial score were detected to show the synovial changes in rat joints after TMJOA induction and macrophage depletion. Changes in rat cartilage after TMJOA induction and macrophage depletion were shown by safranin fast green staining. The bone-related parameters of rats' joints were evaluated by micro-computed tomography analysis. The TMJOA model induced by Complete Freund's Adjuvant injection and disc perforation aggravated synovial hyperplasia and showed a significant up-regulation of expression of F4/80-, CD86-, and CD206-positive cells. F4/80, CD86, and CD206 staining levels were significantly decreased in macrophage depletion rats, whereas the synovitis score further increased and cartilage and subchondral bone destruction was slightly aggravated. Macrophages were crucially involved in the progression of TMJOA, and macrophage depletion in TMJOA synoviocytes promoted synovitis and cartilage destruction.

CFA-treated mice induce hyperalgesia in healthy mice via an olfactory mechanism.

BACKGROUND: Social interactions with subjects experiencing pain can increase nociceptive sensitivity in observers, even without direct physical contact. In previous experiments, extended indirect exposure to soiled bedding from mice with alcohol withdrawal-related hyperalgesia enhanced nociception in their conspecifics. This finding suggested that olfactory cues could be sufficient for nociceptive hypersensitivity in otherwise untreated animals (also known as "bystanders"). AIM: The current study addressed this possibility using an inflammation-based hyperalgesia model and long- and short-term exposure paradigms in C57BL/6J mice. MATERIALS & METHOD: Adult male and female mice received intraplantar injection of complete Freund's adjuvant (CFA) and were used as stimulus animals to otherwise naïve same-sex bystander mice (BS). Another group of untreated mice (OLF) was simultaneously exposed to the bedding of the stimulus mice. RESULTS: In the long-term, 15-day exposure paradigm, the presence of CFA mice or their bedding resulted in reduced von Frey threshold but not Hargreaves paw withdrawal latency in BS or OLF mice. In the short-term paradigm, 1-hr interaction with CFA conspecifics or 1-hr exposure to their bedding induced mechanical hypersensitivity in BS and OLF mice lasting for 3 hrs. Chemical ablation of the main olfactory epithelium prevented bedding-induced and stimulus mice-induced mechanical hypersensitivity. Gas chromatography-mass spectrometry (GC-MS) analysis of the volatile compounds in the bedding of experimental mice revealed that CFA-treated mice released an increased number of compounds indicative of disease states. DISCUSSION AND CONCLUSION: These results demonstrate that CFA-induced inflammatory pain can modulate nociception in bystander mice via an olfactory mechanism involving dynamic changes in volatile compounds detectable in the rodent bedding. SIGNIFICANCE: Social context can influence nociceptive sensitivity. Recent studies suggested involvement of olfaction in this influence. In agreement with this idea, the present study shows that the presence of mice with inflammatory pain produces nociceptive hypersensitivity in nearby conspecifics. This enhanced nociception occurs via olfactory cues present in the mouse bedding. Analysis of the bedding from mice with inflammatory pain identifies a number of compounds indicative of disease states. These findings demonstrate the importance of olfactory system in influencing pain states.

miR­3120/Hsc70 participates in forced swim stress­induced mechanical hyperalgesia in rats in an inflammatory state.

The heat shock cognate 71 kDa protein (Hsc70) is a stress­inducible ATPase that can protect cells against harmful stimuli. Transient receptor potential vanilloid 1 (TRPV1) is a well­documented nociceptor. Notably, Hsc70 can inhibit TRPV1 expression and function, suggesting that Hsc70 may have pain regulation potential. However, the role of Hsc70 in stress­induced hyperalgesia remains unclear. In the present study, the participation of Hsc70 and its regulator microRNA (miR)­3120 were investigated in forced swim (FS) stress­induced mechanical hyperalgesia in rats in an inflammatory state. Complete Freund's adjuvant (CFA) hind paw injection was performed to induce inflammatory pain in rats (CFA rats). Furthermore, in FS + CFA rats, FS stress was performed for 3 days before CFA injection. The levels of Hsc70, miR­3120 and their downstream molecule TRPV1 were measured in the dorsal root ganglion (DRG) with western blotting, immunofluorescence, reverse transcription­quantitative polymerase chain reaction and fluorescence in situ hybridization. The results revealed that FS stress significantly exacerbated CFA­induced mechanical pain. Furthermore, CFA upregulated Hsc70 and TRPV1 expression, which was partially inhibited or further enhanced by FS stress, respectively. In FS + CFA rats, intrathecal injection of a lentiviral vector overexpressing Hsc70 (LV­Hsc70) could decrease TRPV1 expression and improve the mechanical pain. Additionally, the expression levels of miR­3120, a regulator of Hsc70, were markedly upregulated on day 3 following FS stress. Finally, miR­3120 was identified to be colocalized with Hsc70 and expressed in all sizes of DRG neurons. In CFA rats, DRG injection of miR­3120 agomir to induce overexpression of miR­3120 resulted in similar TRPV1 expression and behavioral changes as those caused by FS stress, which were abolished in the presence of LV­Hsc70. These findings suggested that miR­3120/Hsc70 may participate in FS stress­induced mechanical hyperalgesia in rats in an inflammatory state, possibly via disinhibiting TRPV1 expression in the DRG neurons.

From co-delivery to synergistic anti-inflammatory effect: Studies on chitosan-stabilized Janus emulsions having chloroquine phosphate and flavopiridol in Complete Freund's Adjuvant induced arthritis rat model.

For the first time, the co-delivery of chloroquine phosphate and flavopiridol by intra-articular route was achieved to provide local joint targeting in Complete Freund's Adjuvant-induced arthritis rat model. The presence of paired-bean structure onto the dispersed oil droplets of o/w nanosized emulsions allows efficient entrapment of two drugs (85.86-96.22 %). The dual drug-loaded emulsions displayed a differential in vitro drug release behavior, near normal cell viability in MTT assay, better cell uptake (internalization) and better reducing effect of mean immunofluorescence intensity of inflammatory proteins such as NF-κB and iNOS at in vitro RAW264.7 macrophage cell line. The radiographical study, ELISA test, RT-PCR study and H & E staining also indicated a reduction in joint tissue swelling, IL-6 and TNF-α levels diminution, fold change diminution in the mRNA expressions for NF-κB, IL-1ß, IL-6 and PGE2 and maintenance of near normal histology at bone cartilage interface respectively. The results of metabolomic pathway analysis performed by LC-MS/MS method using the rat blood (plasma) collected from disease control and dual drug-loaded emulsions treatment groups revealed a new follow-up study to understand not only the disease progression but also the formulation therapeutic efficacy assessment.

Associations of tissue damage induced inflammatory plasticity in masseter muscle with the resolution of chronic myalgia.

Gene plasticity during myogenous temporomandibular disorder (TMDM) development is largely unknown. TMDM could be modeled by intramuscular inflammation or tissue damage. To model inflammation induced TMDM we injected complete Freund's adjuvant (CFA) into masseter muscle (MM). To model tissue damage induced TMDM we injected extracellular matrix degrading collagenase type 2 (Col). CFA and Col produced distinct myalgia development trajectories. We performed bulk RNA-seq of MM to generate gene plasticity time course. CFA initiated TMDM (1d post-injection) was mainly linked to chemo-tacticity of monocytes and neutrophils. At CFA-induced hypersensitivity post-resolution (5d post-injection), tissue repair processes were pronounced, while inflammation was absent. Col (0.2U) produced acute hypersensitivity linked to tissue repair without inflammatory processes. Col (10U) generated prolonged hypersensitivity with inflammatory processes dominating initiation phase (1d). Pre-resolution phase (6d) was accompanied with acceleration of expressions for tissue repair and pro-inflammatory genes. Flow cytometry showed that immune processes in MM was associated with accumulations of macrophages, natural killer, dendritic and T-cells, further confirming our RNA-seq findings. Altogether, CFA and Col treatments induced different immune processes in MM. Importantly, TMDM resolution was preceded with muscle cell and extracellular matrix repairs, an elevation in immune system gene expressions and distinct immune cell accumulations in MM.

Spinal apolipoprotein E is involved in inflammatory pain via regulating lipid metabolism and glial activation in the spinal dorsal horn.

INTRODUCTION: Inflammation and nerve injury promote astrocyte activation, which regulates the development and resolution of pain, in the spinal dorsal horn. APOE regulates lipid metabolism and is predominantly expressed in the astrocytes. However, the effect of astrocytic APOE and lipid metabolism on spinal cellular function is unclear. This study aimed to investigate the effect of spinal Apoe on spinal cellular functions using the complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model. METHODS: After intraplantar injection of CFA, we assessed pain behaviors in C57BL6 and Apoe knockout (Apoe-/-) mice using von Frey and Hargreaves' tests and analyzed dorsal horn samples (L4-5) using western blotting, immunofluorescence, quantitative real-time polymerase chain reaction, and RNA sequencing. RESULTS: The Apoe levels were markedly upregulated at 2 h and on days 1 and 3 post-CFA treatment. Apoe was exclusively expressed in the astrocytes. Apoe-/- mice exhibited decreased pain on day 1, but not at 2 h, post-CFA treatment. Apoe-/- mice also showed decreased spinal neuron excitability and paw edema on day 1 post-CFA treatment. Global transcriptomic analysis of the dorsal horn on day 1 post-CFA treatment revealed that the differentially expressed mRNAs in Apoe-/- mice were associated with lipid metabolism and the immune system. Astrocyte activation was impaired in Apoe-/- mice on day 1 post-CFA treatment. The intrathecal injection of Apoe antisense oligonucleotide mitigated CFA-induced pain hypersensitivity. CONCLUSIONS: Apoe deficiency altered lipid metabolism in astrocytes, exerting regulatory effects on immune response, astrocyte activation, and neuronal activity and consequently disrupting the maintenance of inflammatory pain after peripheral inflammation. Targeting APOE is a potential anti-nociception and anti-inflammatory strategy.

A study on the therapeutic potential of graphene titanate nanocomposite for treating chemically induced arthritis in rats.

Nanotechnology holds substantial promise in the innovative therapies for rheumatoid arthritis (RA). The current study was designed to synthesize and characterize a new graphene titanate nanocomposite (GTNc) and explore its anti-arthritic, anti-inflammatory, and antioxidant potencies against Complete Freund's adjuvant (CFA)-induced arthritis in rats, as well as investigate the underlying molecular mechanisms. Our characterization methods included XRD, FT-IR, SEM, EDX, zeta potential, practical size, and XRF to characterize the novel GTNc. Our findings revealed that arthritic rats treated with GTNc exhibited lower levels of RF, CRP, IL-1ß, TNF-α, IL-17, and ADAMTS-5, and higher levels of IL-4 and TIMP-3. In arthritic rats, GTNc reduced LPO levels while increasing GSH content and GST antioxidant activity. Additionally, GTNc decreased the expression of the TGF-ß mRNA gene in arthritic rats. Histopathological investigation showed that GTNc reduced inflammatory cell infiltration, cartilage degradation, and bone destruction in joint injuries caused by CFA in the arthritic rats. Collectively, the anti-arthritic, anti-inflammatory, and antioxidant properties of GTNc appear promising for future arthritis treatments and bone disability research.

Anti-arthritic and Antioxidant Effects of Trehalose in an Experimental Model of Arthritis.

BACKGROUND: The purpose of the present study was to study the potential anti-arthritic and antioxidant effects of trehalose in an experimental model of complete Freund's adjuvant (CFA)-induced arthritis. METHODS: Arthritis was induced via subcutaneous injection of CFA (0.1) into the right footpad of each rat. Trehalose (10 mg/kg per day) and indomethacin (5 mg/kg) as a reference drug were intraperitoneally injected into CFA-induced arthritic rats from days 0 to 21. Changes in paw volume, pain responses, arthritic score, and oxidative/antioxidative parameters were determined. RESULTS: Trehalose administration could significantly decrease arthritis scores (p <0.01) and paw edema (p <0.001), and significantly increase the nociceptive threshold (p <0.05) in CFA-induced arthritic rats. Trehalose also significantly reduced the pro-oxidant-antioxidant balance values when compared to CFA treatment alone. In addition, no significant difference was found between the trehalose group and indomethacin as a positive control group. CONCLUSION: The current study suggests that trehalose has a protective effect against arthritis, which may be mediated by antioxidative effects of this disaccharide.

Protective effect of fustin against adjuvant-induced arthritis through the restoration of proinflammatory response and oxidative stress.

Rheumatoid arthritis causes irreparable damage to joints. The present research sought to check fustin's anti-arthritic efficacy against the complete Freund's adjuvant-induced arthritis paradigm in animals by altering the inflammatory response. In the rats, complete Freund's adjuvant was used to trigger arthritis and they received fustin at 50 and 100 mg/kg for 21 days. At regular intervals, the hind paw volume and arthritic score were assessed. After the trial period, hematological, antioxidant, pro-inflammatory cytokines, and other biochemical parameters were estimated. Fustin-treated rats showed the down-regulation of hind paw volume, arthritic score, and altered hematological parameters (TLC, DLC (neutrophil, lymphocyte, monocyte, eosinophil, basophil)). Furthermore, fustin significantly mitigates proinflammatory cytokine (reduced interleukin, tumor necrosis factor-a (TNF-α), IL-6, IL-1ß), oxidative stress (attenuated malondialdehyde (MDA), catalase (CAT), glutathione (GSH), superoxide dismutase (SOD)), attenuated production of prostaglandin E2 and myeloperoxidase (MPO) and improved nuclear factor erythroid 2-related factor (Nrf2) action. Fustin led to the benefit in arthritis-prone animals elicited by complete Freund's adjuvant via pro-inflammatory cytokine.

Research on the improvement effect of Saposhnikovia divaricata (Trucz.) Schischk on rheumatoid arthritis based on the "component-target-pathway" association.

OBJECTIVE: To investigate the therapeutic effect and mechanism of the traditional Chinese medicine Saposhnikovia divaricata (Trucz.) Schischk in rats with complete Freund's adjuvant-induced rheumatoid arthritis (RA). METHODS: The chemical targets and RA targets of Saposhnikovia divaricata (Trucz.) Schischk were acquired by the network pharmacological method. The complete Freund's adjuvant-induced rat RA model was used to further explore the mechanism of Saposhnikovia divaricata (Trucz.) Schischk in improving RA. Pathological changes in the volume of toes, body weight and synovial tissues of joints as well as serum inflammatory factor levels before and after the intervention of Saposhnikovia divaricata (Trucz.) Schischk were investigated. The key metabolic pathways were screened by correlations between metabolites and key targets. Finally, a quantitative analysis of key targets and metabolites was experimentally validated. RESULTS: Saposhnikovia divaricata (Trucz.) Schischk administration increased body weight, mitigated foot swelling and downregulated inflammatory cytokine levels in model rats. The histopathology showed that treatment with Saposhnikovia divaricata (Trucz.) Schischk can induce inflammatory cell infiltration and synovial hyperplasia and obviously reduce cartilage injuries, thus improving arthritis symptoms in rats. According to the network pharmacology-metabonomics association analysis results, the purine metabolic signaling pathway might be the key pathway for RA intervention with Saposhnikovia divaricata (Trucz.) Schischk. Targeted metabonomics, Western blotting (WB) and reverse transcription-polymerase chain reaction (RT‒PCR) assays showed that the recombinant adenosine deaminase (ADA) mRNA expression level and metabolic level of inosine in Saposhnikovia divaricata (Trucz.) Schischk administration group were lower than those of the model group. This reflected that Saposhnikovia divaricata (Trucz.) Schischk could improve RA by downregulating ADA mRNA expression levels and the metabolic level of inosine in the purine signaling pathway. CONCLUSION: Based on the "component-disease-target" association analysis, this study concludes that Saposhnikovia divaricata (Trucz.) Schischk improves complete Freund's adjuvant-induced RA symptoms in rats mainly by downregulating ADA mRNA expression levels in the purine metabolic signaling pathway, mitigating foot swelling, improving the levels of serum inflammatory factors (IL-1ß, IL-6 and TNF-α), and decreasing the ADA protein expression level to intervene in purine metabolism.

A-80426 suppresses CFA-induced inflammatory pain by suppressing TRPV1 activity via NFκB and PI3K pathways in mice.

OBJECTIVES: Pain is associated with many circumstances, including inflammatory reactions, which arise from modification of the features of signaling pathways. α2-adrenergic receptor antagonists are widely utilized in narcosis. Here, the authors focused on the narcotic effect of A-80426 (A8) on Complete Freund's Adjuvant (CFA) injections-triggered chronic inflammation pain in WT and TRPV1-/- mice and explored whether its antinociceptive impact was modulated via Transient Receptor Potential Vanilloid 1 (TRPV1). METHOD: CFA with or without A8 was co-administered to the mice, which were categorized randomly into four groups: CFA, A8, control, and vehicle. Pain behaviors underwent evaluation through mechanical withdrawal threshold, abdominal withdrawal reflex, and thermal withdrawal latency of WT animals. RESULTS: Quantitative polymerase chain reaction revealed that inflammation-promoting cytokines (IL-1ß, IL-6, and TNF-α) were upregulated in Dorsal Root Ganglion (DRG) and Spinal Cord Dorsal Horn (SCDH) tissues of WT animals. A8 administration reduced the pain behaviors and production of pro-inflammatory cytokines; however, this effect was significantly reduced in TRPV1-/- mice. Further analysis showed that CFA treatment reduced the TRPV1 expression in WT mice and A8 administration increased its expression and activity. The co-administration of SB-705498, a TRPV1 blocker, did not influence the pain behaviors and inflammation cytokines in CFA WT mice; however, SB-705498 the effect of A8 in WT mice. In addition, the TRPV1 block decreased the NFκB and PI3K activation in the Dorsal Root Ganglia (DRG) and Spinal Cord Dorsal Horn (SCDH) tissues of WT mice. CONCLUSIONS: Together, A8 exerted a narcotic impact on CFA-supplemented mice via the TRPV1-modulated NFκB and PI3K pathway.

Lariciresinol protects rats from complete Freund's adjuvant induced arthritis in rats via modulation of transforming growth factor-ß and nuclear factor kappa B pathway: An in vivo and in silico study.

Rheumatoid arthritis (RA) is a severe inflammatory auto-immune disorder affecting millions of people across the globe. The current therapeutic options are not adequate to address the complications of RA. Therefore, the present study was conducted to elucidate the protective effect of lariciresinol, a lignan, against Complete Freund's adjuvant (CFA)-induced arthritis in rats. The results of the study showed that lariciresinol improves paw swelling and arthritic scores in rats as compared to CFA rats. Lariciresinol also showed a significant reduction in rheumatoid factor, C-reactive protein, tumor necrosis factor-α, interleukin (IL)-17, and tissue inhibitor of metalloproteinases-3 level with a simultaneous increase in IL-4 level. The burden of oxidative stress was also reduced in CFA rats, as shown by reduced MDA levels and increased SOD and GPx after the administration of lariciresinol. In a Western blot analysis, lariciresinol showed a significant reduction of transforming growth factor-ß and nuclear factor-κB (NF-κB) protein levels in CFA rats. To understand the binding characteristic of lariciresinol with NF-κB, molecular docking analysis was conducted, which showed Larciresinol interacted with the active site of NF-κB. Our study demonstrated the significant protective effect of lariciresinol against RA via multi-target action.

Evaluation of therapeutic potential of ivermectin against complete Freund's adjuvant-induced arthritis in rats: Involvement of inflammatory mediators.

Rheumatoid arthritis is a chronic systemic inflammatory disease with genetic manifestations. According to recently published case reports, patients taking corticosteroid medication for the management of rheumatoid arthritis develop strongloidiasis and are at high risk of developing associated infections. This study explored the antiarthritic role of ivermectin, a drug used in the treatment of strongyloides and to compare its results with dexamethasone. Thirty-two male Wistar rats were randomly divided into four groups: control, diseased, dexamethasone, and ivermectin groups. Rheumatoid arthritis in all rats except the control group was induced by using complete Freund's adjuvant. After 7 days of rheumatoid arthritis induction, animals were treated with dexamethasone 5 mg/kg and ivermectin 6 mg/kg. Body weight, visual arthritic score, total leukocyte count, differential leukocyte count, proinflammatory genes, and histopathological findings were used to assess the effects of ivermectin on rheumatoid arthritis. Treatment with ivermectin showed a significant reduction in inflammatory cells levels, body weight, and visual arthritic score, indicating an improvement in the degree of inflammation as compared with the diseased group. Treatment with ivermectin and dexamethasone significantly reduced the augmentation in the mRNA expression levels of IL-17, TLR-2, TNF, and NF-κB as a result of arthritic development. Ivermectin treatment also showed a significant reduction in the severity of inflammation and destruction of joints and showed comparable effects to dexamethasone, a corticosteroid used for the treatment of rheumatoid arthritis. Ivermectin has significant antiarthritic properties and can be a novel treatment agent for the management of rheumatoid arthritis patients suffering from strongyloidiasis.

Bone marrow-derived mesenchymal stem cells attenuate complete Freund's adjuvant-induced inflammatory pain by inhibiting the expression of P2X3.

Bone marrow-derived mesenchymal stem cells (BMSCs) show a good property for pain treatment by modulating inflammatory response. However, the underlying therapeutic effect and related mechanism of BMSCs on inflammatory pain remain unclear. Therefore, we explored the function and potential mechanism of BMSCs performing in a complete Freund's adjuvant (CFA)-induced inflammatory pain model in this study. Here, BMSCs were injected into the CFA-treated rats, and we used behavioural tests to evaluate the changes in hypersensitivity. High-throughput sequencing was used to screen out the hub genes. Molecular biology experiments were performed to detect the level of P2X3 or inflammatory mediators in rats and observed the distribution of P2X3 in neural cells. Furthermore, the function of the P2X3 was explored via inhibitor and activator experiments. Finally, we found that BMSCs alleviated hyperalgesia and spinal levels of pro-inflammatory factors in CFA-treated rats. High-throughput sequencing showed that P2X3 and P2X7 were identified as hub genes, and only the expression level of P2X3 was significantly down-regulated after BMSCs treatment. Immunohistochemistry showed that P2X3 mainly colocalized with microglia and astrocytes. The levels of P2X3 and pro-inflammatory factors were all significantly reduced after BMSC injection. Moreover, similar attenuation was found in the CFA-treated rats after injecting the P2X3 inhibitor, and a P2X3 antagonist reversed the attenuation induced by the BMSCs. These findings suggest that BMSCs exerted a therapeutic effect on inflammatory pain by inhibiting the expression of P2X3 and the excessive production of inflammatory mediators was associated with an increased P2X3 level and BMSC therapy reverse these effects.

Electroacupuncture Alleviates CFA-Induced Inflammatory Pain via PD-L1/PD-1-SHP-1 Pathway.

Inflammatory pain is difficult to treat clinically, but electroacupuncture (EA) has been demonstrated to be effective in alleviating inflammatory pain. Programmed cell death ligand-1 (PD-L1) and its downstream signal, Src homology region two domain-containing phosphatase-1 (SHP-1) have a critical role in relieving inflammatory pain. However, whether the PD-L1/PD-1-SHP-1 pathway mediates the analgesic and anti-inflammatory effects of EA in inflammatory pain remains unclear. Here, we observed that EA reversed the complete Freund's adjuvant (CFA)-induced hyperalgesia. EA reduced the expression of IL-6, iNOS, and NF-κB pathway in dorsal root ganglia (DRG) on day 7 after CFA injection but had no effect on the expression of IL-6, iNOS, and NF-κB PP65 on day 21 after CFA injection. Moreover, EA upregulated the protein levels of the PD-L1/PD-1-SHP-1 pathway on day 7 and day 21 after CFA injection. Furthermore, EA upregulated PD-L1 expression in calcitonin gene-related peptide (CGRP)+ but not in isohaemagglutinin B4 (IB4)+ and NF200+ neurons on day 7 and day 21 after CFA injection. Intrathecal injection of the PD-L1/PD-1 inhibitor BMS-1 (50 or 100 µg) blocked the EA-induced analgesic effect, significantly increased IL-6 and iNOS levels, and reduced the levels of PD-L1/PD-1-SHP-1. BMS-1 (50 or 100 µg) significantly reduced the expression of PD-L1 in IB4+, CGRP+, and NF200+ neurons. Our results show that EA's anti-inflammatory and analgesic effects are associated with activating the PD-L1/PD-1-SHP-1 pathway and suppressing its regulated neuroinflammation. This study provides a new potential therapeutic target for treating inflammatory pain.

Caspase-11 contributes to pain hypersensitivity in the later phase of CFA-induced pain of mice.

Chronic pain is a common disease that severely disrupts the quality of life. Persistent neuroinflammation and central sensitization play important roles in its pathogenesis. Caspase-11 is a critical modulator of inflammation of central nervous system. However, its role in chronic pain remains elusive. In this study, chronic pain and acute pain were induced via injecting complete Freund's adjuvant (CFA) and 5 % formalin into the plantar of the right hind paw of wild-type (WT) and Caspase-11 deficient (Caspase-11-/-) mice, respectively. In WT mice, CFA injection significantly decreased the hind paw mechanical pain threshold in Von Frey test on 1-7 days after injection and increased the caspase-11 level of ipsilateral dorsal horn of spinal cord on day 2 and day 5 after injection. Compared to the WT mice, Caspase-11-/- mice showed significantly higher mechanical pain threshold in the later phase of CFA-induced pain, but not in the early phase, and had no significant difference in 5 % formalin induced licking and flinching behavior. In addition, the microglial activation, and the mRNA levels of caspase-1 and IL-18 in the spinal cord of Caspase-11-/- mice restored to baseline on the day 5 after CFA injection, but not in WT mice. Our data indicated that Caspase-11 contributed to persistent inflammation in ipsilateral dorsal horn of spinal cord, and consequently pain hypersensitivity in the later phase of CFA-induced pain.

Nimbolide attenuates complete Freund's adjuvant induced arthritis through expression regulation of toll-like receptors signaling pathway.

Nimbolide is an active constituent of Azadirachta indica and is known for its anti-inflammatory, anti-oxidant, immune-modulatory, and anti-cancer effects. Few studies suggest that nimbolide treatment influences the responses to rheumatoid arthritis, but the underlying molecular mechanisms involved are not yet well established. Therefore, the present study was designed to determine the effect of nimbolide on expression regulation of toll-like receptors to attenuate rheumatoid arthritis. The rheumatoid arthritis model was established by injecting complete Freund's adjuvant (CFA) intra-dermally into the sub-plantar region of the left hind paw of rats. Nimbolide (20 mg/kg) and piroxicam (10 mg/kg) were given to arthritic rats. Rats treated with nimbolide showed a significant reduction in inflammatory cells, rheumatoid factor, ESR, and improved the body weight. The results indicated that nimbolide possesses the capacity to attenuate rheumatoid arthritis by downregulating toll-like receptors, IL-17, IL-23, HSP70, and IFN-γ expression levels. Nimbolide treatment showed significant reduction in the severity of inflammation and destruction of joints and showed comparable effects to piroxicam, which is a standard non-steroidal anti-inflammatory drug used for the treatment of rheumatoid arthritis. It can be concluded that nimbolide can be considered as a potential candidate for therapeutic targeting of the toll-like receptors pathway in rheumatoid arthritis.

Efficacy and mechanism of the antinociceptive effects of cannabidiol on acute orofacial nociception induced by Complete Freund's Adjuvant in male Mus musculus mice.

OBJECTIVES: The objectives were to investigate the efficacy and mechanisms of cannabidiol on orofacial nociception induced by Complete Freund's Adjuvant (CFA) in male Mus musculus mice. DESIGN: For the study of efficacy, mice were divided into seven groups: sham; inflammation; and cannabidiol 0.5, 1, 3, 5, and 10 mg. For the study of mechanisms of cannabidiol, mice were divided into six groups: sham, inflammation, calcitonin gene-related peptide (CGRP) antagonist with and without cannabidiol, and vanilloid receptor 1 antagonist with and without cannabidiol. Spontaneous pain-like behaviors, trigeminal nociception, and trigeminal modulating activity were investigated. RESULTS: CFA injected in the right masseter muscle significantly induced spontaneous pain-like behaviors and the trigeminal nociceptive pathway. This effect was inhibited by injection of 1, 3, 5, and 10 mg of cannabidiol. The 50 % inhibitory concentration of cannabidiol on antinociception was found to be 3 mg/kg. In addition, there was no difference in spontaneous pain-like behaviors with vanilloid receptor 1 antagonist injected before treatment with cannabidiol compared to saline control. Reduced c-fos expression was observed in the trigeminal nucleus caudalis and periaqueductal gray in the group injected with CGRP antagonist before treatment with cannabidiol. CONCLUSION: The antinociceptive effects of cannabidiol induced by acute orofacial nociception is mediated by vanilloid receptor 1 but not by CGRP. Cannabidiol can act with peripheral nonpeptidergic neurons and can be used as an alternative drug or as a synergistic medication in pain treatment.

Neutrophil activation and NETosis are the predominant drivers of airway inflammation in an OVA/CFA/LPS induced murine model.

BACKGROUND: Asthma is one of the most common chronic diseases that affects more than 300 million people worldwide. Though most asthma can be well controlled, individuals with severe asthma experience recurrent exacerbations and impose a substantial economic burden on healthcare system. Neutrophil inflammation often occurs in patients with severe asthma who have poor response to glucocorticoids, increasing the difficulty of clinical treatment. METHODS: We established several neutrophil-dominated allergic asthma mouse models, and analyzed the airway hyperresponsiveness, airway inflammation and lung pathological changes. Neutrophil extracellular traps (NETs) formation was analyzed using confocal microscopy and western blot. RESULTS: We found that the ovalbumin (OVA)/complete Freund's adjuvant (CFA)/low-dose lipopolysaccharide (LPS)-induced mouse model best recapitulated the complex alterations in the airways of human severe asthmatic patients. We also observed OVA/CFA/LPS-exposed mice produced large quantities of neutrophil extracellular traps (NETs) in lung tissue and bone marrow neutrophils. Furthermore, we found that reducing the production of NETs or increasing the degradation of NETs can reduce airway inflammation and airway hyperresponsiveness. CONCLUSION: Our findings identify a novel mouse model of neutrophilic asthma. We have also identified NETs play a significant role in neutrophilic asthma models and contribute to neutrophilic asthma pathogenesis. NETs may serve as a promising therapeutic target for neutrophilic asthma.