Complete Freund's Adjuvant Research Articles

Fixed oil from the Caatinga palm Syagrus coronata with effect on Complete Freund's Adjuvant (CFA)-induced arthritis.

Syagrus coronata, a native palm tree in the Caatinga domain, produces fixed oil (ScFO) used therapeutically and dietary by Northeast Brazilian communities. This study evaluated its anti-inflammatory potential of CFA-induced arthritis and its effect on behavioral parameters. In the acute model, ScFO at 25, 50, and 100mg/kg showed edematogenic effects similar to indomethacin at 4mg/kg (p > 0.05). In the arthritis model, 100mg/kg ScFO treatment was comparable to indomethacin (4mg/kg) (p > 0.05). TNF-α and IL-1β levels were significantly reduced in ScFO-treated groups at 25, 50, and 100mg/kg, and the indomethacin group (4mg/kg) versus the positive control (p > 0.05). Radiographs showed severe soft-tissue swelling and bone deformities in the control group, while the 100mg/kg ScFO group had few alterations, similar to the indomethacin group. Histopathological analysis revealed intense lymphocytic infiltration in the control group, mild diffuse lymphocytic infiltration in the indomethacin group, and mild lymphoplasmacytic infiltration with focal polymorphonuclear infiltrates in the 100mg/kg ScFO group. Behavioral analysis showed improved exploratory stimuli in ScFO and indomethacin-treated mice compared to the positive control (p > 0.05). ScFO demonstrated anti-inflammatory effects in both acute and chronic arthritis models, reducing edema and pro-inflammatory cytokines, and improved exploratory behavior due to its analgesic properties.

The analgesic effects of Yu-Xue-Bi tablet (YXB) on mice with inflammatory pain by regulating LXA4-FPR2-TRPA1 pathway

BackgroundOxylipins including lipoxin A4 (LXA4) facilitate the resolution of inflammation and possess analgesic properties by inhibiting macrophage infiltration and transient receptor potential (TRP) protein expression. Yu-Xue-Bi Tablet (YXB) is a traditional Chinese patent medicine used to relieve inflammatory pain. Our previous research has shown that the analgesic effect of YXB is related to inhibiting peripheral inflammation and regulating macrophage infiltration, but the mechanism is not yet clear. The purpose of this study is to explore the mechanisms of YXB on mice models with Complete Freund’s Adjuvant (CFA)-induced inflammatory pain from the perspective at the resolution of inflammation.MethodsMechanical allodynia thresholds and heat hypersensitivity were measured using the Von Frey test and the hot plate test respectively. The open field test and the tail suspension test were employed to measure anxiety and depressive behaviors respectively. The expression of CD68+ and the proportion of F4/80+CD11b+ cells were measured by immunofluorescence staining and flow cytometry. The expression of transient receptor potential ankyrin 1(TRPA1) was measured by immunofluorescence staining and western blotting. Oxylipins omics analysis provided quantitative data on oxylipins in the paws, and enzyme linked immunosorbent assay (ELISA) was used to measure the levels of LXA4 there. Immunofluorescence staining was used to perform the expression of Leukotriene A4 hydroxylase (LTA4H) in the paws of mice. The impact of injecting the formyl peptide receptor 2(FPR2) antagonist WRW4 and the TRPA1 agonist AITC into the left paws was observed, focusing on the expression of mechanical allodynia thresholds, the expression of CD68+, TRPA1 in the paws, and Calcitonin gene-related peptide (CGRP) in the L5 spinal dorsal horn.ResultsYXB elevated mechanical allodynia thresholds, alleviated heat hypersensitivity and anxiety and depressive behaviors in CFA mice. It significantly reduced the number of CD68+ and proportion of F4/80+CD11b+ within the paws, thereby decreasing macrophage infiltration. Additionally, it diminished the expression of TRPA1 in the paws and TRPV1 in the DRG, leading to an inhibition of peripheral sensitization. Through quantitative analysis, it was found that YXB could modulate DHA-derived oxylipins and LXA4. ELISA results indicated that YXB elevated the levels of LXA4 and inhibited the expression of LAT4H in the paws. Furthermore, the pro-resolution and analgesic effects of YXB were hindered after administration of the FPR2 antagonist. Compared with the AITC group, YXB showed no significant improvement in anti-inflammatory and analgesic effects.ConclusionsYXB can regulate the oxylipins of paws in CFA mice to promote the resolution of inflammation. The LXA4-FPR2-TRPA1 pathway is a key mechanism for the resolution of inflammation and analgesic effects.

Targeting next-generation PDE4 inhibitors in search of potential management of rheumatoid arthritis and psoriasis

Immune-mediated inflammatory diseases (IMIDs) comprise a broad spectrum of conditions characterized by systemic inflammation affecting various organs and tissues, for which there is no known cure. The isoform-specific inhibition of phosphodiesterase-4B (PDE4B) over PDE4D constitutes an effective therapeutic strategy for the treatment of IMIDs that minimizes the adverse effects associated with non-selective PDE4 inhibitors. Thus, we report a new class of isoquinolone derivatives as next-generation PDE4 inhibitors for effective management of rheumatoid arthritis (RA) and psoriasis. Among the series, 8 compounds i.e. 1e, 1l, 1m, 1n, 1o, 2m, 2o and 3o showed promising PDE4B inhibition (>80 %) in vitro with IC50 ∼ 1.4–6.2 µM. The compound 1l was identified as an initial hit and was pursued for further studies. According to structure–activity relationship (SAR), an allyl group at C-4 position improved PDE4B inhibition. The correlation between in vitro activity data and binding affinities obtained via molecular docking suggested that the high-affinity binding to PDE4B is a prerequisite for the effective inhibition of PDE4B. Notably, the hit 1l showed selectivity towards PDE4B over PDE4D in vitro. Furthermore, 1l treatment (30 mg/kg) in the adjuvant-induced arthritis (AIA) rat model induced by complete Freund’s adjuvant (CFA) demonstrated anti-arthritic potential via ameliorating paw swelling and body weight, narrowing joint space, reducing excessive immune cells infiltration and pannus formation in addition to reducing mRNA expression of pro-inflammatory cytokines such as TNF-α and IL-6 in synovial tissues of experimental rats. Additionally, 1l reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes. The treatment of these cells with 1l markedly reduced the protein levels of Ki67 and mRNA levels of pro-inflammatory cytokines e.g. IL-17A and TNF-α suggesting its potent anti-psoriatic potential. Furthermore, 1l did not show any significant adverse effects when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at the tested concentrations (1–100 µM) and the NOAEL (no-observed-adverse-effect level) was found to be 100 µM. Thus, with promising anti-inflammatory effects both in vitro and in vivo along with PDE4B selectivity with an acceptable safety margin, 1l emerged as a new and promising inhibitor for further studies.

Phosphotungstic acid-enhanced micro-computed tomography and RNA sequencing provide a new perspective on temporomandibular joint arthritis induced by complete Freund's adjuvant and collagen-induced arthritis in rat models

Background/PurposeTemporomandibular joint (TMJ) arthritis causes inflammation and degradation of the mandibular condylar cartilage and subchondral bone. Complete Freund's adjuvant (CFA) and collagen-induced arthritis (CIA) are models for studying TMJ arthritis. While micro-computed tomography (micro-CT) is crucial for three-dimensional (3D) bone analysis, it has limitations in imaging nonmineralized tissues. Phosphotungstic acid (PTA) enhances soft tissue contrast. However, research on the 3D imaging of mandibular condylar cartilage and the molecular mechanisms of CFA- and CIA-induced arthritis remains unclear. This study aimed to investigate the bone and PTA-stained cartilage in the mandibular condyle using 3D reconstruction and explore the characteristics of enriched gene ontology terms underlying CFA- and CIA-induced TMJ arthritis in rat models. Materials and methodsRat mandibular condyles were collected from control, CFA, and CIA groups. Live micro-CT created 3D bone structures, and PTA-enhanced micro-CT constructed 3D mandibular condylar cartilage. Gene ontology enrichment analysis identified enriched gene ontology terms from differentially expressed genes through RNA sequencing. ResultsMajor deformities in cartilage volume and bone morphology were observed in the arthritis-induced groups. The CIA group exhibited significant correlations between cartilage volume and bone parameters changes. Gene ontology enrichment analysis indicated fewer terms with upregulated differentially expressed genes related to inflammation and immune response in the CIA group than in the CFA group. ConclusionThis study reveals distinct responses between CFA- and CIA-induced TMJ arthritis models. The CIA group exhibited strong correlations between cartilage volume and bone parameter changes and had less pronounced inflammation and immune response than the CFA group.

Pain-suppressed consumption of highly palatable liquid in rats.

This study determined whether consumption of a highly palatable liquid is a reliable measure of inflammatory pain and antinociception in male and female rats. After a 10-day acquisition period, the impact of intraplantar oil vs. complete Freund adjuvant (CFA) on consumption of vanilla-flavored Ensure was assessed, with a sipper tube height 12 or 19 cm above the floor. CFA significantly decreased Ensure consumption, which completely recovered within 4-7 days to levels in oil-treated controls; neither sex nor sipper tube height significantly influenced Ensure consumption. CFA also significantly suppressed Ensure consumption in rats not exposed to the 10-day acquisition period, but only in males. To test the predictive validity of Ensure consumption as a measure of pain, separate rats were pretreated with a vehicle, an opioid, a nonsteroidal anti-inflammatory drug, or a cannabinoid the day after CFA treatment. Morphine and ibuprofen significantly attenuated CFA-suppressed drinking in at least one sex, and tetrahydrocannabinol did not. Neither ibuprofen nor tetrahydrocannabinol significantly altered drinking in oil-injected, 'pain-free' controls, but morphine increased drinking. These results demonstrate that CFA decreases consumption of a highly palatable liquid regardless of previous exposure (training) to the consumption procedure, but only in males. Although standard analgesics attenuate CFA-suppressed drinking, nonspecific hyperphagic effects can confound the interpretation of results. Thus, consumption of a highly palatable liquid is not an optimal measure for candidate analgesic screening.

Study on the anti-inflammatory mechanism of moxibustion in rheumatoid arthritis in rats based on phospholipaseA2 signaling inhibition by Annexin 1.

To determine whether moxibustion had an anti-inflammatory effect on rheumatoid arthritis (RA) by regulating Annexin 1 expression and interfering with the phospholipaseA2 signaling pathway. Thirty male Sprague-Dawley rats were randomly categorized into five groups (six rats per group): blank control (CON) group, RA model (RA) group, moxibustion (MOX) group, Annexin 1 lentiviral intervention (RNAi-Anxa1) group, and Annexin 1 lentiviral intervention + moxibustion (RNAi-Anxa1 + MOX) group. The rats in the RNAi-Anxa1 and the RNAi-Anxa1 + MOX groups were injected with the lentiviral vector-mediated RNAi-Anxa1 into the rat foot pad. An experimental RA rat model was established by injecting Freund's complete adjuvant (FCA) into the RA, MOX, RNAi-Anxa1, and RNAi-Anxa1 + MOX groups. Rats in the MOX and RNAi-Anxa1 + MOX groups received moxibustion treatment. After modeling, using moxibustion "Shenshu (BL23)" and "Zusanli (ST36)", each point is 5 times, bilateral alternating, once a day, 6 times for a course of treatment, between the courses of rest for a one day. A total of three treatment courses were conducted. Both bilateral pad thicknesses were measured using Vernier calipers on experimental days 1, 7, 14, 21, and 28. The expression of cPLA2α signaling in the synovium of diseased joints was observed using Western blot. The pathology of the rat ankle synovium was observed using hematoxylin-eosin (HE) staining. Interleukin (IL)-1β, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) were detected using enzyme-linked immunosorbent assay. Moxibustion increased the levels of Annexin 1 and decreased the inflammatory response in rats with RA. After increasing the expression of Annexin 1, the phosphorylated expression of cPLA2α was inhibited, the serum levels of IL-1β, PGE2, and LTB4 decreased, and the level of IL-10 increased. In moxibustion treated RA rats after the Annexin 1 lentiviral intervention, the serum levels of IL-1β, PGE2, LTB4, and IL-10 were almost unchanged. Moxibustion enhanced the negative regulation of the cPLA2α signaling pathway, increased the synovial Annexin 1 expression, inhibited the cPLA2α signaling pathway, indirectly inhibited the expression of downstream inflammatory factors, and played a role in reducing inflammation.

Neurturin GF Enhances the Acute Cytokine Response of Inflamed Skin

Epidermal keratinocytes, immune cells and sensory nerves all contribute to immune balance and skin homeostasis. Keratinocyte release of growth factors, neuromodulators and immune activators is particularly important because each can evoke local (skin) and systemic (i.e., immune and neural) responses that can initiate and exacerbate skin pathophysiology. From studies of skin and neural growth factors, we hypothesized that neurturin (Nrtn), a member of the glial cell line-derived neurotrophic factor (GDNF) family that is expressed in skin, has particular importance in this process. Here we examine how elevation of Nrtn in skin keratinocytes impacts early cytokine expression in response to complete Freund's adjuvant (CFA)-mediated inflammation. Mice that overexpress Nrtn in keratinocytes (NrtnOE mice) and WT mice injected with Nrtn exhibit an enhanced level of TNFα and IL-1β cytokines in the skin, a response previously shown to support healing. In vitro assays suggest one source of the Nrtn-induced TNFα increase is from keratinocytes, which are shown to express Nrtn and mRNAs encoding the Nrtn receptors GFRα2, Ret, Itgβ1 and NCAM. These findings support the contribution of keratinocyte-derived Nrtn as an autocrine/paracrine factor that acts as a first line defense molecule that regulates the initial cytokine response to inflammatory challenge.

The Combined Effect of Curcumin and Crocin on the Reduction of Inflammatory Responses in Arthritic Rats.

The present study evaluated the anti-arthritic impact of combined crocin and curcumin on Adjuvant Induced Arthritis (AIA) in rats. The arthritis model was induced in rats by injecting Complete Freund's adjuvant (CFA) into the right hind paw and was subsequently treated with crocin and curcumin. Evaluation of anti- arthritic activity was carried out using paw swelling, hematological parameters, biochemical parameters, inflammatory cytokines, and histopathology of rats. The results showed increased paw swelling, increased serum markers levels, including CRP, RF, ALP, ALT, and AST, and inflammatory cytokines (ILlβ and TNFα) along with histology changes (cartilage and bone degradation) in arthritic rats when compared to the normal group. Crocin, curcumin and crocin + curcumin administration at different doses (especially combination at 40 mg/kg and 30 mg/kg, respectively), as well as MTX, revealed a suitable therapeutic effect on AIA rats. Moreover, both phytochemicals and their combination at different doses showed effective anti- arthritic effects owing to their anti-inflammatory effects. Crocin and curcumin, either alone or in combination, can be a suitable treatment modality for rheumatoid arthritis.

Apical periodontitis as an aggravating factor for the severity of rheumatoid arthritis: An animal study.

The present study investigated the influence of apical periodontitis (AP) on the severity of rheumatoid arthritis (RA) using a Wistar rat model. Forty male Wistar rats were distributed across four groups (n = 10) based on the induction of RA and AP: Control, RA, AP, and RA + AP. RA was induced through two immunisations with type II collagen emulsified in incomplete Freund's adjuvant, followed by one immunisation with complete Freund's adjuvant. After 21 days of RA induction, AP was induced by exposing the pulp of four molars. Animals were euthanized after 28 days of pulp exposure. Through the experiment, visual and behavioural assessments tracked RA development and the knees and hind paw joints were measured. Micro-computed tomography scans of knees and hind paws, as well as mandibles and maxillae, were conducted to evaluate RA severity and the presence of AP, respectively. Serum samples were collected to analyse proinflammatory cytokines (IL-1β, IL-2, IL-17, and TNF-α). Non-parametric data were analysed using the Kruskal-Wallis test followed by Student-Newman-Keuls test, while one-way anova followed by Tukey's test was performed for parametric data. A significance level of 5% was employed. All molars submitted to access cavity developed AP. All joints subjected to arthritis induction developed the disease, with AP + RA demonstrating a higher arthritis severity when compared to the RA group (p < .05). RA + AP group displayed a significantly larger hind paw and knee circumference compared to the RA group (p < .05). Micro-CT images of RA and RA + AP groups revealed joints with erosions and bone deformities, with a significantly lower bone surface density, lower trabecular number and higher trabecular separation in the hind paw and a significantly lower percent bone volume and higher trabecular separation in the knees of RA + AP group compared to RA group (p < .05). RA + AP group exhibited a significantly higher level of TNF-α and a lower level of IL-2 compared to all other groups (p < .05). Both RA and RA + AP groups had significantly higher IL-17 levels (p < .05), while there was no significant difference in IL-1β levels among the groups (p > .05). The findings from this study underscore a possible relationship between apical periodontitis and the exacerbation of rheumatoid arthritis.

Soluble E-Cadherin Contributes to Airway Inflammation in Severe Asthma.

Impaired airway epithelial barrier and decreased expression of E-cadherin are key features of severe asthma. As a gatekeeper of the mucosa, E-cadherin can be cleaved from the cell surface and released into the apical lumen as a soluble form (sE-cadherin).This study was aimed to investigate the role of sE-cadherin in severe asthma.Induced sputum was obtained from healthy subjects and patients with asthma. Two murine models of severe asthma were established using either TDI (toluene diisocyanate) or OVA (ovalbumin)/CFA (complete Freund's adjuvants). The role of sE-cadherin in severe asthma was evaluated by intraperitoneal injection of DECMA-1, a neutralizing antibody against sE-cadherin. Mice or THP-1-derived macrophages were treated with recombinant sE-cadherin to explore the pro-inflammatory mechanism of sE-cadherin.Severe asthma patients had a significantly higher sputum sE-cadherin level than the health subjects with mild to moderate asthma, which were positively correlated with sputum HMGB1 level and glucocorticoid dosage required for daily control. Allergen exposure markedly increased sE-cadherin level in the bronchoalveolar lavage fluid in mice. Treatment of DECMA-1 significantly attenuated allergen-induced airway inflammation and hyperresponsivenes in both models of severe asthma. While exposure to recombinant sE-cadherin dramatically up-regulated VEGF expression in THP-1-derived macrophages, and increased neutophlil and eosinophil infiltration into the airway as well as the release of VEGF and IL-6 in mice, both of which can be suppressed by pharmacological inhibition of ERK signaling.Taken together, our data indicated that sE-cadherin contributed to the airway inflammation of severe asthma in an ERK-depedent pathway.

Multidimensional Analgesia of Acupuncture by Increasing Expression of MD2 in Central Nervous System.

To investigate changes of myeloid differentiation factor 2 (MD2) in inflammation-induced pain and acupuncture-mediated analgesia. Mice were randomly divided into three groups by a random number table method: saline group (n=16), complete Freund's adjuvant (CFA) group (n=24) and CFA+electroacupuncture (EA) group (n=26). Inflammation-induced pain was modelled by injecting CFA to the plantar surface of the hind paw of mice and EA was applied to bilateral Zusanli (ST 36) to alleviate pain. Only mice in the CFA+EA group received EA treatment (30 min/d for 2 weeks) 24 h after modelling. Mice in the saline and CFA groups received sham EA. von-Frey test and Hargreaves test were used to assess the pain threshold. Brain and spinal tissues were collected for immunofluorescence staining or Western blotting to quantify changes of MD2 expression. CFA successfully induced plantar pain and EA significantly alleviated pain 3 days after modelling (P<0.01). Compared with the CFA group, the number of MD2+/c-fos+ neurons was significantly increased in the dorsal horn of the spinal cord 7 and 14 days after EA, especially in laminae I - IIo (P<0.01). The proportion of double positive cells to the number of c-fos positive cells and the mean fluorescence intensity of MD2 neurons were also significantly increased in laminae I - IIo (P<0.01). Western blotting showed that the level of MD2 was significantly decreased by EA only in the hippocampus on day 7 and 14 (both P<0.01) and no significant changes were observed in the cortex, thalamus, cerebellum, or the brainstem (P<0.05). Fluorescence staining showed significant decrease in the level of MD2 in periagueductal gray (PAG) and locus coeruleus (LC) after CFA injection on day 7 (P<0.01 for PAG, P<0.05 for LC) and EA significantly reversed this decrease (P<0.01 for PAG, P<0.05 for LC). The unique changes of MD2 suggest that EA may exert the analgesic effect through modulating neuronal activities of the superficial laminae of the spinal cord and certain regions of the brain.

Humanized-Aquaporin-4-Expressing Rat Created by Gene-Editing Technology and Its Use to Clarify the Pathology of Neuromyelitis Optica Spectrum Disorder.

Conventional rodent neuromyelitis optica spectrum disorder (NMOSD) models using patient-derived immunoglobulin G (IgG) are potentially affected by the differences between the human and rodent aquaporin-4 (AQP4) extracellular domains (ECDs). We hypothesized that the humanization of AQP4 ECDs would make the rodent model lesions closer to human NMOSD pathology. Humanized-AQP4-expressing (hAQP4) rats were generated using genome-editing technology, and the human AQP4-specific monoclonal antibody (mAb) or six patient-derived IgGs were introduced intraperitoneally into hAQP4 rats and wild-type Lewis (WT) rats after immunization with myelin basic protein and complete Freund's adjuvant. Human AQP4-specific mAb induced astrocyte loss lesions specifically in hAQP4 rats. The patient-derived IgGs also induced NMOSD-like tissue-destructive lesions with AQP4 loss, demyelination, axonal swelling, complement deposition, and marked neutrophil and macrophage/microglia infiltration in hAQP4 rats; however, the difference in AQP4 loss lesion size and infiltrating cells was not significant between hAQP4 and WT rats. The patient-derived IgGs bound to both human and rat AQP4 M23, suggesting their binding to the shared region of human and rat AQP4 ECDs. Anti-AQP4 titers positively correlated with AQP4 loss lesion size and neutrophil and macrophage/microglia infiltration. Considering that patient-derived IgGs vary in binding sites and affinities and some of them may not bind to rodent AQP4, our hAQP4 rat is expected to reproduce NMOSD-like pathology more accurately than WT rats.

Irisin alleviates CFA-induced inflammatory pain by modulating macrophage polarization and spinal glial cell activation

Although the potent anti-inflammatory effects of irisin have been documented in various inflammatory disorders, its efficacy against inflammatory pain remains unexplored. Herein, we examined the therapeutic effects of irisin in a mouse model of inflammatory pain induced by complete Freund’s adjuvant (CFA). Mice were divided into three groups: normal control, CFA-injected (CFA), and CFA plus irisin-treated (CFA+Irisin). The irisin-treated group exhibited a gradual reduction in mechanical allodynia and thermal hyperalgesia when compared with the CFA group. Moreover, treatment with irisin significantly upregulated the expression of M2 macrophage markers (interleukin [IL]-4 and IL-10) and downregulated M1 macrophage markers (IL-1β, IL-6, and tumor necrosis factor-α) in the local paw tissue, dorsal root ganglion, and spinal cord tissue. However, there was no significant difference in the total number of F4/80+ macrophages in the paw tissue and dorsal root ganglion, indicating phenotypic exchange. Treatment with irisin also downregulated the expression of the glial cell activation-related markers Iba-1 and GFAP in the spinal cord tissue. To elucidate the underlying mechanisms, we detected the expression of Toll-like receptor 4 (TLR4), MyD88, and interferon regulatory factor 5 (IRF5) in paw tissues, dorsal root ganglion, and spinal tissues, revealing that irisin could downregulate the expression of these proteins. Irisin alleviated inflammatory pain by modulating local tissue inflammation and peripheral and central neuroinflammation and reducing glial cell activation and M2 macrophage polarization by modulating the TLR4–MyD88–IRF5 signaling pathway. Accordingly, irisin is a promising candidate for treating inflammatory pain in various diseases.

Reg2 treatment is protective but the induced Reg2 autoantibody is destructive to the islets in NOD mice

Regenerating family protein 2 (Reg2) is a trophic factor which stimulates β-cell replication and resists islet destruction. However, Reg2 also serves as an islet autoantigen, which makes it complicated to judge the effectiveness in treating diabetes. How Reg2 treatment behaves in non-obese diabetic (NOD) mice is to be investigated. NOD mice were treated with recombinant Reg2 protein, Complete Freund’s adjuvant (CFA) + PBS and CFA+Reg2 vaccinations, CFA+PBS- and CFA+Reg2-immunized antisera, and single chain variable fragment (scFv)-Reg2 and mIgG2a-Reg2 antibodies. Glycemic level, bodyweight, serum Reg2 antibody titer, glucose tolerance, and insulin secretion were determined. Islet morphological characteristics, insulitis, cell apoptosis, islet cell components, and T cell infiltration were analyzed by histological examinations. The autoantigenicity of constructed Reg2C and Reg2X fragments was determined in healthy BALB/c mice, and the bioactivity in stimulating cell proliferation and survival was assessed in insulinoma MIN6 cells. Reg2 administration alleviated diabetes in NOD mice with improved glucose tolerance and insulin secretion but elevated serum Reg2 autoantibodies. Histomorphometry showed reduced inflammatory area, TUNEL signal and CD8 + T cell infiltration, and increased β-cell proportion in support of the islet-protective effect of Reg2 treatment. CFA+PBS and CFA+Reg2 immunizations prevented diabetic onset and alleviated insulitis while injections of the antisera offered mild protections. Antibody treatments accelerated diabetic onset without increasing the overall incidence. Reg2C fragment depletes antigenicity, but reserves protective activity in streptozotocin (STZ)-treated MIN6 cells. In conclusion, Reg2 treatment alleviates type 1 diabetes (T1D) by preserving islet β-cells, but induces Reg2 autoantibody production which poses a potential risk of accelerating diabetic progression.

Down-regulation of NF-κB signalling by methanolic extract of Viola odorata (L.) attenuated in vivo inflammatory and angiogenic responses.

Intractable inflammation plays a key role in the progression of autoimmune diseases such as rheumatoid arthritis. Oedema and angiogenesis are the hall marks of chronic inflammation. The current study was aimed to investigate the pharmacological effects of the methanolic extract of Viola odorata (Vo.Me)on inflammation induced oedema and angiogenesis, and to identify the active principles and explore the molecular mechanisms thereof. Various models of inflammation were utilized in rats, including carrageenan- and histamine-induced acute oedema, as well as chronic models of Complete Freund's Adjuvant (CFA)-induced arthritis and cotton pellet-induced granuloma. Anti-angiogenic activity was evaluated by CAM assay followed by quantification of phytoconstituents through HPLC.Effect ofVo.Me treatment on the expression of various mediators (PGE-2 andNO) and genes (IL-1β, TNF-α,NF-κB, and COX-2) were explored byqPCR and ELISA assays.HPLCanalysis showed the presence of quercetin, chlorogenic acid, gallic acid, benzoic acid, m-coumaric acid, p-coumaric acid, synergic acid, caffeic acid, vanillic acid, sinapic acid, and cinnamic acid in Vo.Me. Significant dose-dependent inhibition of rats' paw oedema was observed in the Vo.Me administered groups (p < 0.05)in both acute and chronic inflammatory models. Moreover, at a dosage of 500mg/kg, Vo.Me exhibited a comparable anti-inflammatory effect to indomethacin (p > 0.05). Additionally, Vo.Me demonstrated a remarkable anti-granulomatous activity. Histopathological findings demonstrated amelioration of inflammation in animal paws which were treated with Vo.Meand indomethacin. CAM assay also displayed significant inhibitory effect of Vo.Me on the blood vasculature growth. Vo.Me treatment also causedrelatively less gastric irritation and hepatic damage as compared toindomethacin.At amolecular level, thedown-regulation of NF-κB signalling leading tothedecreased activation of pro-inflammatory mediators (such as IL-1β, TNF-α, and COX-2) and their downstream molecules including prostaglandin E-2 (PGE-2) and nitric oxide (NO), is suggested to be responsible for these diverse anti-inflammatory effects. These findings confirmed the promising anti-inflammatory and anti-angiogenic activities of Vo.Me, which warrant bench-to-bedside translational studies to assess its safety and suitability for clinical usage.

Analgesia by fascia manipulation is mediated by peripheral and spinal adenosine A1 receptor in a mouse model of peripheral inflammation

The role of adenosine receptors in fascial manipulation-induced analgesia has not yet been investigated. The purpose of this study was to evaluate the involvement of the adenosine A1 receptor (A1R) in the antihyperalgesic effect of plantar fascia manipulation (PFM), specifically in mice with peripheral inflammation. Mice injected with Complete Freund’s Adjuvant (CFA) underwent behavioral, i.e. mechanical hyperalgesia and edema. The mice underwent PFM for either 3, 9 or 15 min. Response frequency to mechanical stimuli was then assessed at 24 and 96 h after plantar CFA injection. The adenosinergic receptors were assessed by systemic (intraperitoneal, i.p.), central (intrathecal, i.t.), and peripheral (intraplantar, i.pl.) administration of caffeine. The participation of the A1R was investigated using the 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1R subtype antagonist. PFM inhibited mechanical hyperalgesia induced by CFA injection and did not reduce paw edema. Furthermore, the antihyperalgesic effect of PFM was prevented by pretreatment of the animals with caffeine given by i.p., i.pl., and i.t. routes. In addition, i.pl. and i.t. administrations of DPCPX blocked the antihyperalgesia caused by PFM. These observations indicate that adenosine receptors mediate the antihyperalgesic effect of PFM. Caffeine’s inhibition of PFM-induced antihyperalgesia suggests that a more precise understanding of how fascia-manipulation and caffeine interact is warranted.

Peripheral CaV2.2 channels in skin regulate prolonged heat hypersensitivity during neuroinflammation.

Neuroinflammation can lead to chronic maladaptive pain affecting millions of people worldwide. Neurotransmitters, cytokines, and ion channels are implicated in neuro-immune cell signaling but their roles in specific behavioral responses are not fully elucidated. Voltage-gated CaV2.2 channel activity in skin controls rapid and transient heat hypersensitivity induced by intradermal capsaicin via IL-1α cytokine signaling. CaV2.2 channels are not, however, involved in mechanical hypersensitivity that developed in the same animal model. Here, we show that CaV2.2 channels are also critical for heat hypersensitivity induced by the intradermal (id) Complete Freund's Adjuvant (CFA) model of chronic neuroinflammation that involves ongoing cytokine signaling for days. Ongoing CFA-induced cytokine signaling cascades in skin lead to pronounced edema, and hypersensitivity to sensory stimuli. Peripheral CaV2.2 channel activity in skin is required for the full development and week-long time course of heat hypersensitivity induced by id CFA. CaV2.2 channels, by contrast, are not involved in paw edema and mechanical hypersensitivity. CFA induced increases in cytokines in hind paws including IL-6 which was dependent on CaV2.2 channel activity. Using IL-6 specific neutralizing antibodies, we show that IL-6 contributes to heat hypersensitivity and, neutralizing both IL-1α and IL-6 was even more effective at reducing the magnitude and duration of CFA-induced heat hypersensitivity. Our findings demonstrate a functional link between CaV2.2 channel activity and the release of IL-6 in skin and show that CaV2.2 channels have a privileged role in the induction and maintenance of heat hypersensitivity during chronic forms of neuroinflammation in skin.

Berberine hydrochloride alleviates chronic prostatitis/chronic pelvic pain syndrome by modifying gut microbiome signaling.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is highly prevalent worldwide and poses a significant threat to men's health, particularly affecting young men. However, the exact causes and mechanisms behind CP/CPPS remain unclear, leading to challenges in its treatment. In this research, a CP/CPPS rat model was established with complete Freund's adjuvant (CFA), and berberine hydrochloride was administered through daily gavage to assess its therapeutic effects. The alterations in the gut microbiome induced by CP/CPPS and berberine hydrochloride were investigated through 16S ribosomal RNA sequencing of cecum content and colonic epithelial cells. To investigate the impact of the gut microbiome on CP/CPPS, a pseudo germ-free rat model was established, and fecal microbiome transplantation (FMT) was performed on these rats. In all, berberine hydrochloride demonstrated effective reduction of inflammation and oxidative stress in the prostate, offering significant therapeutic advantages for CP/CPPS. Through analysis of the gut microbiome using 16S ribosome RNA sequencing, distinct differences were observed between CP/CPPS rats and control rats, and Clostridium butyricum was identified as a key bacteria. Pseudo germ-free rats that underwent FMT from CP/CPPS rats or rats treated with berberine hydrochloride displayed varying levels of inflammatory cytokine production, oxidative stress, and activity of associated signaling pathways. In conclusion, the therapeutic potential of berberine hydrochloride in addressing CP/CPPS is highly significant. The gut microbiome has emerged as a critical factor in the development of CP/CPPS and plays a pivotal role in mediating the therapeutic effects of berberine hydrochloride.

S1P/S1PR1 signaling is involved in the development of nociceptive pain.

Pain is a complex perception involving unpleasant somatosensory and emotional experiences. However, the underlying mechanisms that mediate its different components remain unclear. Sphingosine-1-phosphate (S1P), a metabolite of sphingomyelin and a potent lipid mediator, initiates signaling via G protein-coupled receptors (S1PRs) on cell surfaces. It serves as a second messenger in cellular processes such as proliferation and apoptosis. Nevertheless, the neuropharmacology of sphingolipid signaling in pain conditions within the central nervous system remains largely unexplored and controversial. Chronic nociceptive pain models were induced in vivo by intraplantar injection of 20μL complete Freund's adjuvant (CFA) into the left hind paws. We assessed S1P and S1PR1 expression in the spinal cords of CFA model mice. Functional antagonists of S1PR1 or S1PR1-specific siRNA were administered daily following CFA model establishment. Paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL) were measured to evaluate mechanical allodynia and thermal hyperalgesia, respectively. RT-PCR assessed interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels. Western blotting and immunofluorescence were used to analyze glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), STAT3, ERK, and p38 MAPK protein expression. In the chronic nociceptive pain model induced by CFA, S1P and S1PR1 expression levels were significantly elevated, leading to activation of spinal cord glial cells. S1PR1 activation also promoted MMP2-mediated cleavage of mature IL-1β. Additionally, S1PR1 activation upregulated phosphorylation of STAT3, ERK, and p38 MAPK in glial cells, profoundly impacting downstream signaling pathways and contributing to chronic nociceptive pain. The S1P/S1PR1 axis plays a pivotal role in the cellular and molecular mechanisms underlying nociceptive pain. This signaling pathway modulates glial cell activation and the expression of pain-related genes (STAT3, ERK, p38 MAPK) and inflammatory factors in the spinal dorsal horn. These findings underscore the potential of targeting the S1P system for developing novel analgesic therapies.

N-methyl-D-aspartate Receptor Subunits 2A and 2B Mediate Connexins and Pannexins in the Trigeminal Ganglion Involved in Orofacial Inflammatory Allodynia during Temporomandibular Joint Inflammation.

Temporomandibular joint osteoarthritis (TMJOA) is a severe form of temporomandibular joint disorders (TMD), and orofacial inflammatory allodynia is one of its common symptoms which lacks effective treatment. N-methyl-D-aspartate receptor (NMDAR), particularly its subtypes GluN2A and GluN2B, along with gap junctions (GJs), are key players in the mediation of inflammatory pain. However, the precise regulatory mechanisms of GluN2A, GluN2B, and GJs in orofacial inflammatory allodynia during TMJ inflammation still remain unclear. Here, we established the TMJ inflammation model by injecting Complete Freund's adjuvant (CFA) into the TMJ and used Cre/loxp site-specific recombination system to conditionally knock out (CKO) GluN2A and GluN2B in the trigeminal ganglion (TG). Von-frey test results indicated that CFA-induced mechanical allodynia in the TMJ region was relieved in GluN2A and GluN2B deficient mice. In vivo, CFA significantly up-regulated the expression of GluN2A and GluN2B, Gjb1, Gjb2, Gjc2 and Panx3 in the TG, and GluN2A and GluN2B CKO played different roles in mediating the expression of Gjb1, Gjb2, Gjc2 and Panx3. In vitro, NMDA up-regulated the expression of Gjb1, Gjb2, Gjc2 and Panx3 in satellite glial cells (SGCs) as well as promoted the intercellular communication between SGCs, and GluN2A and GluN2B knocking down (KD) altered the expression and function differently. NMDAR regulated Gjb1 and Panx3 through ERK1/2 pathway, and mediated Gjb2 and Gjc2 through MAPK, PKA, and PKC intracellular signaling pathways. These findings shed light on the distinct functions of GluN2A and GluN2B in mediating peripheral sensitization induced by TMJ inflammation in the TG, offering potential therapeutic targets for managing orofacial inflammatory allodynia.