Complete Freund's Adjuvant Research Articles

Procaine regulates the STAT3/CCL5 axis and inhibits microglia M1 polarization to alleviate CFA rats pain behavior.

Neuropathic pain (NP) caused by sciatic nerve injury can significantly impact the quality of life of patients. The M1 phenotype of microglia has been reported to promote the progression of NP. Procaine is a lipid-soluble local anesthetic drug that exerts narcotic analgesic effects. Nevertheless, the detailed effect of procaine in NP is not clear. In order to explore the role of procaine in the polarization of NP microglia, HAPI cells were exposed to LPS to polarize into M1 type. In addition, the number of the M1 phenotype of HAPI cells was assessed using flow cytometry. The binding site between CCL5 and STAT3 was explored using the dual luciferase assay. Furthermore, in vivo experiments were applied for testing the impact of procaine on NP.LPS significantly inhibited HAPI cell viability, which was reversed by procaine. Consistently, procaine alleviated LPS-induced upregulation of inflammatory factors. Additionally, it significantly inhibited HAPI cells M1 polarization induced by LPS. Meanwhile, overexpression of STAT3 was able to promote HAPI cells M1 polarization through binding with the CCL5 promoter region and activating the PI3K/Akt signaling. Procaine could alleviate the painful behavior of complete Freund's adjuvant (CFA) rats by modulating the STAT3/CCL5 axis and inhibiting microglia M1 polarization. In conclusion, procaine alleviated the painful behavior of CFA rats via regulating the STAT3/CCL5 axis and inhibiting microglia M1 polarization. Hence, the research might provide a novel agent for NP treatment.Significance statement Neuropathic pain (NP) refers to pain caused by damage to the somatosensory system, which can be caused by brain or spinal cord injury and have a serious impact on the patient's quality of life. The M1 phenotype of microglia plays a crucial role in promoting the progression of NP. In this study, we investigated the specific mechanism of local anesthetic procaine in improving NP by inhibiting microglia polarization towards M1 type. Our findings may provide a new drug for NP treatment.

Therapeutic Dose of Zinc Aspartate and Zinc Citrate Attenuates Disease Activity Indices in Rheumatoid Arthritis.

Zinc aspartate and zinc citrate have been used as zinc supplements in different health conditions. Taking into consideration their anti-inflammatory, immunomodulatory, anti-oxidant and antimicrobial properties, the present study has been designed to analyse the effect of zinc aspartate and zinc citrate treatment at therapeutic dose level on disease severity index, haematological, serological, antimicrobial and radiological markers of rheumatoid arthritis in Wistar rats. Bactericidal potential of the two organic zinc compounds was analysed in vitro in clinically isolated Escherichia coli. Arthritis was induced in male Wistar rats by intradermal injection of an emulsion containing collagen type II and Complete Freund's Adjuvant (CFA) containing 1mgmL-1 Mycobacterium tuberculosis H37Ra. Zinc aspartate and zinc citrate were orally administered after the onset of the disease for 4weeks. Ameliorative effect of zinc aspartate and zinc citrate was evaluated by analysing indices of severity and disease activity markers of rheumatoid arthritis. The liver and kidney function tests were performed to evaluate any possible adverse effect of compounds. Antimicrobial activity of the zinc compounds was assessed in clinically isolated E. coli by MTT assay. Zinc aspartate and zinc citrate equivalent to a therapeutic dose of 50mg/day of elemental zinc attenuated the clinical characteristic of rheumatoid arthritis in the animal model of arthritis, collagen-induced arthritis (CIA). Both zinc salts also exhibited antimicrobial effects against E. coli. The selected dose of zinc aspartate and zinc citrate showed no adverse effects in treated rats. This study highlights the potentiality of zinc compounds as antiarthritic agents and also point to its preventive effects on microbial growth that has been observed in rheumatoid arthritis patients due to their increased sensitivity for bacterial infection.

Nanoparticle containing recombinant excretory/secretory-24 protein of Haemonchus contortus enhanced the cellular immune responses in mice

Haemonchus contortus poses a global challenge as a parasite affecting small ruminants, yet the problem of absence of an effective vaccine against H. contortus infection still exists. This investigation sought to appraise the immunological reaction induced by recombinant H. contortus excretory/secretory-24 (rHcES-24) in combination with complete Freund’s adjuvant (CFA) and bio-polymeric nanoparticles (NPs) within a murine model. In this study, rHcES-24 was encapsulated in poly(d, l-lactide-co-glycolide) (PLGA) and chitosan (CS) NPs, administered subcutaneously to mice. Researchers analyzed the NPs using scanning electron microscope (SEM) and assessed lymphocyte proliferation, specific antibodies, cytokines, T cell proliferation (CD3e+CD4+, CD3e+CD8a+), and phenotypic alteration in splenocytes (CD11c+CD83+, CD11c+CD86+) through flow cytometry to understand the immune response. The results demonstrated that the administration of nanovaccines (NVs) prompted immune responses towards Th1 pathway. This was indicated by notable enhancements in the production of specific antibodies, heightened cytokine levels, and a robust proliferation of lymphocytes observed in mice that received the NVs compared to control groups. Remarkably, mice vaccinated with the antigen-loaded NPs formulations exhibited considerably higher proportions of splenic dendritic cells (DCs) and T cells in comparison to those receiving the traditional adjuvant or the control groups. Incorporating HcES-24 protein into NPs effectively conferred immunity against H. contortus, paving the way for developing a targeted and commercial vaccine.

Pharmacology and Articular Cartilage Regeneration: A New Strategy for Osteoarthritis

ABSTRACT Background: Osteoarthrosis is a slowly progressive, multifactorial, and non-inflammatory degenerative disease affecting synovial joints and is typically associated with aging or previous joint damage. Method: Fifty male Sprague Dawley rats were utilized in the study and divided into five groups, with ten rats in each group. The experiment was divided into two phases: in Phase 1, ten animals received 50 μL of saline in the left tibiotarsal joint and served as the control group (SAL), while the other four groups (n = 40) received a single dose of 50 μL complete Freund’s adjuvant (CFA) to induce monoarthritis. In Phase II, after the induction of monoarthritis, from the fourth week onward, the rats in the four CFA-induced monoarthritis groups were treated with 20 μl of gamma-amino butyric acid (GABA), serotonin (SERO), or a neurotransmitter cocktail (NTC) which were given intra-articularly once for three consecutive weeks, while one group was kept as an experimental control. The animals were monitored throughout the experiment, for ankle diameter, gait, and radiological assessment, and were then euthanized and subjected to histopathological evaluation at the conclusion of the study. Results: The study found that there were statistically significant differences in the degree of inflammation between the CFA group and the groups treated with GABA, SERO, and NTC. The CFA group exhibited the most severe and persistent inflammation, whereas the NTC group showed a milder and shorter inflammatory response than the control group (SAL). In terms of gait score, the NTC treatment resulted in a significant reduction in score compared to the treatments with GABA and SERO administered separately. The radiological scores revealed that NTC was the most effective treatment in mitigating radiographic evidence of joint damage, with a significant decrease in bone destruction. The histopathological examination showed that the NTC group had minimal tissue damage or inflammation and had a regenerative effect compared to the other treatment groups. Conclusions: In conclusion, the local administration of neurotransmitters was found to be effective in reversing inflammation, improving gait score, and mitigating radiological changes in CFA-induced arthritis in the murine model.

Electroacupuncture may alleviate inflammatory pain by downregulating the expression of P2Y14 receptor in the primary somatosensory cortex.

Increasing evidence indicated that purinergic signalling involved in electroacupuncture (EA)-induced analgesia. Whether purinergic P2Y14 receptor contributes to EA-mediated analgesia remains unclear. Here, we report that the expression of P2Y14 receptor in the hindlimb region of the primary somatosensory cortex (S1HL) was significantly upregulated on Complete Freund's Adjuvant (CFA)-induced pain model mice, while was downregulated after EA treatment (2Hz frequency, 1mA intensity, and 30min duration) at "Zusanli" (also named ST36 acupoint). EA-mediated analgesia could be reversed by injection of P2RY14 agonist uridine diphosphate glucose (UDPG) into the bilateral S1HL, while prolonged by injection of P2RY14 antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPTN). It suggested that EA may alleviate inflammatory pain by downregulating the expression of P2RY14 in the S1HL.

Inhibition of Phosphodiesterase 10A Alleviates Pain-like Behavior in Mice.

Emerging evidence indicates that cyclic nucleotide phosphodiesterases exert distinct functions in pain processing and that targeting phosphodiesterases might be a novel strategy for pain relief. This study hypothesized that the phosphodiesterase isoform PDE10A might be a target for analgesic therapy. In situ hybridization, immunostaining, cyclic nucleotide enzyme immunoassays, real-time cyclic guanosine monophosphate imaging, and real-time quantitative reverse transcription polymerase chain reaction were performed to investigate the expression and activity of PDE10A in the dorsal root ganglia and spinal cord. Mice of both sexes were assessed in multiple pain models after the administration of specific PDE10A inhibitors. PDE10A is distinctly expressed in nociceptive neurons in the dorsal root ganglia and spinal cord of mice. Incubation of cultured sensory neurons with the PDE10A inhibitor, TAK-063 (150 nM), increased cyclic guanosine monophosphate levels in enzyme immunoassays and real-time imaging at the single-cell level. Strikingly, treatment with TAK-063 (0.3 mg/kg intraperitoneal) ameliorated the pain-like behavior of female and male mice in models of acute nociceptive pain after intraplantar injection of capsaicin (mean ± SD; 8.87 ± 8.78 s [TAK-063] vs. 51.24 ± 36.36 s [vehicle], P = 0.020) or allyl isothiocyanate (2.46 ± 3.43 s [TAK-063] vs. 10.36 ± 4.87 s [vehicle]; P = 0.018). Furthermore, TAK-063 (0.3 mg/kg intraperitoneal) reduced established pain-like behavior in models of inflammatory pain induced by intraplantar injection of zymosan (Two-way ANOVA, group, F(1, 18) = 48.51, TAK-063 vs. vehicle; P ≤ 0.0001) or complete Freund's adjuvant (F(1, 14) = 46.10, TAK-063 vs. vehicle; P ≤ 0.0001), without the development of antinociceptive tolerance. The antinociceptive effects were recapitulated using the PDE10A inhibitor PF-2545920. Collectively, our data support the idea that PDE10A is a suitable target for the development of efficacious analgesic drugs.

Therapeutic potential of hyaluronic acid hydrogel combined with bone marrow stem cells-conditioned medium on arthritic rats’ TMJs

Conditioned media (CM) is derived from mesenchymal stem cells (MSC) culture and contains biologically active components. CM is easy to handle and reduces inflammation while repairing injured joints. Combination therapy of the CM with cross-linked hyaluronic acid (HA) could ameliorate the beneficial effect of HA in treating degenerative changes of articulating surfaces associated with arthritic rats’ temporomandibular joints (TMJs). This study aimed to evaluate the therapeutic potential of HA hydrogel combined with bone marrow stem cells-conditioned medium (BMSCs-CM) on the articulating surfaces of TMJs associated with complete Freund’s adjuvant (CFA)-induced arthritis. Fifty female Sprague-Dawley rats were divided randomly into five equal groups. Rats of group I served as the negative controls and received intra-articular (IA) injections of 50 µl saline solution, whereas rats of group II were subjected to twice IA injections of 50 µg CFA in 50 µl; on day 1 of the experiment to induce persistent inflammation and on day 14 to induce arthritis. Rats of group III and IV were handled as group II and instead, they received an IA injection of 50 µl HA hydrogel and 50 µl of BMSCs-CM, respectively. Rats of group V were given combined IA injections of 50 µl HA hydrogel and BMSCs-CM. All rats were euthanized after the 4th week of inducing arthritis. The joints were processed for sectioning and histological staining using hematoxylin and eosin, Masson’s trichrome and toluidine blue special staining, and immunohistochemical staining for nuclear factor-kappa B (NF-κB). SPSS software was used to analyze the data and one-way analysis of variance followed by post-hoc Tukey statistical tests were used to test the statistical significance at 0.05 for alpha and 0.2 for beta. In the pooled BMSC-CM, 197.14 pg/ml of platelet-derived growth factor and 112.22 pg/ml of interleukin-10 were detected. Compared to TMJs of groups III and IV, TMJs of group V showed significant improvements (P = 0.001) in all parameters tested as the disc thickness was decreased (331.79 ± 0.73), the fibrocartilaginous layer was broadened (0.96 ± 0.04), and the amount of the trabecular bone was distinctive (19.35 ± 1.07). The mean values for the collagen amount were increased (12.29 ± 1.38) whereas the mean values for the NF-κB expression were decreased (0.62 ± 0.15). Combination therapy of HA hydrogel and BMSCs-CM is better than using HA hydrogel or BMSCs-CM, separately to repair degenerative changes in rats’ TMJs associated with CFA-induced arthritis.

Toxicological analysis and anti-inflammatory and antioxidant evaluations of extract, fractions and secoxyloganin obtained from Guettarda viburnoides Cham. & Schltdl. in mice

Ethnopharmacological relevanceGuettarda viburnoides, "veludinho do campo," is traditionally used for the treatment of pain and inflammatory conditions in humans; however, only one scientific study has reported this effect in an ear inflammatory model. Therefore, it is necessary to explore other in vivo models and the chemical composition of this medicinal plant. Aim of the studyA chemical investigation of methanolic extract of G. viburnoides (MEGV) (leaves) led to the isolation of secoxyloganin (GV-1). In addition, the preclinical safety of MEGV (in acute and subacute toxicological models, gavage = p.o.), antioxidants of MEGV, ethyl acetate (EAFGV) and hydromethanolic (HMFGV) fractions were tested using free radical scavenging and lipid peroxidation methodologies, and the anti-inflammatory effects of MEGV, HMFGV and GV-1 (p.o.) were evaluated on carrageenan and complete Freund's adjuvant (CFA) models of inflammation in mice. Materials and MethodsMEGV was obtained from air-dried leaves by maceration with methanol at room temperature. MEGV was then purified by liquid-liquid partitioning, to obtain the EAFGV and HMFGV fractions. Purification of HMFGV afforded GV-1. The quantification of total phenols, flavonoids, flavonols, and condensed tannins was subsequently performed for MEGV. The antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and oxidation of β-carotene were evaluated in MEGV and its fractions. The anti-inflammatory activity of MEGV (3, 30, and 100 mg/kg, p.o.) was assayed in carrageenan-induced models, followed by assessments of MEGV (100 mg/kg, p.o.), HMFGV (3 and 30 mg/kg, p.o.) and GV-1 (3 mg/kg, p.o.) in CFA- induced models in mice (including paw oedema, mechanical allodynia and cold sensitivity). Acute (14 days of MEGV, 2000 mg/kg, p.o.) and subacute (28 days, MEGV 30, 100, and 300 mg/kg, p.o.) toxicity was assessed in female Swiss mice. ResultsThe major compound was secoxyloganin (GV-1). The oral acute toxicity test of MEGV revealed no evidence of toxicity, indicating low toxicity according to the Organization for Economic Cooperation and Development (OECD) guidelines. In the subacute toxicity group, no clinical signs of toxicity or changes in body weight, water consumption, food consumption, or organ weight or morphology were observed after 28 days of gavage with MEGV (30, 100, and 300 mg/kg) compared with those in the control group. MEGV, EAFGV, and HMFGV showed significant free-radical scavenging and lipid peroxidation activities, with IC50 values ≤ 26.38 ± 4.56 μg/mL. In in vivo anti-inflammatory assays, MEGV (3, 30 and 100 mg/kg) reduced carrageenan-induced oedema (2 and 4 h) and hyperalgesia (3 and 4 h). In the CFA model, MEGV (100 mg/kg), HMFGV (30 mg/kg) and GV-1 (3 mg/kg) reduced inflammation (at 3, 4 and 24 h) in all parameters (oedema, mechanical allodynia and cold sensitivity). ConclusionThis study revealed that G. viburnoides has antioxidant and anti-inflammatory properties, and no toxicity was detected after acute or subacute gavage with MEGV, validating its traditional use in the treatment of inflammatory conditions.

PEGylated lipid polymeric nanoparticles for management of rheumatoid arthritis

PEGylation, an extensively used surface modification method for nanoparticles (NPs), continues to hold promise for optimizing therapy for various medical conditions by achieving efficient and selective delivery of multiple therapeutics and nanocarriers. Rheumatoid arthritis is a chronic autoimmune and inflammatory disease treated mainly by disease-modifying antirheumatic drugs (DMARDs) (e.g., leflunomide); however, these drugs suffer from systemic exposure and adverse effects. Leflunomide (LEF) was loaded into PEGylated (PEG)-lecithin chitosan nanoparticles (LCNPs) to enhance its therapeutic efficacy and safety profile. LEF-PEG-LCNPs showed a particle size of 115.31 ±3.24 nm, a zeta potential of 31.3±1.8 mV, an entrapment efficiency of 89.63 ±2.25 %, and an improved release profile than non-pegylated LCNPs and LEF-pure. Fourier transform-infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and transmission electron microscopy (TEM) were also performed. The effects of orally administrated LEF-pure, LEF-marketed, LEF-LCNPs and LEF-PEG-LCNPs against Complete Freund’s Adjuvant (CFA)-induced rheumatoid arthritis (RA) in rats were investigated. LEF-PEG-LCNPs showed more significant inhibition of paw edema (72.74%), rheumatoid factor RF (40.76%), TNF-α (55.6%), IL-6 (47.42%), ALT (80.59%), and AST (39.83%) as compared to positive control. Furthermore, histopathological studies showed a nearly normal appearance of bone trabecular tissue, liver, kidney, lung, and heart. This highlights LEF-PEG-LCNPs as an exceptional anti-rheumatoid arthritis therapy compared to pure and marketed formulations.

GLP-1 and its derived peptides mediate pain relief through direct TRPV1 inhibition without affecting thermoregulation.

Hormonal regulation during food ingestion and its association with pain prompted the investigation of the impact of glucagon-like peptide-1 (GLP-1) on transient receptor potential vanilloid 1 (TRPV1). Both endogenous and synthetic GLP-1, as well as a GLP-1R antagonist, exendin 9-39, reduced heat sensitivity in naïve mice. GLP-1-derived peptides (liraglutide, exendin-4, and exendin 9-39) effectively inhibited capsaicin (CAP)-induced currents and calcium responses in cultured sensory neurons and TRPV1-expressing cell lines. Notably, exendin 9-39 alleviated CAP-induced acute pain, as well as chronic pain induced by complete Freund's adjuvant (CFA) and spared nerve injury (SNI), in mice without causing hyperthermia associated with other TRPV1 inhibitors. Electrophysiological analyses revealed that exendin 9-39 binds to the extracellular side of TRPV1, functioning as a noncompetitive inhibitor of CAP. Exendin 9-39 did not affect proton-induced TRPV1 activation, suggesting its selective antagonism. Among the exendin 9-39 fragments, exendin 20-29 specifically binds to TRPV1, alleviating pain in both acute and chronic pain models without interfering with GLP-1R function. Our study revealed a novel role for GLP-1 and its derivatives in pain relief, suggesting exendin 20-29 as a promising therapeutic candidate.

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.

Biomimetic Curcumin-Loaded Liposomes for the Treatment of Dry Eyes and Meibomian Gland Dysfunction: An In Vivo Study.

Background/Objectives: Meibomian gland dysfunction (MGD) and dry eye syndrome (DES) are common eye diseases characterized by altered tear film stability and inflammation of the ocular surface, causing significant discomfort and possible visual impairment. This study aimed to investigate the efficacy of curcumin-loaded liposomes (Lipo@Cur) compared to cyclosporine A-loaded liposomes (Lipo@CycA) in experimental rabbit models of MGD and DES, with a focus on their ability to improve tear film stability and reduce ocular surface inflammation. Methods: MGD and DES were induced using complete Freund's adjuvant (CFA) and treated to evaluate the effect of liposomal formulations on tear break-up time (TBUT), clinical signs of inflammation (telangiectasia, conjunctival hyperemia, meibomian foramen occlusion), and corneal as well as conjunctival histological cells. Results: Lipo@Cur increased TBUT and reduced the signs of ocular surface inflammation, potentially approaching the effectiveness of clinically approved cyclosporine A encapsulated in liposomes (Lipo@CycA). Histological analysis suggested improvements in corneal epithelial thickness and goblet cell density in the treated groups, which may indicate a reversal of DES-induced damage to the ocular surface. Conclusions: Plant-originated curcumin encapsulated in liposomes offers a promising therapeutic strategy for the management of MGD and DES that may improve patient outcomes by addressing the underlying inflammatory mechanisms of these conditions.

Effects of moxibustion in improving synovitis in adjuvant arthritis rats by regulating synovial GPR43 and peripheral blood Th17/Treg balance

To observe the effects of moxibustion on G protein-coupled receptor 43 (GPR43) and helper T cell 17 (Th17)/regulatory T cell (Treg) balance in rats with adjuvant arthritis (AA), so as to investigate the partial mechanism of moxibustion therapy on rheumatoid arthritis (RA). A total of 24 Wistar rats were randomly divided into a normal group, a model group and a moxibustion group, with 8 rats in each group. The AA rat model was replicated using wind, cold and moisture environmental factors + Freund's complete adjuvant (CFA) injection method. In the moxibustion group, moxibustion was performed on bilateral "Zusanli" (ST36) and "Shenshu" (BL23) for 20 min/time, once daily, for 21 d. The changes of joint swelling degree (JSD) and arthritis index (AI) were observed in each group of rats. Transmission electron microscopy and HE staining were used to examine changes in the cellular structure of the ankle joint synovial tissue in each group. Western blot analysis was employed to detect the expression levels of GPR43 in the synovial tissue of the ankle joints. Flow cytometry was used to measure the percentages of Treg and Th17 in peripheral blood. ELISA was used to determine the contents of interleukin-10 (IL-10) and transforming growth factor-β 1 (TGF-β1) in serum. Compared with the normal group, the JSD and AI in the model group increased before and after treatment (P<0.01), with significant synovial pathological and ultrastruct ural injury observed. Additionally, compared with the normal group, the expression of GPR43 in the synovial tissue decreased (P<0.01), the Treg percentage in peripheral blood decreased (P<0.01) and the Th17 percentage increased (P<0.01), serum IL-10 and TGF-β1 contents decreased in the model group (P<0.01). Compared with the model group, the moxibustion group exhibited a significant reduction in JSD and AI after treatment (P<0.01), the degree of pathological and ultrastruct ural damage in the synovium decreased, the expression of GPR43 in the synovial tissue increased (P<0.05), the Treg percentage in peripheral blood increased (P<0.01) and the Th17 percentage decreased (P<0.01), serum IL-10 and TGF-β1 contents increased (P<0.01). The relative expression of GPR43 in synovial tissue was positively correlated with the percentage of Treg in peripheral blood (r=0.967, P<0.01). Moxibustion can significantly improve the inflammatory response in the synovial membrane of AA rats. The mechanism may be related to moxibustion restoring the Th17/Treg balance through regulating GPR43.

UPLC/HR-ESI-MS/MS and GC/MS profiling of Eriobotrya japonica L. fruit in correlation to its antioxidant, anti-inflammatory, and anti-arthritic effects.

Eriobotrya japonica Lindl. (Loquat) fruit is a subtropical edible fruit originally from China. It grows well in Egypt, but it is not widely known. In the current study, the fruit was extracted with 80% ethanol to get the total ethanol extract (TEE). A part of which was fractionated by dichloromethane to yield polar and nonpolar fractions (PF and NPF). The antioxidant and anti-inflammatory activities of the TEE were in vitro evaluated. The complete Freund's adjuvant (CFA) arthritis model was used to explore the in vivo biological assessment of the anti-arthritic properties in vivo of the TEE, PF, and NPF of the fruit. Additionally, the inspected limbs detached from all animals were subjected to histological inspection. Moreover, GC/MS analysis of the unsaponifiable (USF) and saponifiable (SF) fractions of the NPF was performed. Furthermore, 64 metabolites from various chemical classes were identified using UHPLC/HR-MS/MS analysis of the TEE of the fruit in both positive and negative ionization modes. The positive ionization mode of loquat fruit allowed for the first time the detection of two kinds of lyso-glycerophospholipids (Lyso-GPLs): lyso-glycerophosphoethanolamines (Lyso-PtdEtn) and lyso-glycerophosphocholines (Lyso-PtdCho). The fruit extracts exhibited a notable in vivo anti-arthritic activity by decreasing paw thickness in the treated rats and adjusting the inflammatory mediators. The TEE showed the highest anti-arthritic activity, followed by the PF that showed an observed activity, while the NPF exhibited the lowest activity. Histopathological findings showed a marked improvement in the arthritic condition of the excised limbs. Thus, E. japonica fruit may be considered as a promising natural antioxidant and anti-arthritic agent.

Moxibustion Regulates the Expression of T Cells in Rheumatoid Arthritis Through Tim-3/Gal-9 Signaling Pathway.

To observe the effects of moxibustion on T cells and T cell immunoglobulin and mucin-domain-containing molecule-3/galectin-9 (Tim-3/Gal-9) pathway in rats with rheumatoid arthritis (RA). To further explore the possible anti-inflammatory mechanism of moxibustion in the treatment of RA. Thirty Sprague Dawley rats were randomly divided into three groups, including a control group, an RA model group, and a moxibustion group. An RA model was created through the injection of Freund's complete adjuvant. In the moxibustion group, rats were treated with moxibustion at acupoints of "Shenshu" and "Zusanli." A total of three courses of treatment were conducted. Then the thickness of foot pad was measured, joint pathological changes were observed by hematoxylin-eosin (HE) staining, the proportion of CD4+T and CD8+T in peripheral blood was detected by flow cytometry, the expression levels of Tim-3 and Gal-9 in synovium were detected by polymerase chain reaction (PCR), and the expressions of CD4+T and CD8+T in synovium were detected by immunofluorescence. HE staining showed that the synovial tissue of the control group was smooth and neatly arranged without inflammatory cell infiltration. In the model group, the joint space was narrowed, the synovial tissue had congestion and edema, and a large number of inflammatory cells infiltrated. Compared with the model group, in the moxibustion group, the joint space narrowed with synovium hyperemia and edema, and the level of inflammatory cell infiltration decreased. Flow cytometry showed that compared with the model group, CD4+T expression in the moxibustion group was downregulated, while CD8+T expression was upregulated. PCR results showed that compared with the model group, the expressions of Tim-3 and Gal-9 in the moxibustion group were upregulated. Immunofluorescence results showed that compared with the model group, CD4+T expression in the moxibustion group was decreased, while CD8+T expression was increased. The results demonstrate that moxibustion not only suppressed the expression of CD4+T but also promoted the expression of CD8+T. The anti-inflammatory effect of moxibustion may be related to the regulation of T cell expression through the Tim-3/Gal-9 signaling pathway.

Anti-Inflammatory effect of INSL-3 on experimental arthritis model and LPS-induced macrophage cell line

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease that affects the joints of approximately 1 % of the population worldwide and is seen 2–4 times less in males. INSL3 is a gender-specific peptide hormone produced much higher in males than in females and may have an anti-inflammatory role in RA. So, in this study, it was aimed to determine the possible anti-inflammatory effect and dose of insulin-like factor-3(INSL3) in an experimental Complete Freund’s adjuvant(CFA)-induced RA male rat model and lipopolysaccharide(LPS)-induced macrophage cell line and compare it with prednisolone therapy. For in vivo experiments, 48 male mice were randomly divided into 6 groups with 8 subjects in each group: Control group, Arthritis group, Arthritis + Prednisolone(10 mg/kg) group, Arthritis + INSL3(0.08–0.8–8 μg/day) groups. Joint tissue samples obtained from sacrificed subjects were examined by histochemical and immunohistochemically methods after biometric analyses, arthritis severity scoring, and thermal latency experiments. LPS-induced macrophage cells were also treated with prednisolone(1 µg/ml) and INSL3(50–100-200 nM). Cell viability, cell morphology, and TNF-α and IL-6 immune reactivity were evaluated. According to the data obtained from in vivo analyses, it was seen that INSL3 reduced the paw diameter and arthritis severity scoring, degenerative changes, and inflammation and increased the thermal latency, compared to the arthritis group, although not as much as the prednisolone treatment group. In vitro analyses showed that high doses of INSL3 had positive effects on cell viability, morphology, TNF-α, and IL-6 immune reactivity. In conclusion, it was determined that the anti-inflammatory effect of INSL3 was not as pronounced as prednisolone, but it had a more favorable impact on macrophage cell viability and morphology. It was concluded that INSL3 may be a protective therapeutic agent in combination with existing treatment protocols in treating many autoimmune diseases.

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 (id) capsaicin via IL-1α cytokine signaling. CaV2.2 channels are not, however, involved in mechanical hypersensitivity that developed in the id capsaicin animal model. Here, we show that CaV2.2 channels are also critical for heat hypersensitivity induced by id Complete Freund's Adjuvant (CFA). Id CFA, a model of chronic neuroinflammation, involves ongoing cytokine signaling for days leading to pronounced edema and hypersensitivity to sensory stimuli. Peripheral CaV2.2 channel activity in skin was required for the full development and week-long time course of heat hypersensitivity induced by id CFA but, paw edema and mechanical hypersensitivity were independent of CaV2.2 channel activity. CFA induced increases in several cytokines in hind paw fluid including IL-6 which was also dependent on CaV2.2 channel activity. Using IL-6 specific neutralizing antibodies in vivo 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.Significance statement Neuroinflammation can lead to chronic maladaptive pain. Neurotransmitters, ion channels, cytokines, and cytokine receptors are implicated in neuron-immune signaling, but their importance in mediating specific behavioral responses are not fully elucidated. We show that the activity of peripheral CaV2.2 calcium ion channels in skin play a unique role in the induction and maintenance of heat hypersensitivity in the CFA model of prolonged neuroinflammation, but they are not involved in the development of edema and mechanical hypersensitivity. Blocking peripheral CaV2.2 channel activity reduces local cytokine levels in hind paws injected with CFA including IL-6, and neutralizing IL-6 reduces CFA-induced heat hypersensitivity. Our studies define key signaling molecules that act locally in skin to trigger and maintain heat hypersensitivity during chronic neuroinflammation.

Protective effect of Tecoma stans (L.) Juss.ex Kunth in CFA-induced arthritic rats

Ethnopharmacological relevanceTecoma stans (L.) Juss.ex Kunth (Bignoniaceae) is mainly found in tropical and subtropical regions of Africa and Asia. The leaves, flowers, roots, and bark are used to treat various aliments includes, skin infections, kidney problems, intestinal disorders, jaundice, toothaches, joint pain and repair cracked bones, antidotes for snake, scorpion, and rat bites. Aim of the studyThe objective of the study is to assess the anti-arthritic properties of T. stans leaf using Complete Freund's adjuvant (CFA)-induced rat. Materials and methodThe ethanol extract of T. stans leaf (ETSL) was subjected toGas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for the identification of potential bioactive. The anti-arthritic activity was carried out by administering CFA (0.1 ml) into the sub-plantar surface of the right hind paw. The experimental animals were treated with indomethacin (10 mg/kg) and ETSL (250, 500 mg/kg) once a day orally for fourteen days. The arthritic parameters and hematological and biochemical parameters were evaluated using standard kit reagents. The levels of pro-inflammatory cytokines and inflammatory mediators were measured in blood serum. Antioxidant parameters were assessed in homogenized liver and joint tissues. Radiological and histopathological analysis of joint was performed. A computational molecular docking investigation of the phytoconstituents was conducted against COX-2, IL-1β, IL-6, and TNF-α receptors. ResultsThe ETSL at 500 mg/kg demonstrated significant (p < 0.01) restoration of arthritic parameters, hematological and biochemical indices and oxidative stress in CFA-induced rats which was further supported by radiological histological examination. In addition, there was significant (p < 0.05) reduction observed in pro-inflammatory cytokines, inflammatory mediators and up-regulation of anti-inflammatory cytokines in the treated group. Verbascoside was found to exhibit better biding affinities −10.4, −7.4, −7 and −6.2 kcal/mol against COX-2, IL-1β, TNF-α, and IL-6 respectively, confirmed through in silico study. ConclusionsThe observed outcome suggests that ETSL at a dosage of 500 mg/kg demonstrated notable anti-arthritic effects by suppressing pro-inflammatory cytokines and oxidative stress biomarkers. This effect could potentially be attributed to the presence of bioactive verbascoside identified in the LC-MS analysis.

Trifluoro-Icaritin Ameliorates Neuroinflammation Against Complete Freund's Adjuvant-Induced Microglial Activation by Improving CB2 Receptor-Mediated IL-10/β-endorphin Signaling in the Spinal Cord of Rats.

The underlying pathogenesis of chronic inflammatory pain is greatly complex, but the relevant therapies are still unavailable. Development of effective candidates for chronic inflammatory pain is highly urgent. We previously identified that trifluoro-icaritin (ICTF) exhibited a significant therapeutic activity against complete Freund's adjuvant (CFA)-induced chronic inflammatory pain, however, the precise mechanisms remain elusive. Here, the paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and CatWalk gait analysis were used to determine the pain-related behaviors. The expression and co-localization of pain-related signaling molecules were detected by Western blot and immunofluorescence staining. Our results demonstrated that ICTF (3.0mg/kg, i.p.) effectively attenuated mechanical allodynia, thermal hyperalgesia and improved motor dysfunction induced by CFA, and the molecular docking displayed that CB2 receptor may be the therapeutic target of ICTF. Furthermore, ICTF not only up-regulated the levels of CB2 receptor, IL-10, β-endorphin and CD206, but also reduced the expression of P2Y12 receptor, NLRP3, ASC, Caspase-1, IL-1β, CD11b, and iNOS in the spinal cord of CFA rats. Additionally, the immunofluorescence staining from the spinal cord showed that ICTF significantly increased the co-expression between the microglial marker Iba-1 and CB2 receptor, IL-10, β-endorphin, respectively, but markedly decreased the co-localization between Iba-1 and P2Y12 receptor. Conversely, intrathecal administration of CB2 receptor antagonist AM630 dramatically reversed the inhibitory effects of ICTF on CFA-induced chronic inflammatory pain, leading to a promotion of pain hypersensitivity, abnormal gait parameters, microglial activation, and up-regulation of P2Y12 receptor and NLRP3 inflammasome, as well as the inhibition of CB2 receptor and IL-10/β-endorphin cascade. Taken together, these findings highlighted that ICTF alleviated CFA-induced neuroinflammation by enhancing CB2 receptor-mediated IL-10/β-endorphin signaling and suppressing microglial activation in the spinal cord, and uncovered that CB2 receptor may be exploited as a novel and promising target for ICTF treatment of chronic inflammatory pain.

Astaxanthin Alleviates Chronic Prostatitis via the ERK1/2 Signaling Pathway: Evidence from Network Pharmacology and Experimental Validation.

Astaxanthin (AST) has been widely recognized for its therapeutic potential in chronic inflammatory ailments. This study investigates the therapeutic efficacy and underlying mechanisms of AST in the management of chronic prostatitis (CP). Male Sprague-Dawley (SD) rats were randomly divided into control, complete Freund's adjuvant (CFA), and CFA + AST groups. CFA was used to induce the CP model, and saline was used for the control group. Inflammation of the prostate was detected 28 days after oral administration of AST. qRT-PCR and ELISA were used to detect pro-inflammatory factors in RWPE-1 and WPMY-1 cells. Potential targets of AST for CP were explored by network pharmacology, and related proteins were detected by Western blotting. Oral administration of AST alleviated the increase in prostate stroma and reduced inflammatory cell infiltration in CP rats. The IC50 of AST-treated RWPE-1 and WPMY-1 cells for 48 h were 171 and 212.1 μM, respectively. AST pretreatment reduced IL-6 and IL-8 expression in these cells. PPI network, GO, and KEGG enrichment analyses suggested that the antiinflammatory effect of AST was associated with the ERK1/2 pathway. Western blotting showed that AST inhibited ERK1/2 phosphorylation. In addition, AST and ERK1/2 pathway inhibitors (U0126) synergistically inhibited LPS-induced inflammation in prostate cells. Our study identified the potential of AST in the treatment of CP. However, subsequent randomized controlled trials are needed to validate its clinical efficacy.