Amelioration of melittin on adjuvant-induced rheumatoid arthritis: Integrated transcriptome and metabolome.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease lacking a definitive cure. Although conventional treatments such as dexamethasone and methotrexate are prevalent, their usage is constrained by potential adverse effects. Melittin (MLT) has emerged as a promising natural anti-rheumatic drug; however, studies focusing on the role of MLT in modulating the expression and metabolism of RA-related genes are scarce. METHOD: Arthritis was induced in rats using Complete Freund's Adjuvant (CFA), followed by MLT injections for treatment. Post-treatment, the inflammatory status of each group was assessed, and the mechanistic underpinnings of MLT's ameliorative effects on RA were elucidated through transcriptomic and metabolomic analyses. Additionally, this study conducted qRT-PCR validation of key therapeutic genes and characterized the molecular docking interactions of MLT with key receptor proteins (TNF-α and IL-1ß) using the AutoDock Vina software. RESULT: MLT significantly diminished redness and swelling in affected joints, ameliorated inflammatory cell infiltration, and mitigated joint damage. Integration of transcriptomic and metabolomic data revealed that MLT predominantly regulated the transcription levels of pathways and genes related to cytokines and immune responses, and the metabolic biomarkers of Sphingomyelin, fatty acid, and flavonoid. qRT-PCR confirmed MLT's downregulation of inflammation-related genes such as Il6, Jak2, Stat3, and Ptx3. Molecular docking simulations demonstrated the stable binding of MLT to TNF-α and IL-1ß. CONCLUSION: MLT demonstrated significant efficacy in alleviating RA. This study provides a comprehensive summary of MLT's impact on gene expression and metabolic processes associated with RA.

Antiarthritic potential of ethanolic extract of Ixora coccinea leaves on complete Freund's adjuvant-induced arthritis in animal model.

CONTEXT: Ixora coccinea leaves possess antioxidant, anti-inflammatory, antinociceptive, antimutagenic, and gastroprotective properties. On this background, its antiarthritic potential was evaluated. AIMS: The objective is to evaluate the effect of Ethanolic extract of Ixora coccinea leaves (EEICL) on complete Freund's adjuvant-induced arthritis in rats. SETTINGS AND STUDY DESIGN: PG research laboratory, Pharmacology Department, MKCG Medical College, Berhampur, Odisha. SUBJECTS AND METHODS: Thirty-six Wistar albino rats were randomly distributed into sixgroups (n = 6) as follows: Gr 1 (normal control)-DW p.o, Gr-2 (disease control [DC] - Tween 80 p.o), Gr-3 (piroxicam 0.9 mg/kg p.o), Gr-4 (EEICL-1 g/kg, p.o, Gr 4-EEICL-1.5 g/kg p.o, Gr 5-ED50 (0.82 g/kg) + piroxicam (0.45 mg/kg) p.o. After induction of arthritis, drugs, and vehicles were administered daily from 5th to 25th day. On 0, 5th, 10th, 15th, and 25th day, parameters like body weight, rotarod fall time, paw volume displacement, and arthritis index were measured. On the last day, Erythrocyte sedimentation rate (ESR), tissue malondialdehyde (MDA), and histopathological analysis were done. STATISTICAL ANALYSIS USED: Analysis of parametric data was done by one-way ANOVA and nonparametric data by Kruskal-Wallis test using graph pad prism 7.0. P < 0.05 was considered statistically significant. RESULTS: EEICL (1.5 mg/kg) showed anti-arthritic effect compared with DC. Rotarod fall-off time 137.5 ± 2.5 sec and body weight (139 ± 12.74 g) were increased significantly. The percentage inhibition of paw volume was increased(52%) whereas arthritic score(0.33), ESR(3.51mm/hr), synovial tissue MDA level (0.62±0.13µmol/gm) and Mankin score(2) were reduced significantly as compared to disease control. CONCLUSIONS: EEICL has anti-arthritic potential in rat model.

Obacunone Alleviates Inflammatory Pain by Promoting M2 Microglial Polarization and by Activating Nrf2/HO-1 Signaling Pathway.

Background: Treating inflammatory pain (IP) continues to pose clinical challenge, because of the lack of effective pharmacological interventions. Microglial polarization serves as pivotal determinant in IP progress. Obacunone (OB), a low-molecular-weight compound with a diverse array of biological functions, having reported as an activator of nuclear factor E2-related factor 2 (Nrf2), exhibits anti-inflammatory property. However, it remains uncertain whether OB can alleviate IP by facilitating the transition of microglial polarization from the M1 to M2 state through modulating Nrf2/ heme oxygenase-1 (HO-1) pathway. Methods: We induced an mice IP model by subcutaneously administering Complete Freund's Adjuvant (CFA) into the hind paw. Paw withdrawal latency (PWL) in seconds (s) and paw withdrawal frequency (PWF) were employed to evaluate the establishment of the IP model, while a caliper was used to measure the maximal dorsoventral thickness of the mice paw. Nerve injury was assessed by Hematoxylin-Eosin (HE) Staining. Western blot and got conducted for detection of M1/M2 microglial polarization markers, Nrf2 and HO-1 in spinal cord tissues respectively. Results: In comparison to the control cohort, PWF, M1 phenotype marker iNOS, CD86, paw thickness increased significantly within CFA cohort, while PWL, M2 phenotype marker Arg-1, interleukin-10 (IL-10) decreased in the CFA group. In comparison to model cohort, OB treatment decreased PWF, paw thickness, M1 phenotype marker iNOS, CD86 significantly, while PWL, M2 phenotype marker Arg-1, IL-10, Nrf2, HO-1 increased significantly. The morphological injuries of sciatic nerve in CFA mice were obviously improved by OB treatment. OB inhibited the release of M1-related IL-1ß, CXCL1 but promoted M2-related TGF-ß, IL-10 in serum in CFA mice. The intervention of the Nrf2 inhibitor ML385 mitigated analgesic effect of OB. Conclusion: We demonstrate that OB is able to attenuate inflammatory pain via promoting microglia polarization from M1 to M2 and enhancing Nrf2/HO-1 signal. OB treatment may be a potential alternative agent in the treatment of IP.

The binding of PKCε and MEG2 to STAT3 regulates IL-6-mediated microglial hyperalgesia during inflammatory pain.

Studies have suggested that microglial IL-6 modulates inflammatory pain; however, the exact mechanism of action remains unclear. We therefore hypothesized that PKCε and MEG2 competitively bind to STAT3 and contribute to IL-6-mediated microglial hyperalgesia during inflammatory pain. Freund's complete adjuvant (FCA) and lipopolysaccharide (LPS) were used to induce hyperalgesia model mice and microglial inflammation. Mechanical allodynia was evaluated using von Frey tests in vivo. The interaction among PKCε, MEG2, and STAT3 was determined using ELISA and immunoprecipitation assay in vitro. The PKCε, MEG2, t-STAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, GLUT3, and TREM2 were assessed by Western blot. IL-6 promoter activity and IL-6 concentration were examined using dual luciferase assays and ELISA. Overexpression of PKCε and MEG2 promoted and attenuated inflammatory pain, accompanied by an increase and decrease in IL-6 expression, respectively. PKCε displayed a stronger binding ability to STAT3 when competing with MEG2. STAT3Ser727 phosphorylation increased STAT3 interaction with both PKCε and MEG2. Moreover, LPS increased PKCε, MEG2, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and GLUT3 levels and decreased TREM2 during microglia inflammation. IL-6 promoter activity was enhanced or inhibited by PKCε or MEG2 in the presence of STAT3 and LPS stimulation, respectively. In microglia, overexpression of PKCε and/or MEG2 resulted in the elevation of tSTAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and TREM2, and the reduction of GLUT3. PKCε is more potent than MEG2 when competitively binding to STAT3, displaying dual modulatory effects of IL-6 production, thus regulating the GLUT3 and TREM2 in microglia during inflammatory pain sensation.

Manual acupuncture ameliorates inflammatory pain by upregulating adenosine A3 receptor in complete Freund's adjuvant-induced arthritis rats.

BACKGROUND: Adenosine A3 receptor (A3R) exerts analgesic, anti-inflammatory, and anti-nociceptive effects. In this study, we determined the analgesic mechanism of manual acupuncture (MA) in rats with complete Freund's adjuvant (CFA)-induced arthritis and explored whether MA ameliorates inflammation in these rats by upregulating A3R. METHODS: Sixty Sprague Dawley (SD) rats were randomly divided into the following groups: Control, CFA, CFA + MA, CFA + sham MA, CFA + MA + DMSO, CFA + MA + IB-MECA, and CFA + MA + Reversine groups. The arthritis rat model was induced by injecting CFA into the left ankle joints. Thereafter, the rats were subjected to MA (ST36 acupoint) for 3 days. The clinical indicators paw withdrawal latency (PWL), paw withdrawal threshold (PWT), and open field test (OFT) were used to determine the analgesic effect of MA. In addition, to explore the effect of A3R on inflammation after subjecting arthritis rats to MA, IB-MECA (A3R agonist) and Reversine (A3R antagonist) were injected into ST36 before MA. RESULTS: MA ameliorated the pathological symptoms of CFA-induced arthritis, including the pain indicators PWL and PWT, number of rearing, total ambulatory distance, and activity trajectory. Furthermore, after MA, the mRNA and protein expression of A3R was upregulated in CFA-induced arthritis rats. In contrast, the protein levels of TNF-α, IL-1ß, Rap1, and p-p65 were downregulated after MA. Interestingly, the A3R agonist and antagonist further downregulated and upregulated inflammatory cytokine expression, respectively, after MA. Furthermore, the A3R antagonist increased the degree of ankle swelling after MA. CONCLUSION: MA can alleviate inflammatory pain by inhibiting the NF-κB signaling pathway via upregulating A3R expression of the superficial fascia of the ST36 acupoint site in CFA-induced arthritis rats.

Onosma bracteatum Wall Aqueous-Ethanolic Extract Suppresses Complete Freund's Adjuvant-Induced Arthritis in Rats via Regulation of TNF-α, IL-6, and C-Reactive Protein.

Onosma bracteatum Wall (O. bracteatum) has been used traditionally for the management of arthritis; however, its therapeutic potential warrants further investigation. This study aimed to evaluate the anti-arthritic effects of the aqueous-ethanolic extract of O. bracteatum leaves (AeOB) in a rat model of complete Freund's adjuvant (CFA)-induced arthritis. Rats were treated with AeOB (250, 500, and 750 mg/kg), indomethacin (10 mg/kg), or a vehicle control from days 8 to 28 post-CFA injection. Arthritic score, paw diameter, and body weight were monitored at regular intervals. X-ray radiographs and histopathological analysis were performed to assess arthritic severity. Inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) were quantified by qPCR and icromatography. Phytochemical analysis of AeOB revealed alkaloids, flavonoids, phenols, tannins, Saponins, and glycosides. AeOB also exhibited antioxidant potential with an IC50 of 73.22 µg/mL in a DPPH assay. AeOB and diclofenac exhibited anti-inflammatory and anti-arthritic activities. Rats treated with AeOB at 750 mg/kg and indomethacin showed significantly reduced arthritic symptoms and joint inflammation versus the CFA control. The AeOB treatment downregulated TNF-α and IL-6 and decreased CRP levels compared with arthritic rats. Radiography and histopathology also showed improved prognosis. These findings demonstrate the anti-arthritic potential of AeOB leaves.

Anti-inflammatory and anti-rheumatic effects of Phoenix dactylifera L. (date palm) seed by controlling cytokines and inhibiting JAK1/STAT3 pathway on CFA-induced arthritis rat and its phytochemical profiling.

ETHNOPHARMACOLOGICAL RELEVANCE: Phoenix dactylifera L. (date palm) seed is widely used in Arabian traditional medicine to alleviate several health problems including inflammatory conditions. The herbal tea of date palm seed has been consumed by rheumatoid patients to relief their symptoms. AIM OF THE STUDY: The purpose of this study was to investigate the claimed beneficial use of P. dactylifera L. (Sewy variety) seed (PDS) in the treatment of rheumatoid arthritis (RA) and its mechanism of action as well as to study its phytoconstituents. MATERIALS AND METHODS: The anti-inflammatory and anti-oxidative properties of the non-polar and the polar extracts of PDS were studied using Complete Freund's adjuvant (CFA)-induced arthritis rat model. Paw edema, body weight, total nitrate/nitrite NOX content and cytokine markers were evaluated to monitor the progress of arthritis. Also, histological examination and thermal analysis were conducted. The phytoconstituent profiles of non-polar and polar extracts of PDS were investigated using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The multiple reactions monitoring mode (MRM) of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used to quantify phenolic phytoconstituents in both extracts. RESULTS: According to the findings, the polar and non-polar PDS extracts kept body weight comparable to those of healthy individuals while considerably lowering paw swelling, edema, and neutrophil infiltration. It also reduced the levels of Nuclear Factor Kappa B (NF-κB), Tumor Necrosis Factor Alpha (TNF-α), Interleukin 22, Interleukin 23, Interferon (IFN), Interleukin 17, Interleukin 1ß, Interleukin 6, Interleukin 36, Janus Kinase 1 (JAK1), and Signal Transducer and Activator of Transcription 3 (STAT3). They also reduced the degenerative alterations caused by RA. Thermal research gave additional support for these findings. 83 phytoconstituents were identified in the non-polar PDS extract and 86 phytoconstituents were identified in the polar PDS extract. 74 of the identified phytoconstituents were common in both extracts. 33 phytoconstituents were identified here from P. dactylifera for the first time as far as we know. In MRM-LC-ESI-MS/MS analysis, the major phenolics in both extracts were chlorogenic acid, naringenin, and vanillin. Catechin was only detected in the non-polar PDS extract. On the other hand, apigenin, kaempferol, and hesperetin were only detected in the polar PDS extract. Generally, the polar PDS extract showed higher concentrations of the identified phenolics than the non-polar extract. CONCLUSIONS: The PDS extracts especially the non-polar extract showed significant anti-inflammatory and anti-oxidative properties in the CFA-induced arthritis rat model. PDS might be used to produce RA medicines.

Evaluating the docetaxel effect in an animal model of polyarthritis.

Rheumatoid arthritis (RA) is immune-mediated, inflammatory disease that affects synovial joints, and characterized by inflammatory changes in synovial tissue, cartilage, bone, and less commonly in extra-articular structures. Docetaxel (DTX) is a semi-synthetic anti-neoplastic medication. Peptidyl-arginine deiminase type 4 (PAD4) is expressed in macrophages and neutrophils in RA synovial membrane. Their effectiveness is in producing anti-cyclic citrullinated peptide antibodies (ACPA)-targeted citrullinated neoepitopes. AIM: To evaluate the anti-inflammatory effects of DTX in RA and the effect of methotrexate on PAD4 to investigate its potential as an RA biomarker. METHODS: Forty male Wistar rats were divided into five groups of eight rats. Healthy rats formed the control group. The Second Group to Fifth group were induced with Complete Freund's adjuvant. The third group received DTX at a dosage of 1 mg/kg on alternate days, as determined by a preliminary experiment. The fourth group was given 1 mg/kg/week of methotrexate intraperitoneally. The fifth group was treated with a half dose of DTX and methotrexate simultaneously. RESULTS: Significant Arthritis index and knee joint circumference decrease in the DTX group. No significant difference in body weight, platelet-lymphocyte ratio, and white blood cell count between the groups. Neutrophile lymphocyte ratio showed weak correlation with ACPA, while PAD4 showed good correlation with RA markers. Level of ACPA, PAD4, TNF-α, IL-1ß, and VEGF significantly decreased in the DTX group than induction group (p < 0.05). CONCLUSION: DTX reduces the progression and joint destruction in rats induced by Complete Freund's Adjuvant which may due to inhibition of PAD4, TNF-α, IL-1ß, VEGF, and ACPA. Also, methotrexate exhibited anti PAD4 effect.

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

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

Complete Freund's adjuvant as a confounding factor in multiple sclerosis research.

Complete Freund's adjuvant (CFA) is used as a standard adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model in multiple sclerosis studies. Still, CFA induces glial activation and neuroinflammation on its own and provokes pain. In addition, as CFA contains Mycobacteria, an immune response against bacterial antigens is induced in parallel to the response against central nervous system antigens. Thus, CFA can be considered as a confounding factor in multiple sclerosis-related studies performed on EAE. Here, we discuss the effects of CFA in EAE in detail and present EAE variants induced in experimental animals without the use of CFA. We put forward CFA-free EAE variants as valuable tools for studying multiple sclerosis pathogenesis and therapeutic approaches.

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.

NAMPT/NAD+/PARP1 Pathway Regulates CFA-Induced Inflammatory Pain via NF-κB Signaling in Rodents.

Emerging evidence has implicated nicotinamide adenine dinucleotide (NAD+) metabolism in various inflammatory diseases. In the study, the role of NAD+ metabolism in Complete Freund's Adjuvant (CFA)-evoked inflammatory pain and the underlying mechanisms are investigated. The study demonstrated that CFA induced upregulation of nicotinamide phosphoribosyltransferase (NAMPT) in dorsal root ganglia (DRG) without significant changes in the spinal cord. Inhibition of NAMPT expression by intrathecal injection of NAMPT siRNA alleviated CFA-induced pain-like behavior, decreased NAD+ contents in DRG, and lowered poly-(ADP-ribose) polymerase 1 (PARP1) activity levels. These effects are all reversed by the supplement of nicotinamide mononucleotide (NMN). Inhibition of PARP1 expression by intrathecal injection of PARP1 siRNA alleviated CFA-induced pain-like behavior, while elevated NAD+ levels of DRG. The analgesic effect of inhibiting NAMPT/NAD+/PARP1 axis can be attributed to the downregulation of the NF-κB/IL-1ß inflammatory pathway. Double immunofluorescence staining showed that the expression of NAMPT/NAD+/PARP1 axis is restricted to DRG neurons. In conclusion, PARP1 activation in response to CFA stimulation, fueled by NAMPT-derived NAD+, mediates CFA-induced inflammatory pain through NF-κB/IL-1ß inflammatory pathway.

Brain inflammation in experimental autoimmune encephalomyelitis induced in Dark Agouti rats with spinal cord homogenate.

We have recently characterized experimental autoimmune encephalomyelitis (EAE) induced in DA rats with spinal cord homogenate without complete Freund's adjuvant (CFA). The main advantage of this multiple sclerosis model is the lack of CFA-related confounding effects which represent the major obstacles in translating findings from EAE to multiple sclerosis. Here, antigen specificity of the cellular and humoral immune response directed against the central nervous system was explored. The reactivity of T and B cells to myelin basic protein, myelin oligodendrocyte glycoprotein, and ß-synuclein was detected. Having in mind that reactivity against ß-synuclein was previously associated with autoimmunity against the brain, the infiltration of immune cells into different brain compartments, i.e. pons, cerebellum, hippocampus, and cortex was determined. T cell infiltration was observed in all structures examined. This finding stimulated investigation of the effects of immunization on DA rat behavior using the elevated plus maze and the open field test. Rats recovered from EAE displayed increased anxiety-like behavior. These data support CFA-free EAE in DA rats as a useful model for multiple sclerosis research.

Neonatal nociceptive stimulation results in pain sensitization, reduction of hippocampal 5-HT1A receptor, and p-CREB expression in adult female rats.

Painful invasive procedures are often performed on newborns admitted to intensive care units (ICU). The acute and long-term effects caused by these stimuli can be investigated in animal models, such as newborn rats. Previous studies have shown that animals subjected to nociceptive stimuli in the neonatal period show sex-specific behavioral changes such as signs of anxiety or depression. Under the same conditions, neonatal stimuli also provoke an increase in the rate of neurogenesis and cell activation in the hippocampal dentate gyrus. So, this study aims to identify the possible roles of central monoamines, receptor expression (5-HT1A), and signaling factors (p-CREB) underlying the long-term effects of neonatal nociceptive stimulation. For this, noxious stimulation was induced by intra-plantar injection of Complete Freund´s adjuvant (CFA) on the postnatal day 1 (P1) or 8 (P8). Control animals were not stimulated. On P75 the behavioral tests were conducted (hotplate and elevated plus maze), followed by sacrifice and molecular studies. Our results showed that neonatal nociceptive stimulation alters pain sensitization specially in females, while stimulation on P1 increases pain threshold, P8-stimulated animals respond with reduced pain threshold (P < 0.001). Hippocampal expression of 5-HT1A receptor and p-CREB were reduced in P8 F group (P < 0.001) in opposition to the increased utilization rate of dopamine and serotonin in this group (P < 0.05). This study shows sex- and age-specific responses of signaling pathways within the hippocampus accompanied by altered behavioral repertoire, at long-term after neonatal painful stimulation.

A Preterm Rat Model for Pain Studies.

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Effect of electroacupuncture on inflammatory response and immune cells in mice with chronic inflammatory pain. / ç”µé’ˆå¯¹æ ¢æ€§ç‚Žæ€§ç—›å°é¼ ç‚Žæ€§ååº”å’Œå ç–«ç»†èƒžçš„å½±å“.

OBJECTIVES: To observe the effects of electroacupuncture(EA) on local inflammatory mediators and macrophage polarization, and immune cells in the spleen of mice with chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in the hind paw, so as to investigate the immunoinflammatory regulatory mechanisms of EA in relieving pain and swelling in mice with chronic inflammatory pain. METHODS: Thirty C57BL/6 mice were randomly divided into control, model, and EA groups, with 10 mice in each group. Chronic inflammatory pain model were established by subcutaneous injection of 20 µL CFA solution in the left hind paw for 7 consecutive days. After modeling, mice in the EA group received EA at bilateral "Zusanli"(ST36) for 20 min (2 Hz/100 Hz, 1 mA) once a day for 18 consecutive days. Mechanical pain threshold, heat pain thresholds, and paw thickness were measured before and after mode-ling, and after interventions. Western blot was used to detect the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and NOD-like receptor protein 3 (NLRP3) in the paw tissue. Immunohistochemistry was used to detect the positive expression of M1-type macrophage marker inducible nitric oride synthase (iNOS) and M2-type marker CD206 in the paw, and flow cytometry was used to detect the proportion of F4/80+ CD11b+ macrophages, Ly6G+ CD11b+ neutrophils, and CD25+ Foxp3+ regulatory T cells (Treg) in the spleen. RESULTS: Compared with the control group, mechanical pain and heat pain thresholds were significantly reduced(P<0.000 1), while paw thickness, expressions of IL-1ß, TNF-α, and NLRP3 in the paw, and positive expression of M1 macrophage marker iNOS in the paw, the proportions of macrophages and neutrophils in the spleen were significantly increased (P<0.000 1, P<0.001) in the model group. Compared with the model group, mechanical pain threshold and heat pain thresholds, CD206 positive expression in the paw, and Treg cell proportion in spleen were significantly increased (P<0.01), while paw thickness, the expressions of IL-1ß, TNF-α and NLRP3 in the paw, as well as the positive expression of M1 macrophage marker iNOS in the paw, the proportions of macrophages and neutrophils in the spleen were significantly reduced (P<0.001, P<0.01, P<0.05)in mice of the EA group after intervention. CONCLUSIONS: EA may alleviate pain and swelling in mice with chronic inflammatory pain by regulating the numbers of macrophages, neutrophils, and Treg cells, as well as promoting M2 polarization of local macrophages and inhibiting the release of pro-inflammatory cytokines.

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.

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

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

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.