Subcutaneous Air Pouch Research Articles

Anti-Inflammatory, Antinociceptive, and LC-MS Metabolic Profile from Pseudotrimezia juncifolia (Klatt) Lovo & A. Gil.

Pseudotrimezia juncifolia (Klatt) Lovo & A. Gil (Iridaceae) is a popularly known species with primarily ornamental economic interest. It has traditional uses as purgative, in conditions related to the menstrual cycle, for blood purification, as wound healing, and as anti-inflammatory. The anti-inflammatory and antinociceptive activities of the decoction from its aerial stems, corms, and stamens are described here with dereplication studies on LC-MS/MS supported by the GNPS platform, where phenolic compounds were annotated and correlated with its biological activity. The decoction was evaluated in chemical (formalin and capsaicin) and thermal (hot plate) induced nociception or carrageenan-induced inflammation in mice. Decoction (at 10, 30, or 100 mg/kg doses) significantly reduced formalin- or capsaicin-induced nociception. All doses also demonstrated an antinociceptive effect in the hot plate model increasing the time the animal spent in responding to thermal signal. Naloxone partially reversed the antinociceptive effect. An anti-inflammatory effect was observed since a reduction in cell migration, protein extravasation interleukin-1, and tumor necrosis factor production induced by carrageenan in the subcutaneous air pouch was quantified. Metabolomic analyses showed a predominance of phenolic substances, mainly flavonoids and chlorogenic acids. The literature showed that these two groups have significant anti-inflammatory and analgesic activity, and chemical data corroborate the pharmacological results observed.

Low-level LASER therapy accelerates fungal lesions cicatrization by increasing the production of Th1 and Th2 cytokines.

Our study focused on the resolution of lesions caused by fungal infection using a subcutaneous air pouch model of infection. We evaluated cell profile and cytokines, fungi viability, and the presence of fibroblasts and fibrocytes at the site of infection. Inoculation of P. brasiliensis (Pb) was performed using a subcutaneous air pouch model and the LLLT irradiation was performed on alternate days on the rear paws of mice for 10days, after which the cells from the air pouch were collected and analyzed. In animals irradiated with LLLT, the influx of cells to the air pouch was reduced, but they were more activated and produced pro-inflammatory (IL-12, IL-17 and TNF-α) and neutrophil (PMN) activating cytokines (IL-8, GM-CSF and γ-IFN). A better resolution of the infection, evidenced by the reduction in the number of viable fungi with preserved morphology in the air pouch, and an increase in the number of fibrocytes, indicating a healing profile were also observed. LLLT decreased the influx of PMN, but those presents were highly activated, with increased fungicidal activity. LLLT irradiation also resulted in earlier cicatrization at the site of infection, leading to a better outcome of the infection. These data are favorable to the use of LLLT as a complementary therapy in PCM.

Deficiency of histone deacetylases 3 in macrophage alleviates monosodium urate crystals-induced gouty inflammation in mice

BackgroundGout is caused by monosodium urate (MSU) crystals deposition to trigger immune response. A recent study suggested that inhibition of Class I Histone deacetylases (HDACs) can significantly reduce MSU crystals-induced inflammation. However, which one of HDACs members in response to MSU crystals was still unknown. Here, we investigated the roles of HDAC3 in MSU crystals-induced gouty inflammation.MethodsMacrophage specific HDAC3 knockout (KO) mice were used to investigate inflammatory profiles of gout in mouse models in vivo, including ankle arthritis, foot pad arthritis and subcutaneous air pouch model. In the in vitro experiments, bone marrow-derived macrophages (BMDMs) from mice were treated with MSU crystals to assess cytokines, potential target gene and protein.ResultsDeficiency of HDAC3 in macrophage not only reduced MSU-induced foot pad and ankle joint swelling but also decreased neutrophils trafficking and IL-1β release in air pouch models. In addition, the levels of inflammatory genes related to TLR2/4/NF-κB/IL-6/STAT3 signaling pathway were significantly decreased in BMDMs from HDAC3 KO mice after MSU treatment. Moreover, RGFP966, selective inhibitor of HDAC3, inhibited IL-6 and TNF-α production in BMDMs treated with MSU crystals. Besides, HDAC3 deficiency shifted gene expression from pro-inflammatory macrophage (M1) to anti-inflammatory macrophage (M2) in BMDMs after MSU challenge.ConclusionsDeficiency of HDAC3 in macrophage alleviates MSU crystals-induced gouty inflammation through inhibition of TLR2/4 driven IL-6/STAT3 signaling pathway, suggesting that HDAC3 could contribute to a potential therapeutic target of gout.

Anti-Inflammatory Activity of N'-(3-(1H-indol-3-yl)benzylidene)-2-cyanoacetohydrazide Derivative via sGC-NO/Cytokine Pathway.

The N-acylhydrazone function has been reported as a pharmacophore group of molecules with diverse pharmacological activities, including anti-inflammatory effects. Therefore, this study was designed to evaluate the anti-inflammatory potential of the compound N'-(3-(1H-indol-3-yl)benzylidene)-2-cyanoacetohydrazide (JR19) in vivo. The study started with the carrageenan-induced peritonitis model, followed by an investigation of leukocyte migration using the subcutaneous air pouch test and an assessment of the antinociceptive profile using formalin-induced pain. A preliminary molecular docking study focusing on the crystallographic structures of NFκB, iNOS, and sGC was performed to determine the likely mechanism of action. The computational study revealed satisfactory interaction energies with the selected targets, and the same peritonitis model was used to validate the involvement of the nitric oxide pathway and cytokine expression in the peritoneal exudate of mice pretreated with L-NAME or methylene blue. In the peritonitis assay, JR19 (10 and 20 mg/kg) reduced leukocyte migration by 59% and 52%, respectively, compared to the vehicle group, with the 10 mg/kg dose used in subsequent assays. In the subcutaneous air pouch assay, the reduction in cell migration was 66%, and the response to intraplantar formalin was reduced by 39%, particularly during the inflammatory phase, suggesting that the compound lacks central analgesic activity. In addition, a reversal of the anti-inflammatory effect was observed in mice pretreated with L-NAME or methylene blue, indicating the involvement of iNOS and sGC in the anti-inflammatory response of JR19. The compound effectively and significantly decreased the levels of IL-6, TNF-α, IL-17, and IFN-γ, and this effect was reversed in animals pretreated with L-NAME, supporting a NO-dependent anti-inflammatory effect. In contrast, pretreatment with methylene blue only reversed the reduction in TNF-α levels. Therefore, these results demonstrate the pharmacological potential of the novel N-acylhydrazone derivative, which acts through the nitric oxide pathway and cytokine signaling, making it a strong candidate as an anti-inflammatory and immunomodulatory agent.

Suppression of P2X7R by Local Treatment Alleviates Acute Gouty Inflammation.

Gout is the most common inflammatory arthritis associated with interleukin-1β (IL-1β) accumulation during exacerbation. In this study, we aimed to clarify whether potassium channel antagonists attenuate local inflammation in mice with monosodium urate (MSU)-induced gout. We cultured human macrophage THP-1 cells and evaluated the molecular levels of both IL-1β and potassium channels stimulated with MSU and/or potassium channel antagonists. Acute gout models were generated in IL-1β luciferase transgenic male mice using synovium-like subcutaneous air pouches with MSU injection. Their luciferase activities were monitored following potassium channel blocker treatment using the IVIS Spectrum CT imaging system. The lavages and tissues were extracted from their air pouches, followed by cell counting and pathological analysis. MSU stimulation increased the gene expression levels of pro-IL-1β, P2x7r and Kv1.3, whereas the expression of Kcnq1 was decreased in phorbol 12-myristate 13-acetate-induced THP-1 cells. Both high and low concentrations of the P2x7 receptor inhibitor adenosine 5'-triphosphate (ATP) derivative periodate oxidized ATP (oATP) decreased the production of IL-1β in the supernatant of THP-1 cells. The sixth hour was the peak time of IL-1β luciferase activity after MSU intervention in vivo. oATP ameliorated the synovial IL-1β luciferase activity, reduced inflammatory cell infiltration and alleviated the erosive damage in the cartilage. The anti-inflammatory properties of potassium channel inhibitors, especially of oATP, might point to new strategies for local anti-inflammatory therapy for acute gout.

Galantamine attenuates autoinflammation in a mouse model of familial mediterranean fever.

Autoinflammatory diseases, a diverse group of inherited conditionscharacterized by excessive innate immune activation, have limited therapeutic options. Neuroimmune circuits of the inflammatory reflex control innate immune overactivation and can be stimulated to treat disease using the acetylcholinesterase inhibitor galantamine. We tested the efficacy of galantamine in a rodent model of the prototypical autoinflammatory disease familial Mediterranean fever (FMF). Multiple chronic disease markers were evaluated in animals that received long-term galantamine treatment compared to vehicle. Long-term treatment with galantamine attenuated the associated splenomegaly and anemia which are characteristic features of this disease. Further, treatment reduced inflammatory cell infiltration into affected organs and a subcutaneous air pouch. These findings suggest that galantamine attenuates chronic inflammation in this mouse model of FMF. Further research is warranted to explore the therapeutic potential of galantamine in FMF and other autoinflammatory diseases.

Tibouchina granulosa Leaves Present Anti-Inflammatory Effect.

The ethanol extract (EE) prepared from the leaves of Tibouchina granulosa, and its fraction in ethyl acetate (fEA) were evaluated concerning their capacity to reduce inflammation in different experimental models. fEA was also studied concerning its chemical constituents. EE and fEA were assayed for their anti-inflammatory potential, using formalin-induced licking behavior and carrageenan-induced inflammation into the subcutaneous air pouch (SAP) models. Reduction in polymorphonuclear cells (PMN) activation was performed in freshly isolated PMN. Chromatographic analysis of fEA was performed by HPLC-DAD. Hispiduloside was isolated as the main constituent in fEA, and its quantity was estimated to be 39.3% in fEA. EE (30 mg/kg) significantly reduced the second phase of formalin-induced licking. fEA demonstrated a reduction in leukocyte migration into the SAP. EE and fEA drastically reduced cytokines (TNF-α, IL-1β, and IFN-γ), nitric oxide (NO) production, in vitro PMN migration induced by C5a and IL-8, and TNF-α and IL-1β gene expression. Taken together, our data indicate that either ethanol extract or its fEA fraction from leaves of T. granulosa present an anti-inflammatory effect, contributing to the pharmacological and chemical knowledge of this species and confirming the rationale behind its traditional use.

POP1 inhibits MSU-induced inflammasome activation and ameliorates gout.

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

Models of Inflammation: Carrageenan Air Pouch.

The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this article has been extensively used to identify potential anti-inflammatory drugs. © 2021 Wiley Periodicals LLC. Basic Protocol: Air pouch model in the rat Alternate Protocol: Air pouch model in the mouse.

Characterization of βN-Octadecanoyl-5-hydroxytryptamide Anti-Inflammatory Effect.

Background: N-octadecanoyl-5-hydroxytryptamide (C18-5HT) is an amide that can be obtained by the coupling of serotonin and octadecanoic acid. This study aims to characterize the in vivo and in vitro anti-inflammatory activity of C18-5HT. Methods: A subcutaneous air pouch model (SAP) was used. The exudates were collected from SAP after carrageenan injection to assess cell migration and inflammatory mediators production. RAW 264.7 cells were used for in vitro assays. Results: C18-5HT significantly inhibited leukocyte migration into the SAP as well as nitric oxide (NO) and cytokines production and protein extravasation. We also observed an reduction in some cytokines and an increase in IL-10 production. Assays conducted with RAW 264.7 cells indicated that C18-5HT inhibited NO and cytokine produced. Conclusions: Taken together, our data suggest that C18-5HT presents a significant effect in different cell types (leukocytes collected from exudate, mainly polumorphonuclear leukocytes and cell culture macrophages) and is a promising compound for further studies for the development of a new anti-inflammatory drug.

Brazilian Red Propolis shows antifungal and immunomodulatory activities against Paracoccidioides brasiliensis

Ethnopharmacological relevanceParacoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in South America and especially in Brazil with severe clinical consequences that need broadened therapeutic options.Propolis is a natural resin from bees used in folk medicine for centuries with the first report in the ancient history of Egypt by Eberly papyrus, in Middle-Ages used to wash the newborn's umbilical cord and World War II as antiseptic or antibiotics. Nowadays it is a natural product worldwide consumed as food and traditionally used for oral and systemic diseases as an anti-inflammatory, antimicrobial, antifungal, and other diseases. Brazilian red propolis (BRP) is a new type of propolis with a distinguished chemical profile and biological activities from propolis (green) with pharmacological properties such as antimicrobial, anti-inflammatory, antioxidant, and others. Aim of studyThus, the main purpose of this study was to investigate the direct in vitro and ex vivo effect of BRP on Paracoccidioides brasiliensis. Material and methodsAntifungal activity of different concentrations of BRP on a virulent P. brasiliensis isolate (Pb18) was evaluated using the microdilution technique. Also, mice splenic cells co-cultured with Pb18 were treated with BRP at different times and concentrations (only Pb18 = negative control). Mice were inoculated with Pb18 and treated with different concentrations of BRP (50–500 mg/mL) in a subcutaneous air pouch. In this later experimental model, macroscopic characteristics of the air pouch were evaluated, and cellular exudate was collected and analyzed for cellular composition, mitochondrial activity, total protein reactive oxygen specimens (ROS), and nitric oxide production, as well as the number of viable fungal cells. ResultsThe in vitro experiments showed remarkable direct antifungal activity of BRP, mainly with the highest concentration employed (500 mg/mL), reducing the number of viable cells to 10% of the original inoculum after 72 h incubation. The splenocytes co-cultivation assays showed that BRP had no cytotoxic effect on these cells, on the contrary, exerted a stimulatory effect. This stimulation was also observed on the PMNs at the air pouch, as verified by production of ROS and total proteins and mitochondrial activity. This activation resulted in enhanced fungicidal activity, mainly with the 500 mg/mL concentration of BRP. An anti-inflammatory effect was also detected, as verified by the smaller volume of the BRP-treated air pouch as well as by an earlier shift from neutrophils to mononuclear cells present in the infection site. ConclusionOur results strongly suggest, for the first time in the literature, that Brazilian Red propolis has four protective mechanisms in experimental paracoccidioidomycosis: activating neutrophils, exerting a direct antifungal effect, preventing fungal dissemination, and controlling excessive inflammation process.

Aristolochia trilobata: Identification of the Anti-Inflammatory and Antinociceptive Effects

Aristolochia trilobata, popularly known as “mil-homens,” is widely used for treatment of stomach aches, colic, asthma, pulmonary diseases, diabetes, and skin affection. We evaluated the antinociceptive and anti-inflammatory activities of the essential oil (EO) and the main constituent, 6-methyl-5-hepten-2-yl acetate (sulcatyl acetate, SA). EO and SA (1, 10, and 100 mg/kg, p.o.) were evaluated using chemical (formalin-induced licking) and thermal (hot-plate) models of nociception or inflammation (carrageenan-induced cell migration into the subcutaneous air pouch, SAP). The mechanism of antinociceptive activity was evaluated using opioid, cholinergic receptor antagonists (naloxone and atropine), or nitric oxide synthase inhibitor (L-NAME). EO and SA presented a central antinociceptive effect (the hot-plate model). In formalin-induced licking response, higher doses of EO and SA also reduced 1st and 2nd phases. None of the antagonists and enzyme inhibitor reversed antinociceptive effects. EO and SA reduced the leukocyte migration into the SAP, and the cytokines tumor necrosis factor and interleukin-1 (TNF-α and IL-1β, respectively) produced in the exudate. Our results are indicative that EO and SA present peripheral and central antinociceptive and anti-inflammatory effects.

Correction: Heme oxygenase-1—Dependent anti-inflammatory effects of atorvastatin in zymosan-injected subcutaneous air pouch in mice

[This corrects the article DOI: 10.1371/journal.pone.0216405.].

Heme oxygenase-1-Dependent anti-inflammatory effects of atorvastatin in zymosan-injected subcutaneous air pouch in mice.

Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have previously reported that macrophages treated with statins increased the expression of heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress protein, responsible for the degradation of heme. In the present study, we investigated the effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo. Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae, was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory markers were determined in exudates collected from the pouch 24 hours post zymosan injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice treated with atorvastatin showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of atorvastatin treatment on the modification of their relative proportion. These findings identify HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as an in vivo anti-inflammatory agent.

Serotonin amide with anti‐inflammatory and wound healing properties

Some serotonin amides, as N‐behenoyl‐5‐hydroxytryptamine (C22:0‐5‐HT), are present in the surface wax of coffee beans and few studies show their possible biological activity. Thus, the aim of this work was to evaluate the anti‐inflammatory effect of C20:0‐5‐HT in the model of carrageenan‐induced inflammation into the subcutaneous air pouch (SAP), production of reactive oxygen species (ROS) by PMA‐stimulated leukocytes and MTT cell viability assay healing effect on the contraction model of the lesion in diabetic mice. C22:0‐5‐HT was synthesized by the group of Professor Claudia Rezende, Chemistry Institute, UFRJ. Female Webster mice (20–25g; n= 6–8) received oral administration of C22:0‐5‐HT (0.1, 1, 3 or 10 mg/kg) and were evaluated in model of SAP. Mice received carrageenan injection into the SAP and after 24 h were euthanized and the exsudate from SAP were collected for measurement of cell count. For the evaluation of ROS, leukocytes from the SAP were collected and incubated with C22:0‐5‐HT (0.1, 1 or 3 μM) for 1 h. Cells were incubated with 10 nM phorbol myristate acetate and 2 mM 2′‐7′‐dichlorodihidrofluoescein diacetate. The results were expressed as DCF‐DA fluorescence. Cell viability was measured by the MTT assay using RAW 264.7 mouse macrophages. For the analysis of healing activity female Webster mice (30–35g, n=10–12) received alloxan (65 mg/kg, iv) and after 7 days were anesthetized with ketamine and xylazine. The dorse was trichotomized and an area of 10 mm in diameter was exposed. The animals received topical administration (3 mg/animal) for 14 days and photos were taken on days 0, 3, 7, 10 and 14 to follow the retraction of the lesion. The image was processed in ImageJ program and the results are presented as the mean ± standard deviation of the wound area and expressed as arbitrary units (AU). Statistical significance between groups was determined by ANOVA followed by Bonferroni's test (*p<0.05) and the number of the animal research ethical committee is DFBCICB015‐04/16. C22:0‐5‐HT showed anti‐inflammatory activity in the three higher doses tested with reduction on cell migration (vehicle= 60.8 ± 8 × 106 cells/mL versus 61.7 ± 7 × 106 cells/mL, 31.2 ± 6* × 106 cells/mL, 37.1 ± 7* × 106 cells/mL, and 30.4 ± 6* × 106 cells/mL), with the doses of 0.1, 1, 3, and 10 mg/kg, respectively. It was also observed reduction in levels of ROS by PMA‐stimulated leukocytes in all concentrations tested (vehicle= 197,237.4 ± 8 fluorescence versus 98,852.1 ± 9* fluorescence, 87,951.3 ± 8.* fluorescence, 77,932.1 ± 7* fluorescence) and did not reduce the cell viability. Was observed improvement in healing of the treated mice on days 10 and 14 after injury. Naïve: day 0 = 40.1 ± 3.1 AU; 3 = 29.2 ± 5.1 AU; 7 = 13.1 ± 3.4 AU; 10 = 6.1 ± 2.2 AU; 14 = 3.2 ± 1.1 AU; Diabetic mice: day 0 = 41.3 ± 5.1 AU; 3 = 30.2 ± 5.4 AU; 7 = 20.1 ± 4.4 AU; 10 = 13 ± 3.5 AU; 14 = 7.2 ± 2 AU; Diabetic mice C22:0‐5‐HT‐treated (3 mg/animal) day 0 = 37.4 ± 6 AU; 3 = 30.9 ± 4 AU; 7 = 14.1 ± 3 AU; 10 = 2.7 ± 0.9* AU; 14 = 0.3 ± 0.1* AU. The results demonstrate that C22:0‐5‐HT produces anti‐inflammatory and healing effect. The mechanism(s) still under investigation.Support or Funding InformationThis study was financed in part by the CNPq and FAPERJ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New analogs of LASSBio‐1829Cl with anti‐inflammatory properties

IntroductionInflammation is a benefice response of the immune system in response to an infectious agent or tissue damage. During the inflammatory process, there are vascular and cellular involvement. Most common drugs indicated are the non‐steroidal anti‐inflammatory drugs (NSAID). However, due to the diversity of their side effects the continue search for new substances is still a goal for researchers. In this respect LASSBio‐1829Cl was firstly described with significant anti‐inflammatory property. In view of this activity the new analogues, LASSBio‐2060 and LASSBio‐2061, were synthetized. So, the aim of the present work was to evaluate the anti‐inflammatory effects of LASSBio‐2060 and LASSBio‐2061 using traditional methods of inflammation.MethodsMale Swiss Webster mice (28–32g, n=6) were used in models of formalin‐induced licking or carrageenan‐induced cell migration into the subcutaneous air pouch (SAP) models. Mice were orally treated with LASSBio‐2060 or LASSBio‐2061 (10, 30 or 100 μmol/kg). After 1 hour mice received intraplantar injection of formalin (2.5%, 50 μL) and the time that mice spent licking the formalin‐injected paw was recorded with a chronometer. In SAP model, after oral administration of substances, mice received carrageenan (1%, 1 mL) or saline injection into SAP and 24 hours later mice are euthanized and exudate collected for further measurements. Results are presented as media ± sd. Statistical analysis were performed by ANOVA followed by Bonferroni test (*p<0.05). The protocol for use of animals was approved by CEUA/UFRJ and received the number DFBCICB015‐04/16.ResultsAlthough none of the substance inhibited the 1st phase of formalin‐induced licking both had an effect in 2nd phase as shown: vehicle‐treated group: 161.4±67.1sec; LASSBio‐2060, 30μmol/Kg: 173.2±9.6sec; 100μmol/kg: 92.3±37.2*sec. LASSBio‐2061 10μmol/kg: 128.2±50.8sec; 30μmol/kg: 34.0±10.6*sec; 100μmol/kg: 80.3±43.6*sec. LASSBio‐2060 inhibited leukocyte migration in a dose‐dependent manner. vehicle‐treated group: 96.2±31.6 ×103 cells/μL; 10μmol/kg: 62.3±14.2* ×103 cells/μL; 30μmol/kg: 43.7±17.0* ×103 cells/μL; 100μmol/kg: 31.5±12.0* ×103 cells/μL.. LASSBio‐2061 also inhibited leukocyte migration into the SAP at higher doses: vehicle‐treated group: 79.3±28.9 ×103 cells/μL; 10μmol/kg: 72.5±35×103 cells/μL; 30μmol/kg: 30.2±12.7*×103 cells/μL; 100μmol/kg: 48.4±7.6*×103 cells/μL. Both compounds reduce production of the cytokine IL‐1β. LASSBio‐2060, vehicle‐treated group: 2.356.3±783.4 pg/mL; 30μmol/kg: 1.246.3±411.9* pg/mL; 100μmol/kg: 1.359.5±534.0* pg/mL. LASSBio‐2061, significantly reduced only at its lower dose, vehicle‐treated group: 1.813.66 ± 553.7 pg/mL; 10μmol/kg: 1.024.7± 237.5 pg/mL.ConclusionThe results suggest that both substances reduced cell migration induced by carrageenan and reduced the production of cytokine IL‐1β indicating them as promising anti‐inflammatory.Support or Funding InformationFinancial support: CAPES, CNPq, FAPERJ Institute Vital Brazil (donation of mice) Technical Support: Alan MinhoThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New Analogs of Phenacetin with Anti‐Inflammatory Activity

Phenacetin is a compound with pharmacological activity used in the past with antipyretic and analgesic effects. Three new analogues were synthetized aiming improvements in efficacy and less adverse effects, named 4‐ethoxyaniline, R‐71 and R‐72. Nociception and inflammation have been assessed by the formalin‐induced licking response and carrageenan‐induced cell migration models, respectively. Male Swiss webster mice (22–30 g, n = 6–8) were used for both tests, following animal ethics guidelines. The protocol for animal use was approved by CEUA/UFRJ and received the number DFBCICB015‐04/16. Results are compared with vehicle‐treated group and expressed as mean ± SD and statistical analysis were performed by ANOVA followed by Bonferroni test (*p<0.05). Formalin‐induced licking response. The stimulus is given by the injection of 20 μL of formalin 2.5% in mice's left hind paw. The time the animal spends licking the injected paw is recorded. The animals received oral treatment of 4‐ethoxyaniline, R‐71 or R‐72 at 0.1, 1 or 10 mg/kg, acetylsalicylic acid (200 mg/kg) or morphine (2.5 mg/kg) 60 minutes before formalin injection. To access cell migration, the subcutaneous air pouch (SAP) was produced with a sterile air injection and 6 days later, animals received oral treatment with 4‐ethoxyaniline, R‐71 or R‐72 (0.1, 1 or 10 mg/kg) one hour before the injection of sterile carrageenan (1%) or saline into the SAP. After 24 h, all animals were euthanized, the SAP was washed with 1 mL of saline and the exudate was collected for mediators and protein measurements to investigate mechanism of action. Treatment with the three compounds significantly reduced licking time in neurogenic and inflammatory phases of the licking model. 1st phase: vehicle= 52.7 ± 11.3 seconds (sec); 4‐ethoxyaniline: 23.0 ± 5.5* sec; 24.8 ± 10.2* sec; 24.9 ± 3.5* sec; R‐71: 28.8 ± 6.6* sec; 24.2 ± 7.2* sec; 10.2 ± 2.9* sec; R‐72: 16.7 ± 5.6 * sec; 21.7 ± 7.0 * sec; 10.1 ± 3.8 * sec. 2nd phase: vehicle = 212.2 ± 56.3 sec; 4‐ethoxyaniline: 113.8 ± 11.0* sec; 118.2 ± 45.9* sec; 107.8 ± 32.2* sec; R‐71: 126.9 ± 33.3* sec; 137.8 ± 16.2* sec; 82.3 ± 15.9* sec; R72: 129.5 ± 19.6* sec; 109.7 ± 16.2* sec; 60.4 ± 16.6* sec, respectively at doses of 0.1, 1 and 10 mg/kg. Regarding cell migration, 4‐ethoxyaniline reduced 67%, 79% and 77%; R‐71: 66%, 70% and 71%; R‐72: 67%, 69% and 75% when compared to vehicle that also received carrageenan in the pouch. Protein levels were reduced in 4‐ethoxyaniline 31%, 48% and 64%; R‐71: 3%, 15% and 78%; R‐72: 9%, 28% and 70%. NO measurement of 4‐ethoxyaniline reduced 48%, 39% and 3%; R‐71: 56%, 23% and 18%; R‐72: 56%, 44% and 1%. Levels of IL‐1β were also reduced, 4‐ethoxyaniline: 49%, 41% and 55%; R‐71: 38%, 34% and 63%; R‐72: 59%, 16% and 71% respectively at doses of 0.1, 1 and 10 mg/kg. We can conclude that the analogues presented anti‐inflammatory effects since reduced cell migration, NO production and IL‐1β secretion and could be considered to further assays.Support or Funding InformationFinancial Support: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil/Finance Code 001, CNPq and FAPERJ. Leukocyte migration, protein extravasation, Nitric oxide quantification and IL‐1β measurement of treatment with 4‐ethoxyaniline, R‐71 and R‐72. Leukocyte Migration Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 93.4 ± 33.9 93.4 ± 33.9 93.4 ± 33.9 Dexamethasone 31.0* ± 5,5 31.0* ± 5,5 31.0* ± 5,5 4‐Ethoxyaniline 31.6* ± 5.0 20.4* ± 4.7 22.2* ± 6.9 R‐71 32.1* ±4.8 28.9* ± 16.3 27.6* ± 15.3 R‐72 31.7* ± 6.5 29.9* ± 16.6 24.8* ± 8.1 Protein Extravasation Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 703.35 ± 163.9 703.35±163.9 703.35±163.9 Dexamethasone 453.59*±214.5 453.59* ± 214.5 453.59* ± 214.5 4‐Ethoxyaniline 460.8* ± 84.6 345.2*± 181.1 240.8* ± 45.9 R‐71 646.0 ± 147.9 564.7 ± 94.96 214.2* ± 24.6 R‐72 609.5 ± 158.1 483.4 ± 137.3 198.3* ± 28.9 NO Quantification Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 248.63 ± 64.3 248.63 ± 64.3 248.63 ± 64.3 Dexamethasone 112.3* ± 34.9 112.3* ± 34.9 112.3* ± 34.9 4‐Ethoxyaniline 128.3* ± 59.5 149.7 ± 22.1 241.0 ± 86.0 R‐71 109.4* ± 33.48 190.3 ± 74.75 202.3 ± 101.3 R‐72 108.5* ± 36.4 138.2 ± 42.2 244.4 ± 34.9 Levels IL‐1β Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 4016.2 ± 130.6 4016.2 ± 130.6 4016.2 ± 130.6 Dexamethasone 1109.8* ± 307.0 1109.8* ± 307.0 1109.8* ± 307.0 4‐Ethoxyaniline 2038.2 ± 1480.7 2354.9 ± 779.4 1786.7 ± 437.6 R‐71 2472.8 ± 1308.1 2622.4 ± 753.4 1470.5* ± 671.6 R‐72 1637.7 ± 805 3356.6 ± 246.9 1158.8* ± 419.4 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ibuprofen New Analogs with Anti‐Inflammatory Activity In Vivo

Anti‐inflammatory drugs are among the most prescribed in the world. Ibuprofen is well known as a non‐steroidal anti‐inflammatory drug (NSAID) that has proven anti‐inflammatory effects, but like all NSAID, has adverse effects. Our aim is to evaluate effects of new Ibuprofen analogs (R‐60 and R‐65) on acute inflammation models, which are formalin‐induced paw licking and cell migration to the subcutaneous air pouch with exudate evaluation for mediator's production. Experimental groups (n=6–8, 25–35g) male Swiss webster mice were treated 1 h prior inductions at doses of 0.01, 0.1, 1 or 10 mg/kg. Standard drugs used were Ibuprofen (10 mg/kg, p.o.), acetylsalicylic acid (ASA,200 mg/kg, p.o.) or morphine (2.5 mg/kg, i.p.). The analogues reduced paw licking time in the 1st phase: groups treated with R‐60 0.01 mg/kg licked 41.5 ± 21.6 seconds (sec), 0.1 mg/kg: 33 ± 6.4 sec, 1 mg/kg: 32.1 ± 6.9* sec, 10 mg/kg: 29.7 ± 8.4* sec. In 2nd phase reduced licking: 0.01 mg/kg: 184,4 ± 80,1 sec, 0.1 mg/kg: 198,1 ± 54,2 sec, 1 mg/kg: 207,2 ± 23 sec and 89.4 ± 16.4 *sec when treated with 10 mg/kg. R‐65 reduced licking: 34.9 ± 15* sec at the dose of 0.01 mg/kg in the 1st phase, 0.1 mg/kg: 28 ± 5.7* sec, 1 mg/kg: 25.4 ± 7.5* sec and 10 mg/kg: 17 ± 5.8 * sec. In 2nd phase, 10 mg/kg reduced: 56.1 ± 25.1* sec. Ibuprofen reduced licking time in the 1st phase: 29.3 ± 11.6* sec and second: 97.8 ± 48.4* sec. when compared to vehicle‐treated group: 1st phase 53.8 ± 11.9 sec and 2nd phase 202.9 ± 56.6 sec. Regarding cell migration, treatment reduced leukocyte migration at all doses comparing to the vehicle‐treated group: 73.1±11 × 103 cell/mL. The R‐60 0.01 mg/kg treatment led to a migration of 45.7±13 × 103 cell/mL*; 0.1 mg/kg: 46.8 ± 14.7 × 103 cell/mL*, 1 mg/kg: 42.4 ± 12.3 × 103 cell/mL*, 10 mg/kg: 46.6 ± 13.9 × 103 cell/mL*. When treated with R‐65 0.01 mg/kg 47 ± 15.1 ×103 cell/mL* migrated, 0.01 mg/kg: 42 ± 13.1 × 103 cells/mL*, 1 mg/kg: 45.2 ± 14.8 × 103 cell/mL* and 10 mg/kg: 40.9 ± 6.5 × 103 cell/mL*. Ibuprofen: 40.3 ± 9 ×103 cell/mL*. Nitric oxide production was reduced with 0.01 mg/kg treatment of R‐60: 125.5 ± 90.5 μM, 0.1 mg/kg: 103.6 ± 97.9 μM, 1 mg/kg: 91.8 ± 51.6 μM* and 10 mg/kg: 108.9 ± 34.1 μM. R‐65: 0.01 mg/kg: 163.6 ± 84 μM, 0.1 mg/kg: 129.2 ± 85.4 μM, 1 mg/kg: 70.1 ± 52.5 μM* and 10 mg/kg: 89.2 ± 29.7 μM*. Ibuprofen did not reduce nitrite levels: 141.7 ± 14.6 μM when compared to vehicle group: 193.8 ± 110.4 μM. The analogs reduced IL‐1β production at the highest tested dose (10 mg/kg) when comparing to the vehicle‐treated group (605.4 ± 323.1 ng/mL). R‐60: 255.9 ± 103.1 ng/mL* and R‐65: 237 ± 142.7 ng/mL*. Ibuprofen did not reduce IL‐1β levels at any tested dose. R‐60 and R‐65 analogues demonstrated activity in the peripheral nociception in the formalin‐induced paw licking model, as well as in the reduction of cell migration, nitric oxide and IL‐1β production. This work points its anti‐inflammatory effects, which supports the continuity of the tests and that the compounds may become potential candidates to treat inflammation and pain.Support or Funding InformationThis study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES)/Finance Code 001, CNPq and FAPERJ.Alan Minho (technical support)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

BackgroundArhalofenate acid, the active acid form of arhalofenate, is a non-agonist peroxisome proliferator-activated receptor γ (PPARγ) ligand, with uricosuric activity via URAT1 inhibition. Phase II studies revealed decreased acute arthritis flares in arhalofenate-treated gout compared with allopurinol alone. Hence, we investigated the anti-inflammatory effects and mechanisms of arhalofenate and its active acid form for responses to monosodium urate (MSU) crystals.MethodsWe assessed in-vivo responses to MSU crystals in murine subcutaneous air pouches and in-vitro responses in murine bone marrow-derived macrophages (BMDMs) by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE/Western blot, immunostaining, and transmission electron microscopy analyses.ResultsOral administration of arhalofenate (250 mg/kg) blunted total leukocyte ingress, neutrophil influx, and air pouch fluid interleukin (IL)-1β, IL-6, and CXCL1 in response to MSU crystal injection (p < 0.05 for each). Arhalofenate acid (100 μM) attenuated MSU crystal-induced IL-1β production in BMDMs via inhibition of NLRP3 inflammasome activation. In addition, arhalofenate acid dose-dependently increased activation (as assessed by phosphorylation) of AMP-activated protein kinase (AMPK). Studying AMPKα1 knockout mice, we elucidated that AMPK mediated the anti-inflammatory effects of arhalofenate acid. Moreover, arhalofenate acid attenuated the capacity of MSU crystals to suppress AMPK activity, regulated expression of multiple downstream AMPK targets that modulate mitochondrial function and oxidative stress, preserved intact mitochondrial cristae and volume density, and promoted anti-inflammatory autophagy flux in BMDMs.ConclusionsArhalofenate acid is anti-inflammatory and acts via AMPK activation and its downstream signaling in macrophages. These effects likely contribute to a reduction of gout flares.

Synthesis and Biological Activity of Tetrameric Ribitol Phosphate Fragments of Staphylococcus aureus Wall Teichoic Acid.

A systematically designed and synthesized ribitol phosphate (RboP) oligomer using a series of building blocks, which make up the wall teichoic acid (WTA) of S. aureus, is presented. Based on the use of a solution-phase phosphodiester synthesis, a library of ribitol phosphate tetramers, decorated with d-alanine and N-acetylglucosamine (GlcNAc), were generated. The synthesized RboP tetramers showed increased cytokine levels in mice in a subcutaneous air pouch model.