Carrageenan-induced Mouse Paw Research Articles

Antimicrobial and Anti-inflammatory Effects of a Novel Peptide From the Skin of Frog Microhyla pulchra.

Brevinins are an important antimicrobial peptide (AMP) family identified in the skin of Ranidae frogs and generally contain a characteristic ranabox structure at their C-terminal sequence. Herein a novel AMP named brevinin-2MP has been identified from the skin of the frog Microhyla pulchra by molecular cloning. Brevinin-2MP (GVITDTLKGVAKTVAAELLRKAHCKLTNSC) with a high amphipathic α-helix in sodium dodecyl sulfate solutions can destroy bacterial cell membrane and kill microbes. Furthermore, brevinin-2MP has been found to inhibit the lipopolysaccharide (LPS)-induced expression of pro-inflammatory NO, MCP-1, IL-6, and TNF-α via binding unidentified targets on the cell membrane and consequently suppressing the activation of MAPK/NF-κB signaling cascades induced by LPS in RAW 264.7 cells. Consistently, brevinin-2MP significantly alleviates the acute inflammatory response in carrageenan-induced mice paw. In conclusion, brevinin-2MP with anti-inflammatory and antimicrobial properties will be an ideal candidate drug molecule for bacterial inflammation treatment.

Functional Characterization of a Novel Lipopolysaccharide-Binding Antimicrobial and Anti-Inflammatory Peptide in Vitro and in Vivo.

Antimicrobial peptides (AMPs) are key components of host immune defense of vertebrates against microbial invasions. Here, we report a new AMP (esculentin-1GN) characterized from the skin of the frog Hylarana guentheri. Esculentin-1GN (GLFSKKGGKGGKSWIKGVFKGIKGIGKEVGGDVIRTGIEIAACKIKGEC) with high amphipathic α-helical structure in membrane-mimetic environments has the microbial-killing activity by destruction of the cell membrane. Moreover, esculentin-1GN inhibits LPS-induced expression of proinflammatory nitric oxide, interleukin-1β, interleukin-6, and tumor necrosis factor while it enhances expression of interleukin-10. Furthermore, esculentin-1GN can bind to d-(+)-galacturonic acid and LPS. Meanwhile, esculentin-1GN suppresses the activation of inflammatory response pathway induced by LPS. In addition, esculentin-1GN significantly reduces acute inflammation in carrageenan-induced mice paw. Taken together, the novel LPS-binding esculentin-1GN with antimicrobial and anti-inflammatory activities will be an excellent temple for designing new antibiotic formulations.

PHYTOCHEMICAL ANALYSIS AND IMMUNO-MODULATORY EFFECT OF MORINGA OLEIFERA FLOWERS

Objective: This study evaluates the anti-inflammatory activities of different solvent extracts of Moringa oleifera flowers using carrageenan-induced mice paw edema.Methods: Soxhlet extraction method was employed in this study to extract the crude phytochemicals. Phytochemical analysis testing of Moringa oleifera extracts was performed to identify the presence of various phytoconstituents based on the standard procedures. The anti-inflammatory activity was evaluated using acute inflammatory model carrageenan-induced paw edema. Three different flower extracts (petroleum ether, chloroform and methanol) of Moringa oleifera at the dose level of 500 mg/kg body weight. The anti-inflammatory activity on the different extracts of Moringa oleifera was determined through the α-carrageenan induced left hind paw oedema method in albino mice. 0.05 ml of 1% w/v carrageenan suspension was reconstituted with normal saline (0.9% NaCl) to give a homogenous solution which then be injected into the subplantar tissue of the left hind paw of each mice to induce acute inflammation.Results: Treatment with three different solvent extracts showed significant (p<0.05) inhibition in carrageenan-induced paw edema. Petroleum ether and chloroform extracts were found to be less effective than methanolic extracts when compared to (Indomethacin) reference standard at the dose of 10 mg/kg body weight. The phytochemical results obtained indicates that anti-edematous action of Moringa oleifera flowers exhibited in this study is due to the presence of potent anti-inflammatory phytoconstituents (flavonoid, alkaloid, tannin) in impeding arachidonic acid metabolism and production of reactive free radicals. A significant (p<0.05) increase of left hind paw thickness after the drug injection was noticed in the negative control mice group as time persisted. It showed the highest paw thickness at the fifth hour with 4.72 mm±0.07. Whereas the indomethacin treated group showed the highest percent oedema inhibition amongst all experimental group with 38.60% at the fifth-hour post-carrageenan induction. It exhibited a significant inhibition of 29.02% against the oedema after the third hour of carrageenan injection.Conclusion: The methanolic extract of Moringa oleifera flowers extract has anti-inflammatory activity. This activity was related to the dose and these results collaborate the potential traditional use of the plant in folk medicine.

Antioxidant, Anti‐Inflammatory, and Analgesic Activities of Agrimonia eupatoria L. Infusion

Agrimony (Agrimonia eupatoria L.) (Ae) is used in traditional medicine to treat inflammatory and oxidative related diseases. Therefore, this study focuses on the anti-inflammatory and analgesic potential of Ae infusion (AeI). Phenolic compounds characterization was achieved by HPLC-PDA-ESI/MSn. To evaluate antioxidant potential, 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical, and SNAP assays were used. In vitro anti-inflammatory activity of AeI was investigated in LPS-stimulated macrophages by measuring the NO production. In vivo anti-inflammatory activity was validated using the mouse carrageenan-induced paw edema model. Peripheral and central analgesic potential was evaluated using the acetic acid-induced writhing and hot-plate tests, respectively, as well as the formalin assay to assess both activities. The safety profile was disclosed in vitro and in vivo, using MTT and hematoxylin assays, respectively. Vitexin, quercetin O-galloyl-hexoside, and kaempferol O-acetyl-hexosyl-rhamnoside were referred to in this species for the first time. AeI and mainly AePF (Ae polyphenolic fraction) showed a significant antiradical activity against all tested radicals. Both AeI and AePF decreased NO levels in vitro, AePF being more active than AeI. In vivo anti-inflammatory and analgesic activities were verified for both samples at concentrations devoid of toxicity. Agrimony infusion and, mainly, AePF are potential sources of antiradical and anti-inflammatory polyphenols.

Effect of PEP-1-GLRX-1 on catabolic gene expression in human articular chondrocytes and in mouse carrageenan-induced PAW edema model

Effect of PEP-1-GLRX-1 on catabolic gene expression in human articular chondrocytes and in mouse carrageenan-induced PAW edema model

PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes

Background: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model. Methods: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1β- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model. Results: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1β-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1β- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema. Conclusion: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.

PEP-1-FK506BP12 inhibits matrix metalloproteinase expression in human articular chondrocytes and in a mouse carrageenan-induced arthritis model.

The 12 kDa FK506-binding protein (FK506BP12), an immunosuppressor, modulates T cell activation via calcineurin inhibition. In this study, we investigated the ability of PEP-1-FK506BP12, consisting of FK506BP12 fused to the protein transduction domain PEP-1 peptide, to suppress catabolic responses in primary human chondrocytes and in a mouse carrageenan-induced paw arthritis model. Western blotting and immunofluorescence analysis showed that PEP-1-FK506BP12 efficiently penetrated chondrocytes and cartilage explants. In interleukin-1β (IL-1β)-treated chondrocytes, PEP-1-FK506BP12 significantly suppressed the expression of catabolic enzymes, including matrix metalloproteinases (MMPs)-1, -3, and -13 in addition to cyclooxygenase-2, at both the mRNA and protein levels, whereas FK506BP12 alone did not. In addition, PEP-1-FK506BP12 decreased IL-1β-induced phosphorylation of the mitogen-activated protein kinase (MAPK) complex (p38, JNK, and ERK) and the inhibitor kappa B alpha. In the mouse model of carrageenan-induced paw arthritis, PEP-1-FK506BP12 suppressed both carrageenan-induced MMP-13 production and paw inflammation. PEP-1-FK506BP12 may have therapeutic potential in the alleviation of OA progression. [BMB Reports 2015; 48(7): 407-412]

Anti-inflammatory effects of characterized orange peel extracts enriched with bioactive polymethoxyflavones

In view of the potential of polymethoxyflavones (PMFs) and hydroxylated PMFs (OH-PMFs) as bioactives against inflammation, we prepared six different orange peel extracts (OPEs). The major compounds of these extracts were characterized and quantified by high performance liquid chromatography (HPLC). Effects on inflammation were analyzed by nutrigenomics using a human cell-based TPA-induced monocyte–macrophage differentiation model employing U-937 cells and inflammatory surrogate genes. Dose response and kinetics analysis of OPEs with different chemical profiles revealed less cytotoxic effects of PMFs as compared to OH-PMFs as demonstrated by the MTT-method. Noteworthy, a comparison of two PMF members such as 3,5,6,7,3′,4′-hexamethoxyflavone (HexaMF) and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HeptaMF) exhibited less cytotoxic effects of HeptaMF as compared to HexaMF. A specific OPE enriched with HeptaMF, PMFs and OH-PMFs at low concentrations (10μg/mL) significantly down-regulated the expression of a panel of genes involved in inflammatory response, including COX-2, TNF-α, ICAM-1, NFκB, IL-1β, IL-6, and IL-8 with an inflammatory index of −0.55. The strong anti-inflammatory effects were then validated in a mouse carrageenan-induced paw edema model. Oral intake of OPE reduced paw edema significantly in a dose-dependent manner. Importantly, a dosage of 250mg/kg gave an anti-inflammatory effect comparable to ibuprofen. A preliminary clinical study showed that OPE was well tolerated showing no adverse side effects. In summary, enrichment of phyto extracts such as OPEs with specific polymethoxyflavones as anti-inflammatory bioactives is a promising strategy to find naturally derived extracts that are effective against diseases associated with inflammation.

Margaritaria discoidea (Euphorbiaceae) stem bark extract attenuates allergy and Freund's adjuvant-induced arthritis in rodents.

Background:Various parts of Margaritaria discoidea find use in traditional medicine in the treatment of pain and oedema. This study evaluated the anti-allergic, anti-inflammatory and anti-arthritic effects of a 70% (v/v) aqueous ethanol extract of the stem bark of Margaritaria discoidea, MDE in rodents.Materials and Methods:Systemic anaphylaxis was induced by the injection of compound 48/80 into mice and their survival rate was monitored to evaluate the anti-allergic action of the extract. The effect of MDE assessed on the maximal and total oedema responses in the mouse carrageenan-induced paw oedema was used to evaluate the anti-inflammatory action of the extract while the Freund's adjuvant-induced arthritis model was employed to study the anti-arthritic effects of MDE.Results:MDE dose-dependently increased the time for compound 48/80-induced mortality in mice. MDE suppressed the mean maximal swelling and the total paw swellings induced over 6 h in the carrageenan-induced paw oedema when administered either prophylactically or therapeutically. MDE caused a reduction in serum levels of TNFα and IL-6 and significantly suppressed Freund's adjuvant-induced arthritis.Conclusion:Margaritaria discoidea suppresses allergy and exhibits anti-inflammatory activity in mice. In addition it attenuates Freund's adjuvant-induced arthritis through a reduction in serum levels of TNFα and IL-6 in rats.

Aqueous ethanol extract of the fruit of Xylopia aethiopica (Annonaceae) exhibits anti-anaphylactic and anti-inflammatory actions in mice

Ethnopharmacological relevanceXylopia aethiopica has been traditionally used in the form of the dried fruit decoction to treat bronchitis, asthma, arthritis and rheumatism in Ghana, Nigeria and Cameroon. Aim of the study is to evaluate the anti-anaphylactic and anti-inflammatory effects of a 70% aqueous ethanol extract of the fruits of Xylopia aethiopica. Materials and methodsSystemic anaphylaxis was induced by the injection of either compound 48/80 or lipopolysaccharide, LPS and survival rates of mice monitored for 1h or 7 days respectively while IgE-mediated anaphylaxis in a local allergic reaction was studied in the pinnal inflammation model in mice. Clonidine-induced catalepsy in mice was used to evaluate the indirect antihistamine effect of Xylopia aethiopica, XAE. The effects of XAE assessed on the maximal and total oedema responses in the carrageenan-induced paw oedema in mice was used to evaluate the anti-inflammatory action of the extract. ResultsAdministered at 30, 100, 300 and 1000mgkg−1 p.o., XAE dose dependently suppressed compound 48/80-induced mouse systemic anaphylactic shock and offered 63% protection to mice against LPS-induced endotoxic shock at a dose of 300mgkg−1. In addition, the extract (30–300mgkg−1) in a dose dependent manner significantly inhibited by 23–62% the mouse pinnal inflammation. Clonidine-induced catalepsy in mice was significantly suppressed in a dose and time dependent manner when administered both prophylactically and therapeutically. In the same doses, when administered before the induction of the mouse carrageenan-induced paw oedema, the mean maximal swelling attained during 6h was reduced to 41.02±6.94%, 35.61±4.30%, and 29.09±4.90% of the inflamed control response respectively and total paw swellings induced over the 6h were also dose-dependently and significantly suppressed to 74.84±14.84%, 63.95±9.37%, and 48.13±10.90% of the inflamed control response respectively. Administered after the induction of the carrageenan paw oedema the mean maximal swelling attained during 6h was suppressed to 49.84±3.95%, 43.62±1.01%, and 35.97±1.34% of the inflamed control response respectively while the total paw swellings induced over the 6h were also dose-dependently and significantly suppressed at 100 and 300mgkg−1 to 72.39±4.38% and 60.81±3.25% of the inflamed control response respectively. ConclusionThese findings suggest that XAE inhibits mast cell-dependent immediate allergic reactions and exhibit anti-inflammatory actions through the inhibition of histamine release from mast cells via stabilizing the cell membrane. Our results contribute towards validation of the traditional use of Xylopia aethiopica in the treatment of bronchitis, asthma, arthritis and rheumatism.

Lectin extracted from Canavalia grandiflora seeds presents potential anti-inflammatory and analgesic effects

Neutrophil migration is responsible for tissue damage observed in inflammatory diseases and is also implicated in inflammatory nociception. The use of lectins has been demonstrated to be effective in different activities including anti-inflammatory, antimicrobial, and in cancer therapy. In this study, we addressed the potential use of a lectin from Canavalia grandiflora seeds (ConGF) to control neutrophil migration and inflammatory hypernociception. Pretreatment of the animals intravenously (15 min before) with ConGF inhibited neutrophil migration to the peritoneal cavity in a dose-dependent fashion confirmed by an inhibition of rolling and adhesion of leukocytes by intravital microscopy. Another set of experiments showed that pretreatment of the animals with ConGF inhibited the mechanical hypernociception in mice induced by the i.pl. injection of carrageenan or formalin. This anti-nociceptive effect correlated with an effective blockade of neutrophil influx, as assessed by the hind paw tissue myeloperoxidase levels. Furthermore, ConGF had important inhibitory effects on the mouse carrageenan-induced paw edema. In addition, animals treated with ConGF showed inhibition of cytokines release. In conclusion, we demonstrated that the lectin ConGF inhibits neutrophil migration and mechanical inflammatory hypernociception.

Anti-inflammatory activity of phylligenin, a lignan from the fruits of Forsythia koreana, and its cellular mechanism of action

Aim of the study The fruits of Forsythia koreana have long been used in Chinese medicine to treat inflammatory disorders. However, the pharmacological data is not sufficient to clearly establish a scientific rationale for the anti-inflammatory medicinal use of this plant material, and the search for its active principles has been limited so far. Materials and methods Phylligenin (lignan) was isolated from the fruits of Forsythia koreana and its anti-inflammatory activity was examined. Results and conclusion Phylligenin (1–100 μM) and the methanol extract of Forsythia koreana fruits inhibited cyclooxygenase-2 (COX-2)-mediated prostaglandin E 2 and inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) synthesis from lipopolysaccharide-treated RAW 264.7 cells. In the mechanism study, phylligenin inhibited iNOS expression and nuclear factor-κB (NF-κB) activation but had no effect on COX-2 expression. Moreover, phylligenin significantly inhibited mouse carrageenan-induced paw edema by intraperitoneal administration (22.1–34.7% inhibition at 12.5–100 mg/kg). These pharmacological properties indicate that phylligenin possesses significant anti-inflammatory activity in vitro and in vivo, and may provide the scientific rationale for anti-inflammatory use of the fruits of Forsythia koreana.

Small Molecule Disruption of G Protein βγ Subunit Signaling Inhibits Neutrophil Chemotaxis and Inflammation

G protein betagamma subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) gamma catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gbetagamma-dependent signaling, including Gbetagamma-dependent activation of PI3-kinase gamma and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gbetagamma inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gbetagamma-regulation may be an effective anti-inflammation strategy.

Involvement of guanylate cyclase and potassium channels on the delayed phase of mouse carrageenan-induced paw oedema

Previous studies from this laboratory have shown that administration of nitric oxide (NO) donors reduces the early phase (which peaks at 4 h) of carrageenan-induced paw oedema. The aim of this study was to investigate the influence of NO donors on the delayed phase of the mouse paw oedema, which peaks 48 h after carrageenan injection. Treatment of animals with sodium nitroprusside (1.5, 5 and 10 μmol/kg, subcutaneously (s.c.)) 8 h after the subplantar carrageenan injection (300 μg/paw), reduced (∼50%) the delayed phase of paw oedema and the delayed increase in plasma leakage, as assessed by Evans Blue extravasation. Two other NO donors, S-nitroso- N-acetyl- dl-penicillamine (SNAP) or glyceril trinitrate (both at 28 μmol/kg) yielded an inhibition in paw oedema similar to that of sodium nitroprusside. NO-induced inhibition of the delayed phase of paw oedema was reversed when animals were treated with 1 H-[1,2,4]-oxadiazolo-[4,3- a]quinoxalin-1 (ODQ, a soluble guanylate cyclase inhibitor, 11 μmol/kg, s.c.) or with tetraethylammonium (TEA, a nonselective potassium channel blocker, 300 μmol/kg, s.c.), 30 min before the prophylactic dose of sodium nitroprusside. In conclusion, our results show that a brief exposure to NO donors, even when made several hours after the inflammatory reaction has been triggered, is still able to cause an important reduction on the delayed phase of carrageenan-induced mouse paw oedema and fluid leakage. Moreover, this long-lasting NO antiinflammatory effect appears to be dependent on guanylate cyclase and potassium channels.