Neuroinflammation and hypersensitivity evidenced by the acute and 28-day repeated dose toxicity tests of ostrich oil in mice.

The ostrich oil (OO) has been topically used for decades to treat skin diseases. Its oral use has been encouraged through e-commerce advertising several health benefits to OO without scientific evidence on its safety or effectiveness. This study presents the chromatographic profile of a commercially available OO and its acute and 28-day repeated dose in vivo toxicological profiles. OO anti-inflammatory and antinociceptive effects were also investigated. Omega-9 (ω-9; oleic acid; 34.6%) and -6 (linoleic acid; 14.9%) were detected as OO main constituents. A high single dose of the OO (2 g/kg of ω-9) demonstrated no or low acute toxicity. However, when orally treated with OO (30-300 mg/kg of ω-9) for 28 consecutive days, mice exhibited altered locomotor and exploratory activities, hepatic damage, and increased hindpaw sensitivity accompanied by increased levels of cytokine and brain-derived neurotrophic factor in their spinal cords and brains. Lack of anti-inflammatory or antinociceptive activities was also evidenced in 15-day-OO treated mice. These results indicate that chronic consumption of OO induces hepatic injury, in addition to neuroinflammation and subsequent hypersensitivity and behavioural changes. Thus, there is no evidence to support OO use to treating illness in humans.

Involvement of a neutrophil-mast cell axis in the effects of Piper malacophyllum (C. PESL) C. DC extract and its isolated compounds in a mouse model of dysmenorrhoea.

The effects of Piper malacophyllum (C. Pesl) C. DC extracts and its isolated compounds were analysed in a mouse model of primary dysmenorrhoea (PD). Female Swiss mice (6-8 weeks old) on proestrus were intraperitoneally treated with estradiol benzoate for 3 days, to induce PD. Twenty-four hours later, animals were treated 24 h later with vehicle, plant extract, gibbilimbol B, 4,6-dimethoxy-5-E-phenylbutenolide, mixture of 4,6-dimethoxy-5-E-phenylbutenolide and 4,6-dimethoxy-5-Z-phenylbutenolide, or ibuprofen. One hour later, oxytocin was injected and the numbers of abdominal writhing were counted. Then, mice were euthanized and uteri were collected for morphometrical and histological analyses. The effects of P. malacophyllum in inflammation were investigated in mouse peritoneal neutrophils culture stimulated with LPS or fMLP (chemotaxis and mediator release). Finally, uterus contractile and relaxing responses were assessed. Similar to ibuprofen, P. malacophyllum extract and isolated compounds reduced abdominal writhing in mice with PD. Histology indicated a marked neutrophil and mast cell infiltrate in the uterus of PD animals which was attenuated by the extract. The compounds and the extract reduced neutrophil chemotaxis and inflammatory mediator release by these cells. Reduced TNF levels were also observed in uteri of PD mice treated with P. malacophyllum. The extract did not affect spontaneous uterine contractions nor those induced by carbachol or KCl. However, it caused relaxation of oxytocin-induced uterine contraction, an effect blunted by H1 receptor antagonist. Overall the results indicate that P. malacophyllum may represent interesting natural tools for reliving PD symptoms, reducing the triad of pain, inflammation and spasmodic uterus behaviour.

Sambucus nigra: A traditional medicine effective in reducing inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sambucus nigra L. is a plant of European origin and popularly known as elder, elderberry, black elder, European elder, European elderberry, and European black elderberry, being described in pharmacopoeia of several countries. Its flowers and berries have been used in folk medicine to treat feverish conditions, coughing, nasal congestion, and influenza besides its popular use as anti-inflammatory, analgesic, and diuretic agent. AIM OF THE STUDY: The aim of this investigation was to elucidate the anti-inflammatory and the relaxant effect of the lyophilized aqueous extract obtained from S. nigra's flowers on in vivo and in vitro inflammation assays and on the isolated rat vascular and airway smooth muscle tissue. MATERIAL AND METHODS: The anti-inflammatory activity of the extract was investigated using carrageenan-induced inflammation model in the subcutaneous tissue of male Swiss mice orally treated with S. nigra extract (30, 100, 300 or 600 mg/kg). Leukocyte influx and the secretion of chemical mediators were quantified in the inflamed exudate. Additionally, histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1ß, IL-10 and NO production, and adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry) were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with LPS and treated with the extract (1, 10 or 100 µg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. Finally, the effect of the extract was evaluated in isolated rat aorta and trachea rings. RESULTS: The oral treatment with S. nigra promoted reduction in the neutrophil migration as well as the decrease of TNF, IL-1ß and IL-6 levels in the inflamed exudate. In vitro treatment with S. nigra decreased NO2-, TNF, IL-1ß and IL-6 and promoted increase of IL-10 in LPS-stimulated neutrophils. Similarly, the extract reduced the NO2-, TNF and IL-6 in LPS-stimulated macrophages. Rutin, the major constituent of S. nigra extract reduced NO2-, TNF, IL-1ß, and IL-6 and promoted the increase of IL-10 in LPS-stimulated neutrophils supernatant. The extract also shed CD62L and CD18 expressions. The extract was able to increase the efferocytosis of apoptotic neutrophils by increasing the IL-10 and decreasing the TNF levels. Additionally, the extract reduced the hypersensitivity induced by carrageenan and promoted a relaxant effect in isolated vascular and non-vascular rat tissue. CONCLUSIONS: S. nigra flowers extract presents anti-inflammatory effect by modulating macrophage and neutrophil functions including the production of inflammatory mediators and cell migration, by promoting efferocytosis and consequently the resolution of acute inflammation, besides exerting antinociceptive effects, scientifically proving its popular use as medicinal plant. Allied to the relaxant effect in both vascular and non-vascular smooth muscle tissue, S. nigra extract represents an important tool for the management of acute inflammation.

Anti-Inflammatory and Healing Activity of the Hydroalcoholic Fruit Extract of Solanum diploconos (Mart.) Bohs.

BACKGROUND: Solanum diploconos (Mart.) Bohs is a native Brazilian plant belonging to the Solanaceae family, popularly known as "tomatinho do mato" and poorly investigated. Herein, we presented for the first time evidence for the anti-inflammatory and wound healing activities of S. diploconos fruit hydroalcoholic extract. Material and Methods. In vitro fMLP-induced chemotaxis, LPS-induced inflammatory mediator levels (cytokines by ELISA and NO release by Griess reaction), and adhesion molecule expression (CD62L, CD49d, and CD18, by flow-cytometry) were assessed in neutrophils treated with different concentrations of the extract. Inflammation resolution was measured by the efferocytosis assay and the healing activity by in vivo and in vitro assays. The air pouch model of carrageenan-induced inflammation in Swiss mice was used to investigate the in vivo anti-inflammatory effects of the extract. Leukocyte influx (by optical microscopy) and cytokine release were quantified in the pouch exudates. Additionally, the acute and subacute toxic and genotoxic effects of the extract were evaluated. RESULTS: In vitro, the extract impaired neutrophil chemotaxis and its ability to produce and/or release cytokines (TNFα, IL-1ß, and IL-6) and NO upon LPS stimuli (p < 0.01). LPS-treated neutrophils incubated with the extract presented increased CD62L expression (p < 0.01), indicating a reduced activation. An enhanced efferocytosis of apoptotic neutrophils by macrophages was observed and accompanied by higher IL-10 and decreased TNFα secretion (p < 0.01). In vivo, similar results were noted, including reduction of neutrophil migration, protein exudation, and cytokine release (p < 0.01). Also, the extract increased fibroblast proliferation and promoted skin wound healing (p < 0.01). No signs of toxicity or genotoxicity were observed for the extract. CONCLUSION: S. diploconos fruit extract is anti-inflammatory by modulating neutrophil migration/activation as well macrophage-dependent efferocytosis and inflammatory mediator release. It also indicates its potential use as a healing agent. Finally, the absence of acute toxic and genotoxic effects reinforces its possible use as medicinal product.

Toxicological and anti-inflammatory profile of Synadenium grantii Hook. f. in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Synadenium grantii Hook. f., popularly known as "janaúba" or "leiterinha", is used in the folk medicine to treat gastric disorders, some types of neoplasias and inflammatory diseases. AIM OF THE STUDY: The aim of this study was to show the anti-inflammatory activity of the methanol extract obtained from S. grantii stems and also certify the safety of the extract performing toxicological analysis. MATERIAL AND METHODS: The anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally pre-treated with the S. grantii extract (1, 3 or 10 mg/kg). The leukocyte influx (optical microscopy) and secretion of chemical mediators (TNF, IL-6 and IL-1ß, by enzyme-linked immunosorbent assay) were quantified in the inflamed exudate. The toxicity was investigated using the dose-fixed procedure (acute toxicity) and repeated dose 28-day (subacute toxicity) in mice orally treated with S. grantii extract. The open field and rota-rod test were used to evaluate possible interference of adverse effect of S. grantii on motor coordination, locomotor and exploratory activity. RESULTS: The analysis of the inflammatory exudate of S. grantii-treated mice demonstrated reduction in the polymorphonuclear cells (PMN) migration to the inflamed tissue, as well as the reduction of the pro-inflammatory cytokines TNF and IL-1ß. Furthermore, the acute and sub-acute toxicity studies did not show significant changes in body weight, general behaviour, biochemical parameters, organ weight and liver and kidney histopathological analysis. However, animals acutely treated with S. grantii presented reduction in the number of crosses in relation to the vehicle group, without significant difference in the number of elevations and latency time between the groups in rota-rod test. The obtained results allow to set the NOAEL (Non-observed-adverse-effect level) in 100 mg/kg for this specie of rodent. CONCLUSIONS: Together, the results herein obtained show that S. grantii extract presented anti-inflammatory activity by decreasing the influx of PMN to the inflamed tissue, as well as the cytokines TNF and IL-1ß levels. In addition, S. grantii extract seemed not to present significant acute or subacute toxicity when administered to mice, demonstrating for the first time the safety of this extract, when orally administered.

Effects of Eugenia umbelliflora O. Berg (Myrtaceae)-leaf extract on inflammation and hypersensitivity.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Eugenia species are widely used in popular medicine to treat several diseases, such as arthritis, rheumatism and diabetes. Eugenia umbelliflora O. Berg is popularly known in Brazil as "baguaçu", name also conferred to Eugenia jambolana probably due to their apparent similarity. Although the popular use scientifically proved of E. jambolana as anti-diabetes and also as anti-inflammatory, there are only two scientific studies demonstrating anti-ulcer and bactericide activities of E. umbelliflora leaves extract, without reference to its possible anti-inflammatory activity. AIM OF THE STUDY: The aim of this study was to show the anti-oxidant and anti-inflammatory activity of the methanol extract obtained from E. umbelliflora leaves (EuL) using in vitro and in vivo protocols. MATERIALS AND METHODS: The total phenolic content was evaluated using the folin-Ciocalteu colorimetric method and phloroglucinols content by HPLC. The anti-oxidant activity was evaluated by ORAC, ABTS•+, DPPH, and metal chelation methods. The anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally pre-treated with the EuL (0.3, 1 or 3 mg/kg). The leukocyte influx (optical microscopy) and secretion of chemical mediators (TNF, IL-6, IL-1ß and CXCL1, by enzyme-linked immunosorbent assay) were quantified in the inflamed exudate. Histological analysis of the pouches was also performed. The anti-hypersensitive activity was investigated using carrageenan-induced mechanical hypersensitivity and mice were then evaluated using the von Frey filaments. The Open Field test was used to evaluate possible interference of adverse effect of EuL on locomotor activity that could lead to misinterpretation of the hypersensitivity evaluation. RESULTS: The EuL demonstrated important and moderate reducing capacity on ABTS•+ and DPPH assays, respectively, but with slight activity in ORAC test. It reflects low protection against cell damage. The EuL also presented 30% of phenolic compounds. The phloroglucinols content of EuL was 25.9 mg/g, 18.4 mg/g and 16.6 mg/g of eugenial C, eugenial D and eugenial E, respectively. The in vivo analysis of the inflammatory exudate of EuL-treated mice demonstrated reduction in the polymorphonuclear cells (PMN) migration to the inflamed tissue, as well as the reduction of the pro-inflammatory cytokine IL-1ß. Histologically, it was observed evident decrease in the oedema, formed essentially by non-haemorrhagic fibrin exudate, as well as PMN infiltrate, when compared with control mice injected with carrageenan. Furthermore, the extract also presented effective reduction of the mechanical hypersensitivity induced by carrageenan without any interference in animal's locomotor and exploratory activity. CONCLUSIONS: Together, the results herein obtained show that EuL presented anti-inflammatory activity by decreasing the influx of PMN to the inflamed tissue, as well as the cytokine IL-1ß level. This anti-inflammatory activity was also accompanied by significant anti-hypersensitive effect. The effects presented by EuL seem not to be correlated with an antioxidant activity. However other extract chemical compounds could be responsible for its important anti-inflammatory effects.

Aleurites moluccanus and its main active constituent, the flavonoid 2″-O-rhamnosylswertisin, in experimental model of rheumatoid arthritis.

ETHNOPHARMMACOLOGICAL RELEVANCE: Aleurites moluccana is used in folk medicine to treat pain, fever, asthma, hepatitis, gastric ulcer and inflammatory process in general, and the nut oil had been topically applied to treat arthritis and other joint pain, however the seeds are classified as toxic for oral use. AIM: Faced with the need for new alternative to treat the symptoms and modify rheumatoid arthritis (RA) the aim of this work was to evaluate the effects of A. moluccanus' leaves dried extract in rats and mice submitted to complete Freund adjuvant (CFA)-induced RA. MATERIAL AND METHODS: Wistar Rats and Swiss mice were submitted to CFA-induced RA in the right hindpaw. They received A. moluccanus extract (orally; p.o.), dexamethasone (subcutaneously), 2″-O-rhamnosylswertisin (p.o.) or vehicle (p.o.), from the 14th day after the CFA injection for up to 8 days. The mechanical hypersensitivity was evaluated using the von Frey filaments and the paw-oedema was measured using a plethysmometer. The rats' injected hindpaw was used to perform the histological analysis. RESULTS: A. moluccanus was able to significantly reduce the mechanical hypersensitivity in both ipsi- and contralateral hindpaws of mice injected with CFA, in a dose dependent manner. Furthermore, the paw-oedema was progressively reduced by A. moluccanus. Similar results were obtained for the positive-control drug dexamethasone and the isolated compound 2″-O-rhamnosylswertisin. Besides the effects mentioned above, the extract was also effective to repair the joint damage in CFA-induced RA rats, including reduction of fibrosis, cartilage degradation and bone erosion scores. CONCLUSION: These results together with the literature data reinforce the anti-hypersensitivity and anti-inflammatory activity of A. moluccanus extract. Part of the observed effects is due to the presence of the compound 2″-O-rhamnosylswertisin. The fact that the extract acted as a disease modifier point this herbal product as a promisor and safe tool to treat RA and other associated chronic diseases.

Effect of Synadenium grantii and its isolated compound on dysmenorrhea behavior model in mice.

OBJECTIVE: This study aimed to evaluate the effects of the methanol extract of Synadenium grantii Hook f. stems and its main isolated compound Query3,4,12,13-tetraacetylforbol-20-phenylacetate (1) on experimental dysmenorrhea in mice. METHODS: Female Swiss mice (n = 6-15) of 6-8 weeks old were used, selected according to the period of the estrous cycle. Animals in proestrus were treated intraperitoneally (i.p.) for 3 days with estradiol. They orally received, on the 4th day, S. grantii extract, the compound 1, ibuprofen or hyoscine butylbromide (Buscopan compound®). Then they were injected (i.p.) with oxytocin 1 h later and individually observed regarding the abdominal writhing for 45 min. The uterus was weighed, photographed and fixed in paraffin for histological analysis. KEY FINDINGS: The extract inhibited the abdominal writhing and similar results were obtained with compound 1 and the positive control drugs Ibuprofen and hyoscine butylbromide. Reduction of uterus volume and histological inflammatory parameters, such as oedema and leukocyte infiltrate, were observed in animals treated with the extract and compound 1. CONCLUSIONS: Our data show promising activity of the extract against dysmenorrhea, indicating important anti-inflammatory activity. Compound 1 appears to be, at least in part, the main responsible for this promising biological effect.

Direct immunofluorescent labeling of cells.

In the direct immunofluorescent labeling technique, fluorochrome-labeled antibodies are used as probes for particular antigens or biomolecules. Cells, usually after appropriate fixation, are incubated with the antibodies to which fluorochromes have been directly conjugated. Following incubation, excess antibody is washed off with PBS and the cells are mounted on coverslips with antifade mounting medium. Immunofluorescent labeled cells are analyzed using a conventional fluorescence microscope or by confocal microscopy. Direct labeling has two major advantages: it requires only a single incubation with the labeled reagent, decreasing the number of steps in the staining procedure; and more importantly, provides minimal nonspecific staining and less background. Additionally, the direct labeling technique allows the use of two or more primary antibodies of the same species or isotype, avoiding the problems with secondary antibody staining. This method has multiple applications: to label simultaneously two or more antigens within the same cell or tissue sections; to characterize the subcellular distribution of biomolecules of interest, by concurrently labeling with antibodies to both the antigen of interest and to a known organelle; to investigate whether several antigens of interest are colocalized; and to phenotype cells, for which no specific markers are available, using an appropriate panel of antibodies.

Alterations in granule matrix and cell surface of focal adhesion kinase-deficient mast cells.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcepsilonRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK(-/-) embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing beta(7) integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcepsilonRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.