Renata Grespan

Effects of Thymol and Carvacrol, Constituents of Thymus vulgaris L. Essential Oil, on the Inflammatory Response.

Thyme (Thymus vulgaris L., Lamiaceae) is an aromatic and medicinal plant that has been used in folk medicine, phytopharmaceutical preparations, food preservatives, and as an aromatic ingredient. The effect of Thymus vulgaris essential oil (TEO) and its isolated constituents thymol and cavacrol (CVL) were studied in the following experimental models: ear edema, carrageenan-induced pleurisy, and chemotaxis in vitro. In the pleurisy model, TEO, CVL, and thymol significantly inhibited inflammatory edema. However, only TEO and CVL inhibited leukocyte migration. In the in vitro chemotaxis experiment, CVL inhibited leukocyte migration, whereas thymol exerted a potent chemoattractant effect. In the ear edema model, CVL (10 mg/ear), applied topically, reduced edema formation, exerting a topical anti-inflammatory effect. Thymol did not reduce edema formation but rather presented an irritative response, probably dependent on histamine and prostanoid release. Our data suggest that the antiinflammatory effects of TEO and CVL are attributable to the inhibition of inflammatory edema and leukocyte migration.

Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFNγ production

IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN γ production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFNγ but was enhanced by prostaglandin E2 (PGE2). IL-23-induced IL-17 production was increased by PGE2 and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFNγ-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFNγ but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNFα, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFNγ production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs.

CXCR2-Specific Chemokines Mediate Leukotriene B4-Dependent Recruitment of Neutrophils to Inflamed Joints in Mice With Antigen-Induced Arthritis

OBJECTIVE To investigate the mechanism underlying neutrophil migration into the articular cavity in experimental arthritis and, by extension, human inflammatory synovitis. METHODS Antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Migration assays and histologic analysis were used to evaluate neutrophil recruitment to knee joints. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay. Antibodies and pharmacologic inhibitors were used in vivo to determine the role of specific disease mediators. Samples of synovial tissue and synovial fluid from rheumatoid arthritis (RA) or osteoarthritis patients were evaluated for CXCL1 and CXCL5 expression. RESULTS High levels of CXCL1, CXCL5, and leukotriene B4 (LTB4) were expressed in the joints of arthritic mice. Confirming their respective functional roles, repertaxin (a CXCR1/CXCR2 receptor antagonist), anti-CXCL1 antibody, anti-CXCL5 antibody, and MK886 (a leukotriene synthesis inhibitor) reduced mBSA-induced neutrophil migration to knee joints. Repertaxin reduced LTB4 production in joint tissue, and neutrophil recruitment induced by CXCL1 or CXCL5 was inhibited by MK886, suggesting a sequential mechanism. Levels of both CXCL1 and CXCL5 were elevated in synovial fluid and were released in vitro by RA synovial tissues. Moreover, RA synovial fluid neutrophils stimulated with CXCL1 or CXCL5 released significant amounts of LTB4. CONCLUSION Our data implicate CXCL1, CXCL5, and LTB4, acting sequentially, in neutrophil migration in AIA. Elevated levels of CXCL1 and CXCL5 in the synovial compartment of RA patients provide robust comparative data indicating that this mechanism plays a role in inflammatory joint disease. Together, these results suggest that inhibition of CXCL1, CXCL5, or LTB4 may represent a potential therapeutic strategy in RA.

A crucial role for TNF‐α in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5

Background and purpose:  Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP‐2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice.

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

In this study, we have addressed the role of H2S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H2S synthesis inhibitors, dl-propargylglycine (PAG) or β-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H2S donors, NaHS or Lawesson’s reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-α, keratinocyte-derived chemokine, and LTB4. Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (KATP+) channel blocker, glybenclamide. Conversely, diazoxide, a KATP+ channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H2S augments neutrophil adhesion and locomotion, by a mechanism dependent on KATP+ channels.

Peroxisome Proliferator-Activated Receptor-γ Ligand, 15-Deoxy-Δ12,14-Prostaglandin J2, Reduces Neutrophil Migration via a Nitric Oxide Pathway

Ligands for peroxisome proliferator-activated receptor γ (PPAR-γ), such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ2-mediated activation of PPAR-γ ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ2 administration decreases leukocyte rolling and adhesion to the inflammated mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS−/− mice were not susceptible to 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ2-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-1 expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ2, whereas 15d-PGJ2 inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ2 suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

Toxicological studies on Stryphnodendron adstringens

This study was carried out to determine the acute toxicity of total barbatimão extract (LD(50)) after oral administration to mice, and its effect on certain biochemical parameters in plasma of rats after 30 days of administration. The LD(50) value of the extract was 2699 mg/kg. A daily oral administration of extracts at 800 and 1600 mg/kg doses for 30 days caused a decrease in body weight, thymic involution, and an increase of plasma glucose and aspartate aminotransferase levels in the animals. The results showed that the extract administered in a prolonged period produced toxic effects in the experimental animals.

Antifungal activity and inhibition of fumonisin production by Rosmarinus officinalis L. essential oil in Fusarium verticillioides (Sacc.) Nirenberg

The chemical composition of Rosmarinus officinalis L. essential oil (REO) was analysed by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The main compounds of the REO were 1.8 cineole (52.2%), camphor (15.2%) and α-pinene (12.4%). The mycelial growth of Fusarium verticillioides (Sacc.) Nirenberg was reduced significantly by 150 μg/mL of REO. Significant microscopic morphological changes were visualised, such as the rupture of the cell wall and the leakage of cytoplasm at 300 μg/mL of REO. At lower concentrations of REO, the effects on the production of ergosterol and the biomass of mycelium varied, as did the effects on the production of fumonisins, but at ≥300 μg/mL of REO, these processes were significantly inhibited, showing the effectiveness of the REO as an antifungal agent. The results suggested that the REO acts against F. verticillioides by disrupting the cell wall and causing the loss of cellular components, subsequently inhibiting the production of fumonisins and ergosterol.

Effect of Zingiber officinale essential oil on Fusarium verticillioides and fumonisin production

The antifungal activity of ginger essential oil (GEO; Zingiber officinale Roscoe) was evaluated against Fusarium verticillioides (Saccardo) Nirenberg. The minimum inhibitory concentration (MIC) of GEO was determined by micro-broth dilution. The effects of GEO on fumonisin and ergosterol production were evaluated at concentrations of 500-5000 μg/mL in liquid medium with a 5mm diameter mycelial disc of F. verticillioides. Gas chromatography-mass spectrometry showed that the predominant components of GEO were α-zingiberene (23.9%) and citral (21.7%). GEO exhibited inhibitory activity, with a MIC of 2500 μg/mL, and 4000 and 5000 μg/mL reduced ergosterol biosynthesis by 57% and 100%, respectively. The inhibitory effect on fumonisin B1 (FB1) and fumonisin B2 (FB2) production was significant at GEO concentrations of 4000 and 2000 μg/mL, respectively. Thus, the inhibition of fungal biomass and fumonisin production was dependent on the concentration of GEO. These results suggest that GEO was able to control the growth of F. verticillioides and subsequent fumonisin production.

Phlebotomine salivas inhibit immune inflammation-induced neutrophil migration via an autocrine DC-derived PGE2/IL-10 sequential pathway

In the present study, we investigated whether saliva from Phlebotomus papatasi and Phlebotomus duboscqi inhibited antigen‐induced neutrophil migration and the mechanisms involved in these effects. The pretreatment of immunized mice with salivary gland extracts (SGE) of both phlebotomines inhibited OVA challenge‐induced neutrophil migration and release of the neutrophil chemotactic mediators, MIP‐1α, TNF‐α, and leukotriene B4 (LTB4). Furthermore, SGE treatment enhanced the production of anti‐inflammatory mediators, IL‐10 and PGE2. SGE treatments failed to inhibit neutrophil migration and MIP‐1α and LTB4 production in IL‐10−/− mice, also failing in mice treated with nonselective (indomethacin) or selective (rofecoxibe) cyclooxygenase (COX) inhibitors. COX inhibition resulted in diminished SGE‐induced IL‐10 production, and PGE2 release triggered by SGE remained increased in IL‐10−/− mice, suggesting that prostanoids are acting through an IL‐10‐dependent mechanism. SGE treatments in vivo reduced the OVA‐induced lymphoproliferation of spleen‐derived cells. Further, the in vitro incubation of bone marrow‐derived dendritic cells (DC) with SGE inhibited the proliferation of CD4+T cells from OVA‐immunized mice, which was reversed by indomethacin and anti‐IL‐10 antibody treatments. Supporting these results, SGE induced the production of PGE2 and IL‐10 by DC, which were blocked by COX inhibition. These effects were associated with the reduction of DC‐membrane expression of MHC‐II and CD86 by SGE treatment. Altogether, the results showed that Phlebotomine saliva inhibits immune inflammation‐induced neutrophil migration by an autocrine DC sequential production of PGE2/IL‐10, suggesting that the saliva constituents might be promising therapeutic molecules to target immune inflammatory diseases.