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1.
Toxicol Sci ; 142(2): 497-507, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25260831

ABSTRACT

Gold nanoparticle (AuNP) bioconjugates have been used as therapeutic and diagnostic tools; however, in vivo biocompatibility and cytotoxicity continue to be two fundamental issues. The effect of AuNPs (20 nm) conjugated with antibody [immunoglobulin G (IgG)], albumin, protein A, PEG4000, and citrate (cit) were evaluated in vitro using primary human cells of the vascular system. AuNP bioconjugates did not cause lysis of human erythrocytes, apoptosis or necrosis of human leukocytes, and endothelial cells in vitro, although AuNPs had been internalized and detected in the cytoplasm. Moreover, the influence of AuNPs on rheological parameters, blood and vessel wall characteristics was investigated in vivo by intravital microscopy assay using male Wistar rats mesentery microcirculation as model. Intravenous injection of AuNP-IgG or cit-AuNP did not cause hemorrhage, hemolysis or thrombus formation, instead suppressed the leukocyte adhesion to postcapillary vessel walls, an early stage of an inflammatory process. Furthermore, AuNP-IgG abrogated the expression of platelet-endothelial cell adhesion molecule-1, chemotaxis, and oxidative burst activation on neutrophils after leukotriene B4 stimulation, a membrane receptor-dependent stimulus, thus confirming their anti-inflammatory effects in vitro. The expression of oxidative burst activation was also suppressed after stimulating AuNP-IgG-treated neutrophils with lipid-soluble phorbol myristate acetate (PMA), confirming the direct intracellular action of AuNP-IgG on the inflammatory process in vitro. Our in vitro and in vivo experimental approaches highlighted the great potentiality of AuNP bioconjugates for therapeutic and diagnostic applications by parenteral routes.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/toxicity , Biocompatible Materials/toxicity , Endothelium, Vascular/drug effects , Gold/toxicity , Metal Nanoparticles/toxicity , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/drug effects , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Blood Flow Velocity/drug effects , Cell Survival/drug effects , Chemotaxis, Leukocyte/drug effects , Endothelium, Vascular/pathology , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/pathology , Gold/chemistry , Gold/pharmacology , Hemolysis/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Immunoglobulin G/immunology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Male , Metal Nanoparticles/chemistry , Neutrophils/drug effects , Neutrophils/immunology , Particle Size , Rats, Wistar , Surface Properties
2.
J Immunol ; 190(11): 5689-701, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23645879

ABSTRACT

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1(-/-) mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.


Subject(s)
Annexin A1/genetics , Anti-Inflammatory Agents/pharmacology , Peptides/pharmacology , Uveitis/genetics , Animals , Annexin A1/administration & dosage , Annexin A1/chemistry , Annexin A1/metabolism , Annexin A1/pharmacology , Anti-Inflammatory Agents/administration & dosage , Aqueous Humor/cytology , Aqueous Humor/immunology , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cytokines/biosynthesis , Cytokines/genetics , Cytokines/immunology , Disease Models, Animal , Endotoxins/adverse effects , Gene Expression Regulation/drug effects , Lipopolysaccharides/immunology , Male , Mice , Mice, Knockout , Models, Biological , NF-kappa B/metabolism , Neutrophil Infiltration/immunology , Neutrophils/drug effects , Neutrophils/immunology , Oligopeptides/pharmacology , Peptides/administration & dosage , Phosphorylation , Protein Transport/drug effects , Rats , Receptors, Formyl Peptide/genetics , Receptors, Formyl Peptide/metabolism , Uveitis/chemically induced , Uveitis/immunology
3.
Eur J Med Chem ; 58: 117-27, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23123728

ABSTRACT

A series of 3-(triazolyl)-coumarins were synthesized and tested as anti-inflammatory agents. It was possible to infer that these compounds do not alter the interaction of LPS with TLR-4 or TLR-2, as the intracellular pathways involved in the TNF-α secretion and COX-2 activity were not affected. Nevertheless, the compounds inhibited iNOS-derived NO production, without affecting the eNOS activity. The outcome of the docking studies showed that π···π interactions with the heme group are important for the iNOS inhibition, thus making compound 3c a promising lead. Moreover, the efficacy of this compound was visualized by the reduced number of neutrophils in the LPS-inflamed subcutaneous tissue. Together, biological and docking data show that triazolyl-substituted coumarins, that can act on iNOS, are a good scaffold to be explored.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Coumarins/pharmacology , Enzyme Inhibitors/pharmacology , Nitric Oxide Synthase Type II/antagonists & inhibitors , Triazoles/pharmacology , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/chemistry , Cells, Cultured , Coumarins/chemical synthesis , Coumarins/chemistry , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Male , Models, Molecular , Molecular Structure , Neutrophils/drug effects , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/metabolism , Rats , Rats, Wistar , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistry
4.
Arch Toxicol ; 86(11): 1773-81, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22717997

ABSTRACT

Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 µM; 2 h) and subsequently incubated with LPS (5 µg/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or ß(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor κB (NF-κB) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure.


Subject(s)
Hydroquinones/toxicity , Lipopolysaccharides/toxicity , Neutrophils/drug effects , Animals , Cells, Cultured , Escherichia coli/chemistry , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Male , NF-kappa B/metabolism , Neutrophil Activation/drug effects , Neutrophils/metabolism , Nitric Oxide/metabolism , Protein Transport/drug effects , Rats , Rats, Wistar , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolism
5.
Toxicology ; 296(1-3): 20-6, 2012 Jun 14.
Article in English | MEDLINE | ID: mdl-22465845

ABSTRACT

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25 ppm; 1 h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas.


Subject(s)
Chemokine CCL2/metabolism , Hydroquinones/toxicity , Immunosuppressive Agents/toxicity , Monocytes/drug effects , Pneumonia/immunology , Animals , Bronchoalveolar Lavage Fluid/cytology , Cell Count , Cell Line, Tumor , Cell Movement/drug effects , Environmental Pollutants/toxicity , Humans , Macrophages, Alveolar/cytology , Male , Mice , Monocytes/immunology , Pneumonia/pathology , Smoke , Nicotiana
6.
Toxicol Lett ; 211(1): 10-7, 2012 May 20.
Article in English | MEDLINE | ID: mdl-22414385

ABSTRACT

Hydroquinone (HQ) is the main oxidative substance in cigarette smoke and a toxic product of benzene biotransformation. Although the respiratory tract is an inlet pathway of HQ exposure, its effect on airway muscle responsiveness has not been assessed. We thus investigated the effects of low dose in vivo HQ-exposure on tracheal responsiveness to a muscarinic receptor agonist. Male Swiss mice were exposed to aerosolised 5% ethanol/saline solution (HQ vehicle; control) or 0.04 ppm HQ (1h/day for 5 days) and tracheal rings were collected 1h after the last exposure. HQ exposure caused tracheal hyperresponsiveness to methacholine (MCh), which was abolished by mechanical removal of the epithelium. This hyperresponsiveness was not dependent on neutrophil infiltration, but on tumour necrosis factor (TNF) secretion by epithelial cells. This conclusion was based on the following data: (1) trachea from HQ-exposed mice presented a higher amount of TNF, which was abrogated following removal of the epithelium; (2) the trachea hyperresponsiveness and TNF levels were attenuated by in vivo chlorpromazine (CPZ) treatment, an inhibitor of TNF synthesis. The involvement of HQ-induced TNF secretion in trachea mast cell degranulation was also demonstrated by the partial reversion of tracheal hyperresponsiveness in sodium cromoglicate-treated animals, and the in vivo HQ-exposure-induced degranulation of trachea connective tissue and mucosal mast cells, which was reversed by CPZ treatment. Our data show that in vivo HQ exposure indirectly exacerbates the parasympathetic-induced contraction of airway smooth muscle cells, mediated by TNF secreted by tracheal epithelial cells, clearly showing the link between environmental HQ exposure and the reactivity of airways.


Subject(s)
Hydroquinones/adverse effects , Respiratory Mucosa/drug effects , Trachea/drug effects , Tumor Necrosis Factor-alpha/metabolism , Animals , Chlorpromazine/pharmacology , Dose-Response Relationship, Drug , Male , Mast Cells/drug effects , Methacholine Chloride/pharmacology , Mice , Muscle Contraction/drug effects , Respiratory Mucosa/metabolism
7.
Toxicol Lett ; 197(3): 211-8, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20658762

ABSTRACT

Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in the airways and is associated with airway inflammatory disorders such as asthma. The purpose of our study was to investigate the effects of exposure to FA on the allergic lung inflammation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for 3 days, and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route. One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB 4, TXB 2, IL-1 beta, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung. Vitamins C, E and apocynin reduced the levels of LTB 4 in BAL-cultured cells of the FA and FA/OVA groups, but increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of intercellular cell adhesion molecule 1 (ICAM-1). In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability. Our data are indicative that the actions of FA involve mechanisms related to endothelium-leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned.


Subject(s)
Capillary Permeability/drug effects , Formaldehyde/toxicity , Hypersensitivity/pathology , Lung Diseases/chemically induced , Lung Diseases/immunology , Air Pollutants/toxicity , Animals , Ascorbic Acid/pharmacology , Bronchoalveolar Lavage Fluid/cytology , Inflammation , Lung/metabolism , Lung Diseases/drug therapy , Male , Phagocytosis , Rats , Rats, Wistar , Respiratory Burst , Vitamin E/pharmacology
8.
Biol Pharm Bull ; 29(11): 2241-5, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17077521

ABSTRACT

The present study evaluated the effect of the crude extract of the leaves of Nectandra falcifolia (NEES) Castiglioni and its fractions in different experimental models of inflammation (paw edema, pleurisy, and ear edema). Carrageenan-induced edema of the paw and pleurisy were evaluated in Wistar rats (180-220 g), which were treated with different doses of the total extract (250, 500 mg.kg-1). Edema of the ear, induced by croton oil, and determination of myeloperoxidase activity were evaluated in Swiss mice (25-35 g). In this experiment, the crude extract of Nectandra falcifolia (Nf) (1.25, 2.5, 5.0, 7.5 mg) and the hexane, chloroform, ethyl-acetate and hydromethanol fractions (5.0 mg) were applied topically, immediately after application of the oil. The crude extract of Nf (500 mg.kg-1) significantly reduced edema of the paw compared to the control group. Similarly, at doses of 250 and 500 mg.kg-1 it significantly reduced the volume of pleural inflammatory exudate compared to the control animals. However, it did not change the number of migrated cells. At doses of 2.5, 5.0 and 7.5 mg, the crude extract significantly inhibited edema of the ear and the influx of neutrophils. The fractions from Nectandra falcifolia (hexane, chloroform, ethyl acetate and hydromethanol) also inhibited edema of the ear. Taken together, the results demonstrated that the crude extract and its fractions administered to animals orally or topically showed an anti-inflammatory effect.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Lauraceae/chemistry , Plant Extracts/therapeutic use , Plant Leaves/chemistry , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Carrageenan/administration & dosage , Carrageenan/toxicity , Croton Oil/administration & dosage , Croton Oil/toxicity , Drug Evaluation, Preclinical/methods , Ear/pathology , Edema/chemically induced , Edema/metabolism , Edema/prevention & control , Hindlimb/drug effects , Hindlimb/pathology , Indomethacin/pharmacology , Indomethacin/therapeutic use , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/prevention & control , Injections, Intradermal , Male , Nitric Oxide/metabolism , Peroxidase/metabolism , Phytotherapy/methods , Plant Extracts/chemistry , Plant Extracts/pharmacology , Pleurisy/chemically induced , Pleurisy/metabolism , Pleurisy/prevention & control , Rats , Rats, Wistar , Solvents/chemistry
9.
Maringá; s.n; 19 dez. 2003. 74 p. tab, graf.
Thesis in Portuguese | LILACS | ID: lil-444411

ABSTRACT

Durante a sepse, grandes quantidades de óxido nítrico (NO) são sintetizadas pela enzima NO sintase tipo II. Em preparações neuromusculares, o NO produz ou acentua a fadiga de transmissão. 'Train-of-four'(TOF) são pulsos elétricos de 2 Hz aplicados ao nervo motor durante 2 segundos (4 contrações musculares) e o quociente entre as tensões musculares produzidas pela quarta (T4) e primeira (T1) contrações musculares (RTOF = T4/T1) é utilizado para avaliação do bloqueio da transmissão neuromuscular. A redução de RTOF é determinada por bloqueio dos receptores nicotínicos (Nn) facilitatórios e por estimulação dos receptores muscarínicos (M2) inibitórios do terminal nervoso motor. Estudos utilizando preparações neuromusculares de animais sépticos tratadas com bloqueadores neuromusculares em doses que reduzem os valores de RTOF sem promover alterações nas amplitudes das contrações musculares a 0,2 Hz não foram executados. Assim, utilizando preparações nervo ciático-músculo tibial anterior de ratos, verificamos que as menores doses de d-tubocurarina (d-TC), pancurônio (PANC) e galamina (GAL) capazes de produzir 30 por cento de redução em RTOF nos animais sham foram, respectivamente: 54,29 ± 2,86 mg/kg (n=7), 69,02 ± 6,13 mg/kg (n=5), 2,94 ± 0,33 mg/kg (n=4). A dose de d-TC foi menor em animais sépticos (34,53 ± 2,76 mg/kg, n=4), que em animais sham, porém os efeitos foram mais intensos e prolongados na sepse. d-TC (26,50 ± 0,68 mg/kg (n=5), promoveu 30 por cento de redução em RTOF nos animais sham submetidos à infusão de nitroprussiato de sódio (10 mg/kg/min). O tratamento com S-metilisotiouréia (SMT, 13 mg/kg) antagonizou a redução de RTOF em animais sépticos. As menores doses de PANC e GAL que reduziram RTOF em 30 por cento foram semelhantes em todos os grupos experimentais estudados e GAL promoveu efeitos similares em todas as condições experimentais. A redução de RTOF produzida por PANC foi menor que a induzida por d-TC e foi antagonizada pela administração de SMT....


Subject(s)
Animals , Rats , Nitric Oxide , Nitric Oxide/administration & dosage , Nitric Oxide/analysis , Nitric Oxide/pharmacology , Sepsis , Pancuronium/administration & dosage , Pancuronium , Tubocurarine
10.
Maringá; s.n; 2003. 74 p. tab, graf.
Thesis in Portuguese | LILACS | ID: lil-436158

ABSTRACT

Durante a sepse, grandes quantidades de óxido nítrico (NO) são sintetizadas pela enzima NO sintase tipo II. Em preparações neuromusculares, o NO produz ou acentua a fadiga de transmissão. 'Train-of-four'(TOF) são pulsos elétricos de 2 Hz aplicados ao nervo motor durante 2 segundos (4 contrações musculares) e o quociente entre as tensões musculares produzidas pela quarta (T4) e primeira (T1) contrações musculares (RTOF = T4/T1) é utilizado para avaliação do bloqueio da transmissão neuromuscular. A redução de RTOF é determinada por bloqueio dos receptores nicotínicos (Nn) facilitatórios e por estimulação dos receptores muscarínicos (M2) inibitórios do terminal nervoso motor. Estudos utilizando preparações neuromusculares de animais sépticos tratadas com bloqueadores neuromusculares em doses que reduzem os valores de RTOF sem promover alterações nas amplitudes das contrações musculares a 0,2 Hz não foram executados. Assim, utilizando preparações nervo ciático-músculo tibial anterior de ratos, verificamos que as menores doses de d-tubocurarina (d-TC), pancurônio (PANC) e galamina (GAL) capazes de produzir 30 por cento de redução em RTOF nos animais sham foram, respectivamente: 54,29 ± 2,86 mg/kg (n=7), 69,02 ± 6,13 mg/kg (n=5), 2,94 ± 0,33 mg/kg (n=4). A dose de d-TC foi menor em animais sépticos (34,53 ± 2,76 mg/kg, n=4), que em animais sham, porém os efeitos foram mais intensos e prolongados na sepse. d-TC (26,50 ± 0,68 mg/kg (n=5), promoveu 30 por cento de redução em RTOF nos animais sham submetidos à infusão de nitroprussiato de sódio (10 mg/kg/min). O tratamento com S-metilisotiouréia (SMT, 13 mg/kg) antagonizou a redução de RTOF em animais sépticos. As menores doses de PANC e GAL que reduziram RTOF em 30 por cento foram semelhantes em todos os grupos experimentais estudados e GAL promoveu efeitos similares em todas as condições experimentais. A redução de RTOF produzida por PANC foi menor que a induzida por d-TC e foi antagonizada pela administração de SMT....


Subject(s)
Animals , Rats , Nitric Oxide , Nitric Oxide/administration & dosage , Nitric Oxide/analysis , Nitric Oxide/pharmacology , Sepsis , Pancuronium/administration & dosage , Pancuronium , Tubocurarine
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