RESUMO
PURPOSE: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. METHODS: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. RESULTS: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). CONCLUSION: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.
Assuntos
Ácido Ascórbico/farmacologia , Queimaduras/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Araquidonato 12-Lipoxigenase/análise , Araquidonato 12-Lipoxigenase/efeitos dos fármacos , Queimaduras/tratamento farmacológico , Células Cultivadas , Estudos Transversais , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 1/efeitos dos fármacos , Oxidases Duais/análise , Oxidases Duais/efeitos dos fármacos , Feminino , Glutationa Peroxidase/análise , Glutationa Peroxidase/efeitos dos fármacos , Glutationa Transferase/análise , Glutationa Transferase/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Masculino , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/efeitos dos fármacos , Peroxirredoxinas/análise , Peroxirredoxinas/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reprodutibilidade dos Testes , Pele/efeitos dos fármacos , Pele/patologia , Estatísticas não Paramétricas , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Adulto JovemRESUMO
Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.
Assuntos
Humanos , Masculino , Feminino , Adulto , Adulto Jovem , Ácido Ascórbico/farmacologia , Queimaduras/patologia , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Valores de Referência , Pele/patologia , Araquidonato 12-Lipoxigenase/análise , Araquidonato 12-Lipoxigenase/efeitos dos fármacos , Queimaduras/tratamento farmacológico , Células Cultivadas , Estudos Transversais , Estatísticas não Paramétricas , Ubiquitina-Proteína Ligases/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/análise , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 1/efeitos dos fármacos , Peroxirredoxinas/análise , Reação em Cadeia da Polimerase em Tempo Real , Oxidases Duais/análise , Oxidases Duais/efeitos dos fármacos , Glutationa Peroxidase/análise , Glutationa Peroxidase/efeitos dos fármacosRESUMO
Inhalation of formaldehyde (FA) during the pregnancy induces oxidative stress in the uterus, and here we hypothesized that this mechanism may be responsible for the impaired immune response detected in the offspring. In order to investigate the protective effects of Vitamin C on the oxidative stress induced by FA in the uterine microenvironment, pregnant Wistar rats were treated with vitamin C (150mg/kg, gavage) or vehicle (distilled water, gavage) 1h before FA exposure (0.92mg/m(3), 1h/day, 5days/week), for 21days, and the 30days old offspring were submitted to LPS injection (Salmonella abortus equi, 5mg/kg, i.p.). The enhanced gene expression of iNOS, COX-1 and COX-2 and decreased gene expression of SOD-2 in the uterus of FA exposed mothers was rescued by Vit C treatment. Moreover, vitamin C rescued the impaired immune response elicited by LPS in the offspring from FA exposed mothers, by increasing the number of blood and bone marrow leukocytes, and augmenting gene expression of IL-6 and reducing mRNA levels of IL-10 and IFN in the lungs. Vitamin C treatment did not rescue the impaired TLR4-NF-kB pathway in the lung of the offspring, suggesting that FA-induced uterine oxidative stress affects other inflammatory pathways activated by LPS in the offspring. Together, data obtained here confirm our hypothesis that FA-induced oxidative stress in the uterine microenvironment modifies the programming mechanisms of the immune defenses of offspring, leading to an impaired host defense.
Assuntos
Ácido Ascórbico/farmacologia , Formaldeído/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Animais , Ciclo-Oxigenase 1/efeitos dos fármacos , Feminino , Expressão Gênica , Interleucinas/biossíntese , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Gravidez , Ratos , Superóxido Dismutase/efeitos dos fármacos , Receptor 4 Toll-Like/efeitos dos fármacosRESUMO
In vitro anti-inflammatory activity of 4 extracts with different polarity from the basidiomycete Navisporus floccosus was evaluated by determination of the inhibition of prostaglandin E2 formation catalyzed by purified cyclooxygenase (COX)-1 and COX-2 enzymes, and of the inhibition of leukotriene (LT) B4 formation in human polymorphonuclear leukocytes. The n-hexane extract showed the highest activity in all 3 assays. Through analysis by gas chromatography coupled with mass spectrometry (GC-MS), 9 fatty acids and fatty acid esters were identified as the major constituents of this extract. As several of them also showed inhibitory activity in the COX and LTB4 formation assays, it can be assumed that the unsaturated as well as the saturated fatty acids, and maybe also the fatty acid esters, present in the extract synergistically contribute to its in vitro anti-inflammatory activity.
Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Araquidônicos/metabolismo , Basidiomycota/química , Ácidos Graxos/isolamento & purificação , Anti-Inflamatórios/análise , Anti-Inflamatórios/isolamento & purificação , Ácidos Araquidônicos/antagonistas & inibidores , Ciclo-Oxigenase 1/efeitos dos fármacos , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/antagonistas & inibidores , Dinoprostona/metabolismo , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas , Hexanos , Humanos , Leucotrieno B4/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , FarmacognosiaRESUMO
The incidence of hematological disorders has increased steadily in Western countries despite the advances in drug development. The high expression of the multi-resistance protein 4 in patients with transitory aspirin resistance, points to the importance of finding new molecules, including those that are not affected by these proteins. In this work, we describe the synthesis and biological evaluation of a series of N,N'-disubstituted thioureas derivatives using in vitro and in silico approaches. New designed compounds inhibit the arachidonic acid pathway in human platelets. The most active thioureas (compounds 3d, 3i, 3m and 3p) displayed IC50 values ranging from 29 to 84 µM with direct influence over in vitro PGE2 and TXA2 formation. In silico evaluation of these compounds suggests that direct blockage of the tyrosyl-radical at the COX-1 active site is achieved by strong hydrophobic contacts as well as electrostatic interactions. A low toxicity profile of this series was observed through hemolytic, genotoxic and mutagenic assays. The most active thioureas were able to reduce both PGE2 and TXB2 production in human platelets, suggesting a direct inhibition of COX-1. These results reinforce their promising profile as lead antiplatelet agents for further in vivo experimental investigations.
Assuntos
Ciclo-Oxigenase 1/química , Fibrinolíticos/síntese química , Fibrinolíticos/farmacologia , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/farmacologia , Tioureia/análogos & derivados , Ácido Araquidônico/metabolismo , Domínio Catalítico/efeitos dos fármacos , Simulação por Computador , Ciclo-Oxigenase 1/efeitos dos fármacos , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/metabolismo , Fibrinolíticos/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores da Agregação Plaquetária/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Tioureia/farmacologia , Tromboxano B2/metabolismoRESUMO
BACKGROUND: The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. OBJECTIVE: To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. METHODS: Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. RESULTS: Platelet counts were markedly diminished in rats administered with either ARPI (± 88%) or busulfan (± 96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. CONCLUSIONS: Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.
Assuntos
Plaquetas/citologia , Bothrops , Carragenina/farmacologia , Venenos de Crotalídeos/farmacologia , Hiperalgesia/induzido quimicamente , Animais , Aspirina/farmacologia , Plaquetas/enzimologia , Bussulfano/toxicidade , Clopidogrel , Ciclo-Oxigenase 1/efeitos dos fármacos , Hiperalgesia/prevenção & controle , Masculino , Ratos , Ratos Wistar , Trombocitopenia/induzido quimicamente , Ticlopidina/análogos & derivados , Ticlopidina/farmacologiaRESUMO
The anti-inflammatory potential of 26 neolignans (14 of the bicyclooctane-type and 12 of the benzofuran-type), isolated from three Lauraceae species (Pleurothyrium cinereum, Ocotea macrophylla and Nectandra amazonum), was evaluated in vitro through inhibition of COX-1, COX-2, 5-LOX and agonist-induced aggregation of rabbit platelets. Benzofuran neolignans were found to be selective COX-2 inhibitors, whereas bicyclooctane neolignans inhibit selectively the PAF-action as well as COX-1 and 5-LOX. The neolignan 9-nor-7,8-dehydro-isolicarin B 15 and cinerin C 7 were found to be the most potent COX-2 inhibitor and PAF-antagonist, respectively. Nectamazin C 10 exhibited dual 5-LOX/COX-2 inhibition.
Assuntos
Inibidores de Ciclo-Oxigenase/química , Lauraceae/química , Inibidores de Lipoxigenase/química , Inibidores da Agregação Plaquetária/química , Animais , Ciclo-Oxigenase 1/efeitos dos fármacos , Ciclo-Oxigenase 2/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/isolamento & purificação , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Lipoxigenase/isolamento & purificação , Inibidores de Lipoxigenase/farmacologia , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , CoelhosRESUMO
OBJECTIVES: Novel 5-benzilidene thiazolidinones have been synthesized and exhibited anti-inflammatory activity. In this work one of the compounds of the thiazolidinone chemical series, (5Z,E)-3-[2-(4-chlorophenyl)-2-oxoethyl]-5-(1H-indol-3-ylmethylene)-thiazolidine-2,4-dione (PG15) was investigated aiming to determine the drug's anti-inflammatory potential in pre-clinical studies. METHODS: Methods used included the in-vitro inhibition of cyclooxygenase-1 and -2, in-vivo evaluation of anti-inflammatory activity by air pouch and peritonitis models and the pharmacokinetic profile after intravenous (3 mg/kg) and oral (3 and 6 mg/kg) dosing to rats. KEY FINDINGS: A two-compartment model with a fast distribution and an elimination half-life of 5.9 +/- 3.8 h described the PG15 plasma profile after intravenous dosing. PG15 showed an erratic and rapid absorption following oral administration with peak concentrations between 0.5 and 1 h. PG15 0.1 microM inhibited more than 30% and 13% of purified cyclooxygenase-1 and -2 activity in vitro, respectively. A lack of dose dependency was observed for the anti-inflammatory effect in the dose range investigated (0.8-50 mg/kg), with a maximum of 67.2 +/- 4.6% inhibition of leucocyte migration in the carrageenan-induced air pouch model obtained with the 3 mg/kg dose, similar to that observed for indometacin 10 mg/kg. CONCLUSIONS: The erratic absorption of PG15 observed after oral dosing could explain the lack of anti-inflammatory dose dependency.
Assuntos
Anti-Inflamatórios/farmacologia , Indóis/farmacologia , Inflamação/tratamento farmacológico , Tiazolidinas/farmacologia , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Ciclo-Oxigenase 1/efeitos dos fármacos , Ciclo-Oxigenase 2/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/administração & dosagem , Inibidores de Ciclo-Oxigenase/farmacocinética , Inibidores de Ciclo-Oxigenase/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Indóis/administração & dosagem , Indóis/farmacocinética , Indometacina/farmacologia , Inflamação/fisiopatologia , Injeções Intravenosas , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Camundongos , Peritonite/tratamento farmacológico , Peritonite/fisiopatologia , Ratos , Ratos Wistar , Tiazolidinas/administração & dosagem , Tiazolidinas/farmacocinética , Distribuição TecidualRESUMO
Pretreatment using celecoxib, a cyclooxygenase (COX) 2 inhibitor, or indomethacin, a nonselective COX inhibitor, reduced lypopolyssaccharide (LPS)-induced leukocyte migration to the rat peritoneal cavity. The effect of celecoxib (12 mg/kg) or indomethacin (2 mg/kg) on neutrophil chemotaxis induced by formyl-methionyl-leucyl-phenylalanine (FMLP) in an in vitro chemotactic assay (Boyden chamber) was investigated. Celecoxib and indomethacin inhibited chemotaxis induced by FMLP (Control=26.6+/-1.45, Celecoxib=12.8+/-3.04, Indomethacin=6.26+/-2.19 cells/field). When observed under intravital microscopy, a mouse cremaster preparation was used to assess the microvasculature to further investigate which step of cell recruitment was affected by these drugs. Celecoxib and indomethacin inhibited leukocyte migration induced by 0.05 microg/kg LPS injected into the cremaster muscle. However, the effect of celecoxib was associated with reduced cell rolling and adhesion, whereas indomethacin was only effective at inhibiting cell adhesion. Furthermore, SC560 pretreatment (a COX-1 selective inhibitor) of normal or LPS-challenged tissues did not alter leukocyte migration or cell adhesion, but it did enhance leukocyte rolling activity in both cases. Taken together, these results indicate that: 1) COX-1 activity is mainly related to leukocyte traffic under physiological conditions, and 2) COX-2 activity is mainly related to cell traffic under inflammatory conditions in vascular beds, suggesting a possible effect of selective COX-2 inhibitors on the expression of adhesion molecules.