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2.
Inflamm Res ; 69(1): 115-130, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31786615

RESUMO

OBJECTIVE: To examine whether free (3-PD-5free) and/or liposomal (3-PD-5lipo) 6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin (3-PD-5) (1) modulate the effector functions of neutrophils from patients with rheumatoid arthritis under remission (i-RA) and with active disease (a-RA), in vitro; and (2) exert anti-inflammatory effect in a rat model of zymosan-induced acute joint inflammation. METHODS AND RESULTS: Incorporation of 3-PD-5 into unilamellar liposomes of soya phosphatidylcholine and cholesterol was efficient (57.5 ± 7.9%) and yielded vesicles with low diameter (133.7 ± 18.4 nm), polydispersity index (0.39 ± 0.06), and zeta potential (- 1.22 ± 0.34 mV). 3-PD-5free (1 µM) and 3-PD-5lipo (3 µM) equally suppressed elastase release and reactive oxygen species generation in neutrophils from healthy subjects and i-RA and a-RA patients, stimulated with immune complexes. 3-PD-5free (20 µM) suppressed the release of neutrophil extracellular traps and chemotaxis in vitro, without clear signs of cytotoxicity. 3-PD-5lipo (1.5 mg/kg, i.p.) diminished joint edema and synovial infiltration of total leukocytes and neutrophils, without changing the synovial levels of TNF-α, IL-1ß, and IL-6. CONCLUSION: Altogether, the results reported herein indicate that 3-PD-5 is a promising modulator of the early stages of acute joint inflammation that can help to diminish not only excessive neutrophil infiltration in the synovia but also neutrophil activation and its outcomes in RA patients.


Assuntos
Anti-Inflamatórios/administração & dosagem , Antirreumáticos/administração & dosagem , Artrite Reumatoide/tratamento farmacológico , Cumarínicos/administração & dosagem , Doença Aguda , Adulto , Idoso , Animais , Artrite Reumatoide/imunologia , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Humanos , Imunomodulação , Lipossomos , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/fisiologia , Ratos Wistar , Adulto Jovem
3.
Mol Immunol ; 116: 80-89, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31630079

RESUMO

Here we report the effects of exogenous and endogenous galectin-1 (Gal-1) in modulating the functional responses of human and murine neutrophils at different stages of activation, i.e. naive, primed, and activated. Exposure to Gal-1 did not induce ROS production in either naive or N-formyl-methionyl-leucyl-phenylalanine-primed (fMLP; 10-9 M) neutrophils. However, Gal-1 elicited a concentration-dependent ROS production in neutrophils activated with fMLP at concentrations ranging from 10-8 M to 10-6 M. Additional fMLP (10-7 M) stimulation of fMLP-activated neutrophils increased ROS production, whose intensity was inversely related to the fMLP concentration used in the first activation step (10-8 M to 10-6 M), and was not influenced by the presence of Gal-1. Naive neutrophils treated with Gal-1 and then exposed to fMLP (10-6 M) or phorbol-12-myristate-13-acetate (10-7 M) produced less ROS, as compared to naive neutrophils not treated with Gal-1. Interestingly, these in vitro Gal-1 effects were associated with Gal-1 carbohydrate-binding activity and the ability to decrease FPR-1 (formyl peptide receptor 1) expression in naive human neutrophils. Conversely, positive ROS modulation by Gal-1 in activated neutrophils was not associated with FPR-1 expression but it was related to its carbohydrate recognition. In vitro, fMLP stimulation of Gal-1-/- mouse neutrophils produced more ROS than fMLP stimulation of Gal-1+/+ neutrophils and this effect may be associated with increased FPR-1 expression. Exogenous Gal-1 induced ROS production in Gal-1-/- mouse neutrophils more effectively than in Gal-1+/+ mouse neutrophils. Compared to Gal-1+/+ mice, Gal-1-/- mice exhibited lower bacterial load in the peritoneal fluid and peripheral blood, thus indicating a greater bactericidal activity in vivo. These findings demonstrate that endogenous Gal-1 restricts ROS generation that correlates with bacterial killing capacity in inflammatory neutrophils. Thus, endogenous and exogenous Gal-1 may either positively or negatively modulate the effector functions of neutrophils according to the cell activation stage.


Assuntos
Galectina 1/farmacologia , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Receptores de Formil Peptídeo/metabolismo , Adulto Jovem
4.
Nat Prod Res ; 33(17): 2521-2525, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29527950

RESUMO

The aerial parts of Baccharis dracunculifolia (BdE) is used in the Brazilian folk medicine to treat inflammatory conditions. Here we examined the ability of free and liposomal BdE to modulate reactive oxygen species generation in human neutrophils in vitro and zymosan-induced acute joint inflammation in Wistar rats. We prepared biocompatible liposomes of soya phosphatidylcholine and cholesterol with low diameter, homogeneous size distribution, and neutral surface charge. Free BdE decreased joint swelling, total leucocyte and neutrophil infiltration, and the synovial levels of tumour necrosis factor-α and interleukins 6 and 1ß. Incorporation of BdE into liposomes preserved its capacity to inhibit the neutrophil superoxide anion and total reactive oxygen species generation, and improved its anti-inflammatory effect in vivo by decreasing the effective BdE dose by nearly sixfold. The same liposome type lowered the effective dose of caffeic acid by nearly sixteenfold. Therefore, incorporation of BdE into phosphatidylcholine-cholesterol liposomes improves its anti-inflammatory effect.


Assuntos
Anti-Inflamatórios/isolamento & purificação , Baccharis/química , Lipossomos/metabolismo , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Brasil , Ácidos Cafeicos/metabolismo , Movimento Celular , Colesterol/metabolismo , Edema/tratamento farmacológico , Humanos , Leucócitos/efeitos dos fármacos , Medicina Tradicional , Neutrófilos/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacologia , Extratos Vegetais/química , Ratos , Ratos Wistar
5.
Rheumatol Int ; 38(6): 1043-1052, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29464314

RESUMO

We examined the functional activity of peripheral blood neutrophils and the complement system activation status in patients with rheumatoid arthritis (RA) undergoing infliximab/methotrexate combined therapy. We studied female RA patients under treatment with infliximab (3-5 mg/kg) and methotrexate (15-25 mg/week) who presented inactive (i-RA; n = 34, DAS-28 ≤ 2.6) or at least moderately active disease (a-RA; n = 29, DAS-28 > 3.2), and age-matched healthy women (n = 38). We measured the levels of reactive oxygen species (ROS) generation (chemiluminescence assay) and membrane expression of FcγRIIa/CD32, FcγRIIIb/CD16, CR1/CD35, and CR3/CD11b receptors (ELISA assay) in neutrophils. We also determined the hemolytic activity of the alternative and classical pathways of the complement system (spectrophotometry), serum levels of C5a and Bb (ELISA assay), and serum chemotactic activity (Boyden chamber). Compared with the control group, i-RA and a-RA patients exhibited: (1) increased neutrophil ROS production and membrane expression of FcγRIIa/CD32, FcγRIIIb/CD16, and CR1/CD35, indicating neutrophil activation; and (2) increased serum chemotactic activity and decreased activity of the alternative complement pathway, indicating systemic complement system activation. The levels of C-reactive protein in a-RA patients were augmented, compared with i-RA patients. Although infliximab/methotrexate combined therapy induced disease remission according to the DAS-28 criteria, both i-RA and a-RA patients still exhibited significant levels of systemic activation of neutrophils and the complement system.


Assuntos
Artrite Reumatoide/imunologia , Ativação do Complemento , Neutrófilos/imunologia , Adulto , Anticorpos Monoclonais/uso terapêutico , Complexo Antígeno-Anticorpo/biossíntese , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Brasil , Feminino , Humanos , Infliximab/uso terapêutico , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo
6.
Free Radic Biol Med ; 115: 421-435, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29248721

RESUMO

In this study, we report the ability of a set of eight 3-phenylcoumarin derivatives bearing 6,7- or 5,7-dihydroxyl groups, free or acetylated, bound to the benzopyrone moiety, to modulate the effector functions of human neutrophils. In general, (i) 6,7-disubstituted compounds (5, 6, 19, 20) downmodulated the Fcγ receptor-mediated neutrophil oxidative metabolism more strongly than 5,7-disubstituted compounds (21, 22, 23, 24), and (ii) hydroxylated compounds (5, 19, 21, 23) downmodulated this neutrophil function more effectively than their acetylated counterparts (6, 20, 22, 24, respectively). Compounds 5 (6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin) and 19 (6,7-dihydroxy-3-[3',4'-dihydroxyphenyl]-coumarin) effectively downmodulated the neutrophil oxidative metabolism elicited via Fcγ and/or complement receptors. Compound 5 also downmodulated the immune complex-stimulated phagocytosis, degranulation of elastase, and production and release of neutrophil extracellular traps, as well as the human neutrophil chemotaxis towards n-formyl-methionyl-leucyl-phenylalanine, without altering the expression level of formyl peptide receptor type 1. Both compounds 5 and 19 did not impair the neutrophil capacity to recognize and kill Candida albicans. Docking calculations revealed that compounds 5 and 19 directly interacted with three catalytic residues - Gln-91, His-95, and Arg-239 - inside the myeloperoxidase active site. Together, these findings indicate that (i) inhibition of reactive oxygen species generation and degranulation of elastase are closely associated with downmodulation of release of neutrophil extracellular traps; and (ii) compound 5 can be a prototype for the development of novel immunomodulating drugs to treat immune complex-mediated inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Cumarínicos/farmacologia , Armadilhas Extracelulares/metabolismo , Neutrófilos/fisiologia , Elastase Pancreática/metabolismo , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismo , Anti-Inflamatórios/química , Células Cultivadas , Cumarínicos/química , Humanos , Imunomodulação , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo
7.
J Bioenerg Biomembr ; 49(6): 423-435, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29128917

RESUMO

Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD+), generating a large mitochondrial membrane potential. The NAD+ carrier utilized the electrochemical proton gradient to drive NAD+ entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.


Assuntos
Aspergillus fumigatus/química , Mitocôndrias/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Saccharomyces cerevisiae/genética , Deleção de Genes , Xenoenxertos , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais , NAD/metabolismo , Proteínas de Transporte de Nucleotídeos , Estresse Oxidativo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
8.
J Pharm Pharmacol ; 69(12): 1829-1845, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28994118

RESUMO

OBJECTIVES: To examine whether the hydroalcoholic extract from Baccharis dracunculifolia leaves (BdE) modulates the human neutrophil oxidative metabolism, degranulation, phagocytosis and microbial killing capacity. METHODS: In-vitro assays based on chemiluminescence, spectrophotometry, flow cytometry and polarimetry were used, as well as docking calculations. KEY FINDINGS: At concentrations that effectively suppressed the neutrophil oxidative metabolism elicited by soluble and particulate stimuli (<10 µg/ml), without clear signs of cytotoxicity, BdE (1) inhibited NADPH oxidase and myeloperoxidase activity; (2) scavenged H2 O2 and HOCl; (3) weakly inhibited phagocytosis; and (4) did not affect neutrophil degranulation and microbial killing capacity, the expression levels of TLR2, TLR4, FcγRIIa, FcγRIIIb and CR3 and the activity of elastase and lysozyme. Caffeic acid, one of the major B. dracunculifolia secondary metabolites, did not inhibit phagocytosis but interfered in the myeloperoxidase-H2 O2 -HOCl system by scavenging H2 O2 and HOCl, and interacting with the catalytic residues His-95, Arg-239 and Gln-91. CONCLUSIONS: BdE selectively modulates the effector functions of human neutrophils, inhibits the activity of key enzymes and scavenges physiological oxidant species. Caffeic acid contributes to lower the levels of oxidant species. Our findings help to unravel the mechanisms by which these natural products exert immunomodulatory action towards neutrophils.


Assuntos
Baccharis/química , Fatores Imunológicos/farmacologia , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Adulto , Ácidos Cafeicos/isolamento & purificação , Ácidos Cafeicos/farmacologia , Citometria de Fluxo , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Humanos , Fatores Imunológicos/isolamento & purificação , Luminescência , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Folhas de Planta , Espectrofotometria
9.
Clin Exp Rheumatol ; 35(2): 247-254, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27908303

RESUMO

OBJECTIVES: Neutrophils play a major role in rheumatoid arthritis (RA) pathogenesis. We aimed to evaluate if neutrophil DNA damage in RA patients is associated with the disease activity, autoantibodies status, carriage of the RA shared epitope (SE) and treatment. METHODS: DNA damage was assessed by alkaline comet assay in peripheral blood (77 patients and 55 healthy controls) and in 10 RA synovial fluid neutrophils. Evaluation of the respiratory burst of 30 patients with RA and 30 healthy controls was done. RESULTS: Compared to controls, RA patients exhibited increased neutrophil DNA damage. RA synovial fluid cells DNA damage was increased when compared to OA synovial fluids cells. In addition, our study shows that anti-TNF-α therapy reduces the frequency of DNA damage. Patients with simple or double dose of shared epitope presented a higher frequency of DNA damage compared to patients without the allele. Positive correlation was found between neutrophil DNA damage and DAS-28 and ROS production. CONCLUSIONS: Our results suggest that an increase of respiratory burst of neutrophils reflects the higher levels of DNA damage in neutrophils and a positive correlation between DNA damage and disease activity shows the importance of oxidative stress in the pathogenesis of RA.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Dano ao DNA , Epitopos/imunologia , Antígenos HLA/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Adulto , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Autoanticorpos/sangue , Estudos de Casos e Controles , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Epitopos/sangue , Epitopos/genética , Feminino , Antígenos HLA/sangue , Antígenos HLA/genética , Humanos , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Índice de Gravidade de Doença , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia
10.
Artigo em Inglês | MEDLINE | ID: mdl-26346244

RESUMO

Rheumatoid arthritis (RA) is a highly disabling disease that affects all structures of the joint and significantly impacts on morbidity and mortality in RA patients. RA is characterized by persistent inflammation of the synovial membrane lining the joint associated with infiltration of immune cells. Eighty to 90% of the leukocytes infiltrating the synovia are neutrophils. The specific role that neutrophils play in the onset of RA is not clear, but recent studies have evidenced that they have an important participation in joint damage and disease progression through the release of proteolytic enzymes, reactive oxygen species (ROS), cytokines, and neutrophil extracellular traps, in particular during frustrated phagocytosis of immune complexes (ICs). In addition, the local and systemic activation of the complement system contributes to the pathogenesis of RA and other IC-mediated diseases. This review discusses (i) the participation of Fcγ and complement receptors in mediating the effector functions of neutrophils in RA; (ii) the contribution of the complement system and ROS-dependent and ROS-independent mechanisms to joint damage in RA; and (iii) the use of plant extracts, dietary compounds, and isolated natural compounds in the treatment of RA, focusing on modulation of the effector functions of neutrophils and the complement system activity and/or activation.

11.
Hum Immunol ; 75(8): 785-90, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24945596

RESUMO

Considering that human neutrophil FcγRIIa and FcγRIIIb receptors interact synergistically with CR3 in triggering neutrophil functional responses, allelic polymorphisms in these receptors might influence such interactions. We assessed whether FcγRIIIb polymorphisms affect FcγR/CR cooperation in mediating the neutrophil oxidative burst (OB), in particular the FcγRIIIb/CR3 cooperation that occurs via lectin-saccharide-like interactions. The OB of human neutrophil antigen (HNA)-1a-, HNA-1b-, and HNA-1a/-1b-neutrophils stimulated with immune complexes, opsonized or not with serum complement, was measured by the luminol-enhanced chemiluminescence assay. Compared with HNA-1a-neutrophils, HNA-1b-neutrophils exhibited reduced FcγR-stimulated OB, but increased FcγR/CR-stimulated OB. It suggests that (i) FcγR and CR cooperate more effectively in HNA-1b-neutrophils, and (ii) the HNA-1b allotype influences the FcγRIIIb cooperation with FcγRIIa, but not with CR3. HNA-1a- and HNA-1b-neutrophils exhibited similar OB responses elicited via CR3 alone or via FcγR/CR-independent pathways. In addition, the level of FcγRIIIb, FcγRIIa, and CR3 expression did not differ significantly among the neutrophil groups studied. Together, these results demonstrate that the HNA-1b allotype influences the functional cooperation between FcγRIIIb and FcγRIIa, and suggest that the difference in the glycosylation pattern between HNA-1a and HNA-1b does not affect the FcγRIIIb cooperation with CR3.


Assuntos
Antígeno de Macrófago 1/genética , Neutrófilos/imunologia , Receptores de IgG/genética , Explosão Respiratória/imunologia , Adulto , Complexo Antígeno-Anticorpo/farmacologia , Proteínas do Sistema Complemento/farmacologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica , Haplótipos , Humanos , Isoantígenos/genética , Isoantígenos/imunologia , Antígeno de Macrófago 1/imunologia , Masculino , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Polimorfismo Genético , Cultura Primária de Células , Receptor Cross-Talk/imunologia , Receptores de IgG/imunologia , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/genética , Transdução de Sinais
12.
J Ethnopharmacol ; 150(2): 655-64, 2013 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-24076472

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The great potential of phytotherapic drugs for treating and preventing inflammatory diseases mediated by increased neutrophil reactive oxygen species (ROS) generation has guided the search for new natural products with antioxidant and immunomodulatory properties. Baccharis dracunculifolia D.C. (Asteraceae), the main botanical source of Brazilian green propolis, is a native plant from Brazil widely used in folk medicine as anti-inflammatory. This study aims: (a) to determine the influence of seasonality on the chemical profile and biological activity of Baccharis dracunculifolia (Asteraceae) leaf extracts (BdE); (b) to analyze the correlation between the major compounds and the ability of BdE to modulate the superoxide anion and total ROS generation by human neutrophils. MATERIALS AND METHODS: The extracts were obtained from leaf samples collected monthly during one year. The superoxide anion and total ROS generation were assessed by the lucigenin (CL-luc)- and luminol (CL-lum)-enhanced chemiluminescence assays. RESULTS: Seasonality influenced more the quantitative than the qualitative chemical profile of B. dracunculifolia, and affected its biological activity. The major compounds identified were caffeic acid, p-coumaric acid, aromadendrin-4'-methyl ether (AME), isosakuranetin and artepillin C. The IC50 values obtained for CL-lum and CL-luc inhibition by BdE ranged from 8.1-15.8 and 5.8-13.3µgmL(-1), respectively, and correlated positively with caffeic acid concentration. CL-luc inhibition correlated negatively with the concentration of artepillin C, AME, isosakuranetin and total flavonoids. The BdE sample from May/07 inhibited CL-lum and CL-luc the most strongly (IC50=8.1 ± 1.6 and 5.8 ± 1.0 µg mL(-1), respectively), and contained the highest ratio of caffeic acid to the other isolated compounds; so, this ratio could be employed as chemical marker for this biological activity of B. dracunculifolia. CONCLUSION: The ability of B. dracunculifolia to inhibit the neutrophil ROS generation depends more on the type and ratio of phenolic compounds and flavonoids than on their high absolute concentrations. Together, our results help select the most appropriate plant material for the production of phytotherapic drugs to be used in the treatment of inflammatory diseases mediated by increased neutrophil activation.


Assuntos
Baccharis , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Adolescente , Adulto , Ácidos Cafeicos/análise , Ácidos Cafeicos/farmacologia , Células Cultivadas , Flavonoides/análise , Flavonoides/farmacologia , Humanos , Neutrófilos/metabolismo , Fenóis/análise , Fenóis/farmacologia , Extratos Vegetais/química , Folhas de Planta , Estações do Ano , Adulto Jovem
13.
Chem Biol Interact ; 206(1): 63-75, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23994743

RESUMO

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.


Assuntos
Antifúngicos/farmacologia , Antioxidantes/farmacologia , Candida albicans/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Umbeliferonas/farmacologia , Ânions/antagonistas & inibidores , Ânions/metabolismo , Antifúngicos/química , Antioxidantes/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Umbeliferonas/química
14.
J Med Food ; 16(8): 692-700, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23905650

RESUMO

Increased neutrophil activation significantly contributes to the tissue damage in inflammatory illnesses; this phenomenon has motivated the search for new compounds to modulate their effector functions. Coumarins are natural products that are widely consumed in the human diet. We have evaluated the antioxidant and immunomodulator potential of five 4-methylcoumarin derivatives. We found that the 4-methylcoumarin derivatives inhibited the generation of reactive oxygen species by human neutrophils triggered by serum-opsonized zymosan or phorbol-12-myristate-13-acetate; this inhibition occurred in a concentration-dependent manner, as revealed by lucigenin- and luminol-enhanced chemiluminescence assays. Cytotoxicity did not mediate this inhibitory effect. The 7,8-dihydroxy-4-methylcoumarin suppressed the neutrophil oxidative metabolism more effectively than the 6,7- and 5,7-dihydroxy-4-methylcoumarins, but the 5,7- and 7,8-diacetoxy-4-methylcoumarins were less effective than their hydroxylated counterparts. An analysis of the biochemical pathways suggested that the 6,7- and 7,8-dihydroxy-4-methylcoumarins inhibit the protein kinase C-mediated signaling pathway, but 5,7-dihydroxy-4-methylcoumarin, as well as 5,7- and 7,8-diacetoxy-4-methylcoumarins do not significantly interfere in this pathway of the activation of the human neutrophil oxidative metabolism. The 4-methylcoumarin derivatives bearing the catechol group suppressed the elastase and myeloperoxidase activity and reduced the 1,1-diphenyl-2-picrylhydrazyl free radical the most strongly. Interestingly, the 5,7-dihydroxy-4-methylcoumarin scavenged hypochlorous acid more effectively than the o-dihydroxy-substituted 4-methylcoumarin derivatives, and the diacetoxylated 4-methylcoumarin derivatives scavenged hypochlorous acid as effectively as the 7,8-dihydroxy-4-methylcoumarin. The significant influence of small structural modifications in the inhibitory potential of 4-methylcoumarin derivatives on the effector functions of neutrophil makes them interesting candidates to develop new drugs for the treatment of inflammatory diseases mediated by increased neutrophil activation.


Assuntos
Cumarínicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Elastase Pancreática/antagonistas & inibidores , Adulto , Antioxidantes/química , Antioxidantes/farmacologia , Células Cultivadas , Cumarínicos/química , Humanos , Neutrófilos/enzimologia , Elastase Pancreática/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Anal Biochem ; 437(2): 130-2, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23499965

RESUMO

This article shows how six vehicles interfere in the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hypochlorous acid (HOCl) and taurine chloramine in vitro. All the tested vehicles inhibited TMB oxidation by HOCl; dimethyl sulfoxide had a remarkable effect at concentrations as low as 0.00005% (v/v). Cremophor EL and ethanol inhibited TMB oxidation by taurine chloramine at concentrations higher than 0.05 and 25% (v/v), respectively; the other vehicles did not affect this reaction. The results will help to guide the choice of solvent for the TMB oxidation assay performed under viable experimental conditions for evaluation of the HOCl and taurine chloramine scavenging ability of drugs.


Assuntos
Benzidinas/química , Dimetil Sulfóxido/química , Ácido Hipocloroso/química , Taurina/análogos & derivados , Etanol/química , Glicerol/análogos & derivados , Glicerol/química , Oxirredução , Quercetina/química , Taurina/química
16.
Drug Deliv ; 19(4): 177-87, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22533556

RESUMO

CONTEXT AND OBJECTIVE: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. MATERIALS AND METHODS: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. RESULTS AND DISCUSSION: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. CONCLUSION: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.


Assuntos
Complexo Antígeno-Anticorpo/fisiologia , Flavonoides/farmacologia , Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Animais , Galinhas , Relação Dose-Resposta a Droga , Feminino , Humanos , Lipossomos , Masculino , Neutrófilos/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo
17.
J Liposome Res ; 22(2): 89-99, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22011316

RESUMO

Liposomes have been employed as potential drug carriers. However, after their in vivo administration, they can be destabilized by proteins of complement system, contributing to the clearance of vesicles from blood circulation. Antioxidant flavonoids such as quercetin have been reported to be beneficial to human health, but their low water solubility and bioavailability limit their enteric administration. Therefore, the development of appropriate flavonoid-carriers could be of great importance to drug therapy. The aim of the present study was to evaluate the activation of human complement system proteins by liposomes composed of soya phosphatidylcholine (SPC) and cholesterol (CHOL) or cholesteryl ethyl ether (CHOL-OET) loaded with quercetin or not. The consumption of complement, via classical (CP) and alternative (AP) pathways, by different vesicles was evaluated using a hemolytic assay and quantitative determination of iC3b and natural antibodies deposited on empty liposomal surfaces by ELISA. The main results showed that empty liposomes composed of large amounts of CHOL consumed more complement components than the others for both CP and AP. Furthermore, replacement of CHOL with CHOL-OET reduced complement consumption via both CP and AP. Incorporation of quercetin did not change CP and AP consumption. Deposition of iC3b, IgG and IgM in vesicles composed of SPC:CHOL-OET at a molar ratio of 1.5:1 was lower compared to the others. Taken together, these observations suggest that liposomes composed of SPC:CHOL-OET at a molar ratio of 1.5:1 are the most appropriate among the vesicles studied herein to be used as a drug carrier system in further investigations.


Assuntos
Ativação do Complemento , Portadores de Fármacos/química , Lipossomos/química , Quercetina/química , Animais , Ensaio de Atividade Hemolítica de Complemento , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lipossomos/sangue , Masculino , Quercetina/sangue , Coelhos , Valores de Referência , Ovinos , Propriedades de Superfície
18.
J Microencapsul ; 28(4): 258-67, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21545317

RESUMO

Considering that antioxidant flavonols have been reported to be beneficial to human health, but that their low water solubility and bioavailability limit their administration through systemic route, the development of suitable flavonol-carriers is of great importance for clinical therapeutics. The aim of this study was to prepare liposomes containing flavonols or not and evaluate their antioxidant activity. Vesicles were obtained by ethanol injection method and characterized in terms of entrapment efficiency, size and zeta potential. Inhibitory activity of liposomal flavonols on reactive oxygen species generation was assessed in vitro using luminol-H(2)O(2)-horseradish peroxidase technique. Antioxidant activity of liposomal flavonols is dependent on concentration and chemical structure of active compound. Quercetin and myricetin are the most active flavonols (IC(50) = 0.6-0.9 µmol/L), followed by kaempferol (IC(50) = 3.0-4.5 µmol/L) and galangin (IC(50) = 4.0-7.0 µmol/L). Our results suggest that antioxidant-loaded liposomes may be promising tools for therapy of diseases where oxidative stress is involved.


Assuntos
Antioxidantes/química , Flavonóis/química , Peróxido de Hidrogênio/química , Luminol/química , Avaliação Pré-Clínica de Medicamentos , Peroxidase do Rábano Silvestre/química , Humanos , Lipossomos
19.
Immunobiology ; 215(6): 475-85, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19720428

RESUMO

Galectins are beta-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (Nphi) from C57BL/6 wild-type (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MTT assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in Nphi culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in Nphi cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Nphi number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates T. gondii-induced Nphi toxicity as well as Nphi degranulation regardless of infection. Furthermore, Gal-3 expression by Nphi was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-alpha later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in Nphi following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in Nphi life span and activation during early T. gondii infection.


Assuntos
Galectina 3/imunologia , Regulação da Expressão Gênica/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Degranulação Celular/genética , Degranulação Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Galectina 3/genética , Galectina 3/metabolismo , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Knockout , Ativação de Neutrófilo/genética , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/genética , Toxoplasmose/metabolismo
20.
Food Chem Toxicol ; 47(1): 163-70, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19022329

RESUMO

The tamarind (Tamarindus indica L.) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC50 (in microg/10(6)cells)=115.7+/-9.7 (LumCL) and 174.5+/-25.9 (LucCL)], than the OZ- [IC50=248.5+/-23.1 (LumCL) and 324.1+/-34.6 (LucCL)] or fMLP-stimulated cells [IC50=178.5+/-12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O2 consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 microg/10(6)cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases.


Assuntos
Frutas/química , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Extratos Vegetais/farmacologia , Tamarindus/química , Antioxidantes/química , Antioxidantes/farmacologia , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Medições Luminescentes , Masculino , NADPH Oxidases/antagonistas & inibidores , Elastase Pancreática/antagonistas & inibidores , Ésteres de Forbol , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo
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