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Master's Dissertation
DOI
https://doi.org/10.11606/D.9.2013.tde-04102013-095142
Document
Author
Full name
Éric Diego Barioni
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2013
Supervisor
Committee
Farsky, Sandra Helena Poliselli (President)
Andrade, Sérgio Faloni de
Martins, Joilson de Oliveira
Title in Portuguese
Caracterização das ações do extrato da inflorescência da Achyrocline satureoides sobre a função de neutrófilos na inflamação
Keywords in Portuguese
Bolsa de ar
LPS
Marcela
Microscopia intravital
Moléculas de adesão
Neutrófilos
receptor toll-like-4
Toxicidade
Abstract in Portuguese
Achyrocline satureoides (Lam) D.C., popularmente conhecida como "marcela", é utilizada popularmente para tratar diversas doenças, como mal estar gástrico e intestinal, inflamações, diabetes e outros. Como os mecanismos de ação do extrato de A. satureoides ainda não são conhecidos, o presente trabalho visou esclarecer os mecanismos antiinflamatórios do extrato bruto hidroalcoólico das inflorescências de A. satureoides, focando na migração e função fagocítica e microbicida de neutrófilos. Para tanto, o extrato hidroalcoólico foi administrado por gavage (50, 100 e 250mg/kg) em ratos Wistar, machos, adultos, e a inflamação foi induzida pela injeção do lipopolisacarídeo de E. coli (LPS; 2mL de solução; 500 µg/mL em PBS) no tecido subcutâneo dorsal (modelo da bolsa de ar). Animais controles receberam volumes equivalentes de veículo do extrato e indometacina (30mg/kg). Foram quantificados o número de neutrófilos (câmara de Neubauer e esfregaços corados por Panótico) e a concentração de Leucotrieno B4 e CINC-1 (ELISA) no foco de lesão; a interação leucócito-endotélio em vênulas da microcirculação mesentérica após estímulo in situ pelo lipopolisacarídeo de E.coli (LPS; 30µg/40µL; por microscopia intravital); a expressão de moléculas de adesão e do toll-like receptor (TLR-4), além da quantificação do burst oxidativo (induzido pelo miristato-acetato de forbol - PMA) e fagocitose em neutrófilos circulantes (citometria de fluxo); análise histológica e quantificação dos marcadores hepáticos (AST, ALT e Gama-GT) e renais (uréia e creatinina) no plasma por espectrofotometria. Os resultados obtidos mostraram que o tratamento com o extrato reduziu o número de neutrófilos e a concentração de LTB4 e CINC-1 na bolsa de ar; reduziu a expressão de TLR4 pelos neutrófilos circulantes e a porcentagem de neutrófilos positivos para L-selectina e β2-integrina; inibiu a adesão e o comportamento rolling de leucócitos ao endotélio microvascular; reduziu o burst induzido por PMA; aumentou o potencial de fagocitose, sem alterar o burst induzido por Staphylococcus aureus, não alterou a morfologia tecidual e a concentração sérica dos marcadores de atividade hepática e renal. Em conjunto, os dados obtidos mostram que dose, aparentemente, não tóxica do extrato de A. satureoides exerce efeito antiinflamatório in vivo frente ao LPS, quantificados pela redução da migração e pelas interferências nas atividades fagocítica e microbicida dos neutrófilos.
Title in English
Characterization of Achyrocline satureoides inflorescence extract actions on the neutrophils role in inflammation
Keywords in English
Adhesion molecules
Air pouch
Intravital microscopy
LPS
Marcela
Neutrophils
Toll-like receptor 4
Toxicity
Abstract in English
Achyrocline satureoides (Lam) D.C., popularly known as "marcela", is popularly used to treat several diseases, such as gastric and intestinal disorders, inflammation, diabetes and others. As the mechanisms of the extract of A. satureoides have not been elucidated, this study aimed to investigate the anti-inflammatory mechanisms of the crude hydroalcoholic extract of the flowers of A. satureoides, focusing on migration and phagocytic and microbicidal neutrophils function. The hydroalcoholic extract was administered by gavage (50, 100 and 250mg/kg) into male Wistar rats, adult, and inflammation was induced by injection of lipopolysaccharide E. coli (LPS; 2 mL of solution; 500µg/mL in PBS) into the dorsal subcutaneous tissue (air pouch model). Control animals received equivalent volume of extract vehicle or indomethacin solution (30mg/kg). It was quantified the numbers of neutrophils (Neubauer chamber and stained smears by Panoptic) and concentrations of leukotriene B4 (LTB4) and CINC-1 (ELISA) in the focus of the injury; the leukocyte-endothelium interactions in mesenteric venules of the microcirculation after stimulation by LPS (30µg/40µL; intravital microscopy); the adhesion molecules and toll-like receptor (TLR-4) expressions and quantification of oxidative burst and phagocytosis in circulating neutrophils (flow cytometry); histological analysis and the hepatic markers (AST, ALT and gamma-GT) and kidney (urea and creatinine) concentrations in plasma by spectrophotometry. Data obtained showed that the treatment reduced the numbers of neutrophils and concentrations of LTB4 and CINC-1 in the subcutaneous tissue; reduced the TLR4 expression by circulating neutrophils and the number of β2-integrin- and L-selectin-positive neutrophils; inhibited the leukocyte adhesion and the rolling behavior to vascular endothelium; reduced the burst evoked by PMA; increased the phagocytosis without changing the burst induced by Staphylococcus aureus; and did not alter the tissue morphology and concentration of hepatic and renal enzymes in the serum. Together, these data suggest that dose, apparently non-toxic, of extract of A. satureoides exerts anti-inflammatory effect in vivo, quantified by the reduced migration and by interference in the phagocytic and microbicidal activities of neutrophils.
 
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Publishing Date
2013-11-18
 
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