EFEITO DO EXTRATO DE Dysphania ambrosioides (L) NA RESPOSTA DE NEUTRÓFILOS HUMANOS.

Abstract

Neutrophils present rapid mechanisms of immune response against pathogens phagocytosis, as and release of potent preformed or not inflammatory mediators. However, exacerbated activation of these cells has been related to worsening of some inflammatory diseases and harmful effects in chronic infections. In this context, regulation of these mechanisms is necessary. Natural products are an interesting possibility, and the plant Dysphania ambrosioides (mastruz) already has several biological activities demonstrated, for example immunomodulation. In this work, we evaluated in vitro effect of crude hydroalcoholic extract of Dysphania ambrosioides (HEDa) on human neutrophils. Peripheral blood neutrophils from healthy donors were treated with HEDa at 5, 50 or 125 μg / mL for one, two or four hours. After treatment by one or four, cells were incubated with Candida albicans-GFP (Green Flourescent Protein) yeasts at a ratio of 1: 2 (cells: yeast) for 30 minutes, and phagocytosis was analyzed by flow cytometry. The HEDa did not interfere in phagocytic activity of neutrophils, regardless of the concentrations and time of treatment. For degranulation assay, neutrophils were treated for one to two hours and labeled with Anti-CD63 (primary granules), Anti-CD66b and Anti-CD15 (secondary granules) and Anti-CD14 (secretory vesicle) antibodies. HEDa stimulated migration of secretory vesicle and secondary granules in a dose dependent manner, in both evaluated times. However, in the one-hour period, only the highest concentration (125 μg / mL) induced exocytosis, and in two hours, this effect was observed in 50 and 125 μg / mL. For oxidative burst, neutrophils treated with HEDa were subsequently stimulated or not with phorbol-12- myristate-13-acetate (PMA); production of reactive oxygen species (ROS) was quantified by luminol-dependent chemiluminescence and lucigenina assay. As a result, the treatment stimulated oxidative burst in a similar way in both times. When neutrophils received a posterior stimulus with PMA, cells treated with HEDa had a reduction in ROS generation, and at 125 μg / mL this reduction was more expressive. Neutrophil Extracellular Traps (NETs) release was evaluated quantitatively (Sytox® Orange fluorescence assay) and qualitative (fluorescence microscopy). HEDa treatment showed a tendency to stimulate NETs, however, it was not significant. When neutrophils were treated with EHDa and stimulated by PMA, the treatment showed potential to regulate this mechanism, similar to oxidative burst results. Treatment with hydroalcoholic extract of D. ambrosioides stimulated the main effector mechanisms of human neutrophils without maintaining this activation at potential deleterious levels to the organism, and presented a modulatory effect in a PMA over-stimulated cells. These results reinforce the perspective of use of mastruz as a therapy, applied in infectious and / or inflammatory diseases.