Atividade imunomoduladora da própolis vermelha de Alagoas em macrófagos murinos ativados por lipopolissacarídeo in vitro

Abstract

Red propolis (RP) is a natural resin, produced by Apis mellifera bees from plant exudate (Dalbergia ecastophyllum), which has several biological properties, including antiinflammatory activity. Inflammation is the body's response mechanism against various injuries; however, this exacerbated process can trigger an inflammatory disease. Thus, the search for new natural immunomodulatory agents is necessary for the development of new therapeutic approaches that modulate the inflammatory process. In this context, the objective of this work was to evaluate the immunomodulatory potential of Alagoas red propolis (ARP) and its constituents isoliquiritigenin (ISL) and formononetin (FMT) in murine macrophages stimulated with lipopolysaccharide (LPS) in vitro. Initially, the maximum non-toxic concentration (CMNT) of the hydroalcoholic extract (HE) of PVA, ISL, and FMT in murine macrophages of the J774A.1 cell line was determined, based on the analysis of cell viability by the MTT assay. Then, the EH immunomodulatory activities of ARP, ISL, or FMT were evaluated by treating LPS-stimulated macrophages under different experimental conditions. The expression of markers F4/80 and CD86 was evaluated by multiparametric flow cytometry and the quantification of pro and antiinflammatory cytokines was evaluated by the Cytometric Bead Array (CBA) methodology in the cell culture supernatant. Activation of MAPKs (mitogen-activated protein kinases) was evaluated by analyzing the phosphorylation of ERK1/2 and p38 proteins by intracellular flow cytometry. The expression of NOS2, Arg1, SOCS3, TGF-β, Lamp2, MIP-1α, Ccne1, and HO-1 genes was evaluated by RT-qPCR. It was observed that treatment with EH of PVA reduced the percentage of double-positive cells (F4/80+ CD86+), decreased the expression of the activating molecule CD86 and the levels of proinflammatory cytokines IL-6 and TNF. Furthermore, an increase in ERK1/2 and p38 phosphorylation and a decrease of NOS2 and SOCS3 expression were detected 48h after treatment. Treatment with ISL significantly reduced the frequency of F4/80+ CD86+ cells, decreased CD86 expression and phosphorylation of ERK1/2 and p38 proteins. IL6 and TNF levels were also reduced after ISL treatment. Furthermore, there was a significant decrease in the expression of NOS2 and SOCS3 genes. On the other hand, FMT treatment did not reduce the percentage of double-positive cells (F4/80+ CD86+) and did not alter the phosphorylation of the ERK1/2 and p38 pathways in LPS-stimulated murine macrophages. However, a reduction in IL-6 and TNF levels was observed in the supernatant of macrophages treated with FMT.In conclusion, in this study, it was detected a promising immunomodulatory activity of ARP and the ISL compound on murine macrophages in vitro. These results contribute to a better understanding of the immunomodulatory activity of ARP and its constituents, favoring the development of new therapies and immunological interventions for the treatment of inflammatory diseases.