Caracterização do potencial anti-inflamatório do triterpeno natural uvaol em macrófagos in vitro

Abstract

Uvaol, urs-12-ene-3,28-diol, is a pentacyclic triterpene found in abundance in olive oil and has recognized with antioxidant properties. Recent studies have reported the inhibitory effects of uvaol on the immunoinflammatory response. Macrophages, particularly, are shown to be crucial cells both for the establishment/propagation and for the inflammatory resolution reactions. However, the actions of this triterpene on the macrophage’s functions have not been investigated until this date. Thus, in this study, we aimed to characterize the anti- inflammatory activities of uvaol in macrophages activated in vitro. Initially, the effect of uvaol on J774 macrophages viability was evaluated by the MTT method. Then, we found that in macrophages stimulated with LPS, uvaol in concentrations unable to induce cytotoxic effects, significantly reduced the secretion of TNF-α, IL-1β, and IL-6, without interfering with the secretion of IL-10. To check whether these effects of uvaol also extended to human cells, we used macrophages derived from human monocytes of the THP-1 cell line. With these cells, we found the same cytotoxicity profile, as well as a similar inhibition profile in the production of mediators, since treatment with uvaol also inhibited the production of TNF-α, IL-1β, IL-6, MCP-1 and IL-8 in cells stimulated with LPS. In J774 macrophages stimulated with LPS, uvaol also inhibited the generation of nitric oxide. When evaluating the its phagocytosis, it was found that treatment with uvaol significantly induced an increase in the percentage of cells that phagocytosed, as well as in the number of particles phagocytosed by cells, clearly demonstrating an improvement in phagocytosis ability. Considering that the pro- inflammatory characteristics of macrophages were inhibited by treatment with uvaol, we sought to quantify the expression of the surface marker CD86, a typical molecule of macrophages of the M1 profile (pro-inflammatory). The results revealed that macrophages treated with uvaol exhibited a significant reduction in the expression of the CD86 marker. Additionally, secretion of the typical cytokine for the M1 profile, IL-6, as well as for the M2 profile, IL-10, in macrophages only submitted to functional differentiation was significantly inhibited by treatment with uvaol. Furthermore, when evaluating the mRNA expression of typical genes for the M1 profile, such as TNF-α and IL-6, and the M2 profile, such as Arg1 and TGF-β, we found that uvaol significantly inhibited the expression of the TNF-α, IL-6 and Arg1 genes, except for the expression of the TGF-β gene. When evaluating the activation of the intracellular signaling pathway of NF-kB, it was found that treatment with uvaol inhibited the translocation of p65-NF-kB, which may justify the anti-inflammatory effects induced by treatment with uvaol in macrophages. Together, these results suggested that uvaol exerts an anti-inflammatory effect on macrophages, decreasing cytokine secretion and increasing phagocytic activity, these phenomena seem to be related to attenuation in the polarization of M1 macrophages and reduction in nuclear translocation of NF-kB, reinforcing its use as a beneficial health agent in inflammatory conditions.