Isolated Polyphenols and Farnesol, Stable in Culture Medium, Function Synergistically in a Hormesial Manner to Modulate LPS-stimulated Raw 264.7 Macrophage Polarization toward the M0 or M2 State under Multiple Paradigms

摘要

Consumption of polyphenol-rich foods, such as dry edible beans, is correlated with positive health outcomes. However, when used in isolation, the health benefits in a food matrix, e.g. from whole foods, that is, not a supplement, either disappear or require substantially more of the compound than can be provided in the diet. Hence, we propose that using purified polyphenols in combination may elicit a synergistic response in the context of altering macrophage polarization when challenged with LPS. After starting with 14 polyphenols, 7 were eliminated as these were not stable in the culture medium and are therefore impossible to ascertain if alterations in cell behavior are due to the phenol or decomposition products. The stable phenols were taken up and effluxed by RAW 264.7 macrophage cells, though the M1 polarized cells trafficked phenols slower than the M0 cells. We had previously determined that farnesol is a potent signaling molecule in macrophages and added it to the remaining 7 polyphenols for screening the ability of the combinations to lower the biomarkers NO and IL-1beta for 28 combinations. Of these, 4 were deemed most effective for further examination, p-coumaric acid:farnesol, astragalin:vanillic acid, 4-vinylguaiacol:vanillic acid, and vanillin:farnesol. These combinations were able to shift macrophage gene expression, especially raising COX2, CARKL, and PPARgamma mRNA while lowering Arg2. The combinations also restored the compromised citric acid cycle that is critical for M1 polarization. Importantly, the most effective concentration was generally 10 nM for both altering gene expression and the organic acid profile. Synergy surface response plot showed that the combined compounds work synergistically at lower concentrations, e.g. below 1000 nM, but become antagonistic above 1000 nM. This hormesis dose response is apparent as the combinations acted synergistically at lower concentrations and became antagonistic at higher concentrations. The significance of this project is the establishment that polyphenols stable in culture medium and/or with farnesol, but not as single compounds, function synergistically in the nanomolar range with a hormesial dose response to protect from, compete with, and treat pro-inflammatory M1 RAW 264.7 macrophage polarization induction by lipopolysaccharide at multiple levels, from gene expression to metabolic changes.