In vitro approach to evaluate the role of gut phenolic metabolites in the modulation of inflammation and atherosclerosis

BACKGROUND: Inflammation is a common process in endothelial dysfunction, characterized by an increase in the expression of endothelial cell adhesion molecules and a decrease of nitric oxide production [1]. Evidence suggests that polyphenols may play a beneficial role in attenuating inflammation with implications on atherosclerosis [2]. However, their mechanism of action is still not entirely understood due to their poor absorption and extensive intestinal and hepatic transformation [3]. OBJECTIVE: This study aims to evaluate the ability of some gut phenolic metabolites (i.e. protocatechuic, gallic, syringic and vanillic acid; PA, GA, SA, VA, respectively) to reduce the adhesion of monocytes (THP-1) to endothelial cells (HUVECs), in a stimulated pro-inflammatory environment, and to decrease the production of cell adhesion molecules (VCAM-1 and E-selectin), as potential markers involved in such modulation. METHODS: Adhesion of THP-1 to HUVECs: Day 1-Preparation of 96 wells plate (2x104 HUVEC per well); Day 2-Labelling of THP-1 cells with CellTrackerTM Green CMFDA, addition of THP-1 (2x105 cells/well) and TNF- (100 ng mL-1) to HUVEC, and incubation for 24h; Day 3-Incubation with PA, GA, VA and SA at different concentrations (from 0.01 till 10μg mL-1) for 24 h; Day 4- Reading of the fluorescence (excitation: 485 nm, emission: 538 nm, mod. F200 Infinite, TECAN Milan, Italy) Evaluation of cell adhesion molecules: The levels of vascular cell adhesion molecules (VCAM-1), and endothelial selectin (E-selectin) were measured in the supernatant by ELISA kits following the manufacture’s instruction. The results derived from three indipendent experiments. RESULTS: GA and PA significantly decreased the adhesion of THP-1 to HUVECs at 1 µg mL-1 (-18.0%; p=0.04 for GA, -29.5%; p=0.03 for PA) and 10 µg mL-1 (-59.3%; p<0.001 for GA, -44.3%; p=0.001 for PrA) compared to TNF-. VA significantly reduced the adhesion only at the maximum concentration (-20.8%; p<0.005). No effect was observed after SA supplementation. CONCLUSION: In conclusion, these preliminary results seem to support the capacity of gut phenolic metabolites to modulate the endothelial cell adhesion process. The reduction in the adhesion process seems to be driven by a decrease of E-selectin levels but not VCAM-1. Ongoing experiments are attempting to clarify the mechanisms of action of each compound involved in the above observations.