Crossing and metabolism of tyrosol and hydroxytyrosol by implementing an in vitro blood-brain barrier model of human primary cells

Abstract

There is mounting evidence that the neuroprotective activities associated with olive oil consumption are related to the presence of the phenolic alcohols tyrosol (Tyr) and hydroxytyrosol (HT). In vitro blood-brain barrier (BBB) models are considered as indispensable platforms for the mechanistic assessment of compound permeability. However, it is important to note that most of these models offer only a limited representation of BBB physiology. The aim of the present study was to develop a human tri-culture (human brain microvascular endothelial cells (HBMECs), astrocytes and pericytes) BBB model to evaluate the permeability of dietary bioactives. In particular, the crossing of Tyr and HT through the BBB and the BBB’s potential to further metabolize these bioactives was evaluated. The set up of the model was focused on HBMECs seeding density and extracellular matrix composition, revealing that 1 × 10⁵ cells and fibronectin coating of the apical transwell surface yielded higher TEER values and improved barrier integrity. Immunocytochemical analysis further confirmed well-defined ZO-1 localisation at cell–cell junctions. After 96h of the establishment of the triculture, the human origin-BBB (ho-BBB) model presented the optimal barrier conditions for permeability studies execution. The transport across the ho-BBB of Tyr and HT (1 and 10 µM) was evaluated by UPLC-MS/MS. Our results prove that the ho-BBB is more permeable to HT (high permeability) than Tyr (medium permeability) determined by calculating transport percentages and apparent permeability coefficients (Papp). This study provides the first evidence that HBMEC cells can metabolize HT, transforming it into HT-3ʹ-sulfate and HT-4ʹ-sulfate.