Modulatory effect of EVOO polyphenols and their metabolites on inflammatory response in Adenocarcinoma Colon Cells (Caco-2) and Human Umbilical Vein EC (HUVEC)

Mediterranean diet is considered a model of healthy nutrition, able to prevent and/or slow down the progression of the most common degenerative diseases, such as Cardiovascular Disease (CVD) and Inflammatory Bowel Disease (IBD), characterized by a chronic inflammation state that alters structure and functionality of the endothelial and intestinal barrier. EVOO(EVOO), the main lipid source in the Mediterranean diet, is rich in bioactive compounds mainly hydroxytyrosol (HT) and tyrosol (Tyr), that have been shown to possess antimicrobial, antioxidant and anti-inflammatory properties. However, it is well known that free forms of phenolic compounds ingested with food are largely metabolized in the intestine and liver and their concentrations after ingestion are too low to explain the biological effects observed in vivo and in vitro models. In particular, HT and Tyr undergo glucuronidation and sulfonation, and their sulphate and glucuronide metabolites are the prevalent forms found in human plasma and urine where they reach relevant concentrations compatible with biological activity. In this context, the aim of the present research project was to evaluate the protective effect of the main phenolic compounds of EVOO, HT and Tyr, and their sulphate and glucuronide metabolites, on the inflammatory response at intestinal level (in Caco-2 cell monolayer) and endothelial level (in HUVEC cell monolayer), using pro-inflammatory stimuli, such as LPS and/or a hyperglycemia (HG) condition. Their modulatory action was evaluated focusing on the alteration of tight junctions (TJ) and the activation of cellular pathways, as mitogen-activated protein kinases (MAPK) and NLRP3 inflammasome, which are linked to chronic inflammatory diseases. The alteration of epithelial/endothelial barrier in Caco-2 and HUVEC monolayers, treated with LPS or HG alone or together with EVOO phenolic compounds and their sulphate and glucuronide metabolites, was evaluated through cell permeability tests (TEER, FITC-Dextran permeability assay) and the determination of the disruption and/or relocation of TJ proteins, in relation to redox-sensitive MAPK modulation and activation of the NLRP3 inflammasome. Obtained data showed that HG and physiopatologically relevant concentration of LPS increase permeability in both Caco-2 and HUVEC monolayers, through the alteration of TJ proteins, occludin, zonulin and JAM-A (Junctional adhesion molecule A), following the activation of pathways involved in the inflammatory process such as p38 and ERK1/2 MAPK and NLRP3 inflammasome. It was also observed that pretreatment with physiologically concentration of HT, Tyr and their sulphated and glucuronidated derivatives induce a protective effect, limiting the alteration of TJ and the activation of MAPK and NLRP3 inflammasome, strengthening the hypothesis that HT and Tyr, as well as their metabolites, may exert a significant role in the maintenance of intestinal and endothelial barrier integrity. Finally, it was observed that HT and Tyr metabolites and their parent compounds exert a comparable effect both in intestinal and EC, by modulating key intracellular signaling pathways involved in the cellular pro-inflammatory response.