New Insight and Knowledge on anti-inflammatory Effectiveness of dietary phenolics (NIKE)

Ellagitannins (ETs), a polyphenolic subclass mainly found in berries, walnuts, and pomegranate, have shown antioxidant, anti-inflammatory and anticancer effects in several in vitro studies. However, these results have not been confirmed in vivo. The gap between in vitro and in vivo studies could depend on the direct exposure of cultured cells to whole plant extracts or to single parent plant phenolic at rather high non-physiological doses. On the contrary, in vivo human internal compartments are exposed to metabolites formed and absorbed after gastrointestinal transit, digestion and metabolic conjugation [1]. NIKE aims to fill the gap between in vitro and in vivo results evaluating the anti-inflammatory effects of ETs with a new comprehensive, high-throughput approach (Figure 1). This approach could be used to study not only ETs, but many different bioactive molecules. Differently from common translational science approach, the dietary intervention will be conducted at the beginning of the project; the rationale of NIKE approach is based on the need to identify the ET-derived metabolites (ETMs), and their concentration in the bloodstream, after consumption of the ET-rich food. The first step will be the assessment of the anti-inflammatory effect of an ET-rich food (a pomegranate juice) in a randomised, placebo-controlled study on volunteers suffering of inflammatory bowel disease (IBD) in clinical remission with high risk of clinical flare. Pomegranate juice will be used as vehicle because its great richness in ETs. The level of faecal calprotectin will be used as surrogate outcome to assess the intestinal inflammation [2]. Presence and concentration of ETMs in plasma and urine samples will be also verified as biomarker of the dietary intake. The gastroenterologists Dr. Luigi Ricciardiello and Dr. Andrea Belluzzi will carry out the patient recruitment at the Gastroenterology Unit, St. Orsola-Malpighi Hospital (Bologna, Italy). As second step, NIKE will unravel the mechanism of anti-inflammatory action of ETs in in vitro studies. Two cell model systems will be used in this part of the project: intestinal cells and blood monocytes, since both cell types are interacting during the intestinal inflammatory response [3]. The tested concentrations will be realistic and representative of those physiologically reached after the intake of dietary quantities of ETs. Particularly: 1. Human colorectal adenocarcinoma cells (Caco-2) will be used to assess the anti-inflammatory effect of ETMs at gut level. The expression of selected genes and proteins related to IBD will be verified with both real-time PCR and western blot analyses. 2. Primary human monocytes will be used to clarify the effects of ETMs on the macrophage polarisation process [4]. To understand the molecular pathways involved, the effects will be evaluated using a microarray technology. The analysis will be focused principally on the expression of inflammatory mediators, which have been identified as involved in IBD. In the third step of the NIKE project, data obtained in Caco-2 cells will be verified ex-vivo in the intestinal biopsies obtained from volunteers recruited in the intervention study. As well, data obtained in monocytes will be compared with cytokines and chemokines level in the blood of volunteers. The correlation among in vitro, ex-vivo and in vivo data will validate the NIKE circular approach. The integral analysis of all results obtained in the project will elucidate the role of ET-rich foods in the secondary prevention of IBD, deepening the existing knowledge on their mechanism/s of action at the molecular, metabolic and genomic levels. References. 1) Espín et al, Evid Based Complement Alternat Med 2013; 2013:270418. 2) Konikoff & Denson, Inflamm Bowel Dis 2006; 12:524-34. 3) Wallace et al, World J Gastroenterol 2014; 20:6-21. 4) Derlindati et al, Food Funct 2012; 3:1144-52.