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Intestinal epithelial cells play an important role in the mucosal inflammatory response. These cells synthesize and secrete inflammatory mediators, and selectively modulate the permeability of the epithelial monolayer thus exposing immune cells to antigens. Although intestinal inflammatory response is crucial to maintain gut structural integrity and function, alteration and dysregulation of inflammatory pathways contribute to tissue damage and ulceration, and are thought to be pivotal factors in the pathogenesis of different inflammatory gut diseases [1]. The limited efficacy of conventional pharmacological therapy in the intestinal inflammatory conditions has fostered research on alternatives and, at the same time, stresses the importance of prevention. In this context, the influence of dietary components, becoming in a physiological close proximity to intestinal cells and then to inflammatory processes within the intestinal mucosa, appears of nutritional and clinical interest [2]. Among natural preventive and complementary approaches to improve inflammatory symptoms, dietary polyphenols represent potential candidates and proanthocyanidins are particularly interesting [3]. For their relatively high concentration in a number of edible plants and their high digestive stability and limited intestinal adsorption [4, 5], proanthocyanidins reach the colon at relatively high concentrations and may have direct effects on the intestinal mucosa through their interaction with the intestinal epithelial cell membranes [6, 7]. The edible pistachio nut has been ranked among the first 50 food products highest in antioxidant potential [8]. A number of data show that the pistachio nut consumption has positive effects in human serum lipid profile and cardiovascular disease (CVD) risk factors [9,10] and significantly improves oxidative status and reduces circulating inflammatory biomarkers [11]. Our previous research provided evidence that a hydrophilic extract from Sicilian pistachio nuts (HPE) contains substantial amounts of polyphenols, including proanthocyanidins, and possesses radical scavenging and antioxidative properties in in vitro models of lipid oxidation [12]. Moreover we also demonstrated that HPE has anti-inflammatory activities in lipopolysaccharide (LPS)-activated macrophages interfering with the NF-kB activation, and that the high molecular weight proanthocyanidin fraction (PF) can play a major role as the bioactive component of HPE [13]. In the present study we investigated the activity of HPE, and of its polymeric proanthocyanidin fraction as well, in an in vitro model of intestinal inflammation, consisting of Caco-2 cells differentiated into epithelial intestinal cells and exposed to the inflammatory actions of interleukin (IL)-1. Our results clearly show that HPE effectively inhibits the inflammatory response in intestinal epithelial cells, and that highly polymeric proanthocyanidin components exhibit qualitative and quantitative effects substantially comparable to those of whole extracts when tested at the same concentration found in the extracts. The protective effects are expressed through a marked decrease in release and expression of inflammatory mediators and occur in parallel with a reduced activation of the nuclear factor-B. Moreover, our results clearly show that HPE can partially prevent the IL-1-induced gap formation with perturbation of the monolayer integrity, as shown by a limited IL-1-induced increase of paracellular permeability. Finally we provide evidence that HPE treatment increases transepithelial electrical resistence of Caco-2 cells monolayer, demonstrating that protective effects of HPE under our conditions occur in parallel with molecular interaction of nut components with the epithelial cells membranes. To assess the physiological relevance of the tested concentration it is worth noting that a single serving of pistachio nut (28.34 g) [USDA National Nutrient Database for Standard Reference] contains around 62,34 mg polymeric proanthocyanidins (cyanidin equivalents). Once diluted in a gastrointestinal volume of 600 mL, this results in a 3,5 M concentration (104 g/ml) (as cyanidin equivalents) of polymeric proanthocyanidins which represent a plausible concentration in the human gut [14]. This concentration is one order higher than the concentrations selected in our cell model and this suggests that our results might be physiologically relevant in the gastrointestinal tract. Data here presented may further remark the potentially beneficial health effects that may arise by daily intake of small quantity of pistachio nut. Widely available, inexpensive and frequently consumed, this nut for its favorable fatty acid profile and high content in bioactive antioxidant compounds, positively influences the plasma lipid parameters and oxidative status, and elicits antinflammatory properties. In this respect, for its high content in large proanthocyanidins, consumption of pistachio nut may exert locally significant beneficial effects to physiology of gastrointestinal tract.

EPA (acido eicosapentaenoico) e DHA (acido decosaesaeoico) sono derivati metabolici degli AGE (acidi grassi essenziali) e in particolare dell'acido linolenico (omega-3)

BETANIN INHIBITS MYELOPEROXIDASE/NITRITE-MEDIATED PEROXIDATION OF HUMAN LOW-DENSITY LIPOPROTEIN M. Allegra, L. Tesoriere, D. Butera, A.M. Pintaudi, M.A. Livrea Dipartimento Farmacochimico Tossicologico e Biologico - Facoltà di Farmacia - Università di Palermo - Via C. Forlanini 1 - 90134 Palermo INTRODUCTION: Betanin, the betalain red pigment occurring in the Cariophillalae order plants, including cactus pear, has recently been reported to posses reducing properties and to behave as lipoperoxyl radical-scavenger in vitro (1). In addition, this phytochemical is bioavailable, accumulates in human LDL after ingestion of cactus pear fruits, and is able to protect LDL against copper-induced oxidation in vitro (2,3). Myeloperoxidase (MPO) has been implicated in the in vivo LDL modification and atherogenesis (4). The enzyme, in the presence of nitrite, generates two powerful oxidizing agents, the tyrosyl radical and the nitrosyl one, both of which promote LDL lipid oxidation (4). Taking all this into account we have decided to investigate weather betanin could counteract MPO/nitrite-induced oxidation of LDL. MATERIALS AND METHODS: Human MPO was purchased from Calbiochem and Glucose oxidase from Sigma. All other chemical and solvents were purchased from Sigma Aldrich or Merck. Preparation of LDL. LDL was prepared from blood serum of healthy volunteers according to Kleinveld et al. (5) with minor modification and stored in the presence of 4mM EDTA at -80°C. Lipid peroxidation of LDL. Reactions were carried out according to Kostyuk et al. (6). RESULTS: Our results, indicate that betanin is able to inhibit the MPO/nitrite-induced LDL lipid peroxidation, in a dose-dependent manner in the range 1 to 10 μM (Fig.1). We have compared the effectiveness of betanin with that of two well-known physiological antioxidants: α-tocopherol and ascorbic acid. As reported (4), α-tocopherol, the most powerful radical-scavenger, is only able to partially protect LDL lipids from oxidation by the MPO/nitrite system and has scarce or no effect on the powerful hydrophilic oxidants generated by nitrite. On the contrary, vitamin C, which is able to scavenge the peroxidase-generated nitrating species, was very efficient in counteracting the MPO/nitrite-sustained lipid peroxidation. Betanin, was even more effective than vitamin C, at inhibiting the oxidative damage to LDL. The IC50 calculated for betanin (1.4 μM) was more than 10-fold lower than that for ascorbic acid (15.6 μM). Nitrite, an oxidation product of nitric oxide metabolism, and MPO are considered mediators of the LDL oxidation process in vivo. Our study shows that betanin, a phytochemical occurring in the cactus pear fruit, is able to protect LDL, in an experimental set-up of physiological relevance. In addition, the molecule acts at micromolar concentrations, and appears much more effective than ascorbic acid. Our data collectively indicate a favourable modulation of the oxidation process of LDL and may contribute to the supposed beneficial effect of cactus fruit (2). 1. Lievrea M.A. et al. (2003) in Herbal Medicines, Marcel Dekker, Inc. 537-556. 2. Tesoriere L. et al. The American Journal of Clinical Nutrition (in press). 3. Tesoriere L. et al. (2003). Free Radical Research 37, 689-696. 4. Carr A.C. et al. (2000). Arterioscler Thromb Vasc Biol. 1716-1723. 5. Kleinveld, H.A. et al. (1992). Clin. Chem. 38, 2066-2072. 6. Kostyuk V.A. et al. (2003) FEBS Letters 537:146-50. 7. Kleinveld H.A. et al. (1992). Clinical Chemistry, 38. 2006-2072. 8. Kostyuk V.A. et al. (2003). FEBS Letters, 146-153.

Inflammation is a complex physiological response that requires the activity of a sophisticated regulatory network involving the activation of specific genes for defense, tissue repair and remodeling. Although transcriptional activation has been shown to be critical in the regulation of inflammatory genes (1) the role of epigenetic phenomena in the modulation of the inflammatory response is now emerging (2). Specifically, it has been recently reported that proinflammatory stimuli induce DNA demethylation in the interleukin IL-1 promoter of human articular chondrocytes (3). IL-1 cytokine, among several proinflammatory agents, represents an essential player in the inflammatory conditions of the gut (4): functioning as the strongest signal transduction to NF-kB, IL-1 increases in intestinal paracellular permeability and over-expression of proinflammatory genes (5). In this tissue, moreover, inflammatory response is crucial to maintain its structural integrity and function, thus, alteration and deregulation of inflammatory pathways contribute to tissue damage and ulceration, and are pivotal factors in the pathogenesis of several inflammatory gut diseases. In the present study we evaluate both wide-ranging and gene-specific epigenetic changes in the inflammatory response of Caco-2 cells differentiated into intestinal epithelial cells and exposed to the inflammatory actions of IL-1β. Our results clearly show that IL-1induces changes in the DNA methylation either at genome and gene level and that the local methylation changes are induced in two pro-inflammatory genes that are IL-1-regulated. In particular, we show that a cell exposure to IL-1β for 24 h induces, in a dose-dependent manner, hypomethylation of genomic DNA in respect to untreated cells. We also observe a reduced DNA methyltransferases activity of cell lysates obtained from IL-1 treated cells. Finally our data show that IL-1 is able to induce hypomethylation of specific CpG sites in IL-6 and IL-8 genes. These preliminary results suggest that IL-1 in intestinal epithelial cells is able to act as an epigenetic modulator towards the entire genome and specific genes. Modulation of epigenetic changes in inflammation may provide a new “reading frame” of the inflammatory diseases molecular basis. Since epigenetic modifications are potentially reversible, a thorough understanding of these changes during inflammatory response opens opportunities to develop efficient agents for specific targets.

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Nucleoside phosphotransferase purified from chicken intestinal mucosa is a multisubunit protein of high molecular weight regulated by many nucleotides. The adsorption of a nucleotide effector to regulatory sites favors the conversion of the enzyme to a stable form and induces a modification of the substrate site, increasing its affinity for substrate donors. We studied the effects exerted by orthophosphate on the reaction and on the stability of the enzyme. P(i) inhibits competitively nucleoside phosphotransferase activity, whichever nucleoside monophosphate is employed as donor. Nucleotide effector (d-TDP) removes entirely the inhibitory effect exerted by different concentrations of P(i). The sigmoidal kinetic response to activation by d-TDP is increased by 100 μM P(i), which raises the interaction coefficient from 2.25 to 3.54. P(i), like nucleotide effectors, stabilizes nucleoside phosphotransferase against heat and enzyme dilution. Although we do not exclude the possibility that P(i) at higher concentrations can also compete effectively with phosphate donors at the catalytic site, we were inclined to think that the findings are compatible with competition between phosphate and nucleotides at regulatory sites.