Effetto pro-ossidante e pro-infiammatorio degli ossisteroli in cellule intestinali: attività modulatoria della frazione fenolica del vino

Because the intestine sits at the interface between the organism and its luminal environment, it represents a critical defense barrier against luminal toxic agents. Thus, in addition to being exposed to luminal nutrients, the intestinal mucosa is constantly challenged by diet-derived oxidants, mutagens, and carcinogens as well as by endogenously generated reactive oxygen species. Among the oxidizing agents cholesterol oxidation products, oxysterols, are particularly relevant; besides being generated endogenously, they may be introduced with the diet. Oxysterols are involved in physiological processes, but they also exert several detrimental effects including cytotoxicity, induction of cell death, carcinogenicity and pro-oxidative and pro-inflammatory activities. These compounds have recently been suggested to be associated with the pathogenesis of inflammatory bowel diseases (IBD), and with a high risk of developing colorectal cancer. There is an increasing interest in the mechanisms of response of the intestinal epithelium to oxidative stress and in the capability of nutritional antioxidants to strengthen endogenous antioxidant defenses. Among dietary-derived antioxidants, phenolic compounds have been described to have a wide range of biological activities and many reports have illustrated their promising role as preventive tools in several acute and chronic disorders. Phenolic compounds are absorbed and extensively metabolized in vivo: however, the percentage of absorption does not exceed a few percent of the ingested dose. It is assumed that dietary polyphenols may display their first antioxidant defense in the digestive tract, where they concentrate, both through their “conventional“ antioxidant capacity, limiting ROS formation and scavenging them, and by an “indirect” antioxidant effect, inducing endogenous protective enzymes and/or regulating cellular process such as inflammation. Wine accounts for a very high proportion of phenolic compounds intake in the Mediterranean diet; typically a glass of red wine contains about 100 mg of compounds generally called polyphenols. We investigated the ability of two wine phenolic extracts, obtained from grape varieties grown in Sardinia, Cannonau (red) and Vermentino (white), to exert a protective action against the pro-oxidative and pro-inflammatory effect of oxysterols in Caco-2 cells, a cell line with enterocyte-like features, as a model system that mimics the insult of dietary oxidized lipids on the intestinal mucosa. Oxidative damage was induced by treating cells with a dietary-representative oxysterols mixture (7-ketocholesterol, 5α,6α-epoxycholesterol, 5β,6β-epoxycholesterol, 7α-hydroxycholesterol and 7β-hydroxycholesterol); malondialdehyde (MDA) production was evaluated as marker of oxidative damage, together with cell death. Changes in the cellular redox state were evaluated determining the glutathione peroxidase (GPx) activity, the variation of GSH concentration, NADPH oxidase 1 (NOX1) activation and ROS generation. Twenty-four hours exposure to oxysterols induced cell death and MDA production, together with a significant loss of GSH, just after 30 min of incubation. An increase in the GPx activity was observed 18 hours after treatment. Pretreatment with both the wine phenolic extracts protected Caco-2 cells from oxidative damage and the level of GSH was preserved; a further increase of the GPx activity was observed in cells pretreated with wine fenolic extracts in agreement with previous studies. Oxysterols oxidizing action was due, at least in part, to the generation of intracellular ROS, as demonstrated by the use of a fluorescent probe. Since the increase of ROS levels by oxysterols in Caco-2 cells has been reported to involve modulation of NOX1, we also measured the activation of this enzyme; oxysterols treatment significantly enhanced the activity of NOX1. Wine phenolic extracts pretreatment reduced ROS formation, probably by a scavenging direct action, but also through the modulation of NOX1 activity, which was significantly inhibited by the extracts. In view of recent studies reporting that oxysterols may induce an increase of ROS production through an enhanced expression of NOX1 in Caco-2 cells in association with an increase of inflammatory cytokine production (IL-6 and IL-8), we investigated the antinflammatory effect of the wine phenolic extracts, by measuring the release of IL-6 and IL-8 by Caco-2 cells treated with the oxysterols mixture. Differentiated Caco-2 cells in fact have been reported to be also an excellent in vitro model of intestinal inflammation, responding to different inflammatory stimuli. Cannonau phenolic extract exerted a significant antinflammatory activity, inhibiting the release of the two interleukins. Finally, we evaluated the signaling pathways modulated by oxysterols and wine phenolic extracts with the aim of clarifying the molecular mechanism involved in the antioxidant and antinflammatory activity. For this purpose the possible modulation of the MAPKs cascade was analyzed in Caco-2 treated with oxysterols. These kinases have been reported to be activated by various stress stimuli, and they have been also implicated in oxysterols induced cytokine secretion and apoptosis. Under our experimental conditions, the oxysterols mixture was not able to modulate ERK phosphorilation, but induced p38 and JNK pathways activation. Pretreatment with both the wine phenolic extracts significantly inhibited p38 and JNK phosphorilation. The results of this study support a potential role for wine phenolic compounds in preventing oxysterols induced oxidative damage and inflammatory cytokine production in Caco-2 cells. We suggest that wine phenolic compounds exerted such a preventive effect through a mechanism involving a direct scavenging of ROS and/or an inhibition of NOX1, which in turn affected ROS production and redox sensitive MAPKs, p38 and JNK, expression and interleukins release. Although further studies are needed, our results point out for the first time a direct antioxidant and antinflammatory action of the phenolic fraction from the red wine Cannonau and the white wine Vermentino on enterocytes exposed to oxidizing species. These data have an interesting biological significance, suggesting the possibility that, together with their metabolites, wine polyphenols may significantly contribute to preserve the integrity of intestinal mucosa against oxidative damage and inflammation related disorders. Our data also support the opinion that total phenolic content, as well as wine’s color, are not essential for the antioxidant activity of a wine extract; although the extract obtained from the red wine Cannonau was effective at lower concentrations and in all the experimental conditions, the extract obtained from the white wine Vermentino showed basically a comparable efficacy