Funcionalidad del aceite de oliva virgen extra en la enfermedad inflamatoria intestinal

Laburpena

Inflammatory bowel diseases (IBD), Crohn¿s disease (CD) and ulcerative colitis (UC), are chronic and inflammatory disorders in the gastrointestinal tract with an increasing prevalence associated with industrialization of nations and a rising in countries developing. (Molodecky et al., 2012). The etiology of IBD is poorly understood, but certainly an increase in inflammatory mediators as well as a dysregulation of the immune system with an uncontrolled T cell activity, play a remarkable role in its pathogenesis. Actually, a large proportion of patients do not respond to conventional therapy, comprising corticosteroids and immunosuppressants, and also new biological therapies gives both the patients and clinicians motives to search for dietary complements, without side effects, to the conventional therapy. In fact, the use of dietary complements and alternative medicine is widely prevalent among IBD patients (Weizman et al., 2012). Actually, the interest in dietary supplements is growing (Braus and Elliott 2009) and nutritional therapy may be considered as supportive treatment for IBD patients without the undesirable effects of classic pharmacotherapy (Stuchlik and Zak 2002). Olive Oil (OO) is the characteristic culinary fat of the Mediterranean area being described as a key bio-active food (Puertollano et al., 2010). According to European regulation exits different olive oil categories, extra virgin olive oil (EVOO) which is obtained from the fruit of the olive tree (Olea europaea L.), solely by mechanical or other physical preparation, under conditions that do not alter its natural composition; olive oil, is the oil obtained by mechanical exclusion but also using solvents or re-esterification processes and olive-pomace oil is the oil obtained by treating olive pomace (the product remaining after the mechanical extraction of olive oil) with solvents or other physical treatments. Traditionally the beneficial effects of EVOO have been ascribed to its MUFA (Bermudez et al., 2011). However, EVOO also contains multiple minor components, present in a small amount (about 1-2 % of oil weight) with important biological properties (Alarcon de la Lastra et al., 2001). They are classified into two fractions: the unsaponifiable fraction (UF), defined as the fraction extracted with solvents after the saponification of the oil and the soluble or phenolic fraction (PE). Phenolic compounds have shown anti-inflammatory, antioxidant, antimicrobial and antiproliferative effects as well as the ability to modulate important cellular signalling pathways (Escrich et al., 2011, Visioli et al., 2011). Current epidemiological and experimental studies support the beneficial role of dietary polyphenols in several gastrointestinal diseases, including IBD (Frontela et al., 2010). Scientific evidence support the potential use of nutraceuticals focused in polyphenols constituents as agents capable to prevent or accelerate healing of gastrointestinal mucosal damage (Sanchez-Fidalgo et al., 2010, Villegas et al., 2011, Rosillo et al., 2012). On the other hand, UF comprises 1¿2% of total olive oil and is constituted by a wide number of compounds with a large biological value such as triterpenic alcohols, sterols, hydrocarbons (squalene), vitamins (¿- and ¿-tocopherols), ß-carotene, phytosterols, pigments, and volatile compounds, among others (Ghanbari et al., 2012). The first studies about the beneficial effects of UF suggested that this fraction may exert important biological activities such as an anti-atherogenic role and favorable effects on endothelial function (Perona et al., 2004, Perona et al., 2006, Acin et al., 2007). Therefore, the objectives of this thesis were: 1. Functionality of AOVE and its PE on in vivo and in vitro inflammation models. 1.A. To determinate the possible protective effect of dietary EVOO PE supplementation on dextrane sulphate sodium (DSS)-induced chronic colitis. 1.B. To evaluate the potential PE anti-inflammatory effect on lipopolysaccharide (LPS)-stimulated peritoneal murine macrophages. 2. Functionality of AOVE and its UF on in vivo and in vitro inflammation models. 2.A. To valorate the effects of dietary UF supplementation on DSS-induced acute colitis. 2.B. To evaluate the potential PE anti-inflammatory effect on lipopolysaccharide (LPS)-stimulated peritoneal murine macrophages. 2.C.. To investigate the effects of UF on blood and intestinal T cells from IBD patients in relapse and remission as well as from healthy subjects. FUNCTIONALITY OF EXTRA VIRGIN OLIVE OIL AND ITS POLIPHENOLIC FRACTION ON IN VIVO AND IN VITRO INFLAMMATION MODELS 1.A. Dietary extra virgin olive oil polyphenols supplementation modulates DSS-induced chronic colitis in mice Six-week-old mice were randomized in four dietary groups: standard diet (SD), EVOO diet, and both enriched with PE (850 ppm) (SD+PE and EVOO+PE). Our results revealed, that dietary supplementation of PE and mainly EVOO enriched with PE reduced the severity and extent of progressive chronic colonic damage induced by a short 5-day (acute phase) exposure of DSS followed by a 3-week rest period in C57BL/6 mice. These results were corroborated by histological findings. Moreover, increased concentrations of inflammatory cytokines such as tumor necrosis factor (TNF)-¿ and chemokines, for example, monocyte chemo-attractant protein (MCP)-1, enhance severity of colitis and neutrophil/macrophage migration (MacDermott 1999, MacGuckin et al., 2009). Our results demonstrate that EVOO, PE or EVOO+PE dietary treatment was able to reduce significantly MCP-1 and TNF-¿ mRNA levels, being the animals group fed with EVOO+PE showed the lowest levels. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in cell proliferation. Chronic inflammation seems to be associated to a higher proliferative activity (Vetuschi et al., 2002). In our study, diet enrichment with EVOO, PE or EVOO+PE notably reduced PCNA immunostaining. Cyclooxygenase (COX)-2 and inducible nitric oxide synthetase (iNOS) are pro-inflammatory proteins that play a pivotal role in mediating inflammation and contribute to DSS-induced inflammation in mice (Sanchez-Fidalgo et al., 2010, Rosillo et al., 2012). Importantly, EVOO, PE or EVOO+PE enrichment significantly down-regulated both proinflammatory protein expressions. Accumulative evidences suggest a strong support for a role in the mitogen-activated protein kinase (MAPKs) mediating IBD, in fact, preclinical studies with MAPK inhibitors have demonstrated significant efficacy repeatedly in experimental colitis models suggesting a possible application in IBD treatment (Hollenbach et al., 2004, Assi et al., 2006, Chromik et al., 2007). Our study revealed that dietary EVOO/PE enrichment diminished the p38 and Jun N-terminal kinase (JNK) MAPKs up-regulation; moreover, PE-enriched EVOO showed an efficiently higher reduction of JNK activation. One of the well-studied transcription factors downstream of MAPKs signalling is the nuclear factor NF¿B. Multiple lines of evidence suggest that NF¿B activation actively contributes to the development and maintenance of intestinal inflammation since this nuclear transcription factor binds to the promoter of many genes that are essential for the activation of immune responses including iNOS, COX-2, and TNF-¿ among others (Schreiber et al., 1998, Wullaert et al., 2011). Our results showed that chronic inflammatory induced by DSS was accompanied by an inhibition of the inhibitory protein of NF¿B (IKB¿) expression in colon. In fact, the highest IKB¿ protein expression levels were detected in those animals fed with EVOO enriched with PE. Peroxisome proliferator-activated receptor gamma (PPAR)¿ is a member in the superfamily of nuclear receptors implicated in the regulation of intestinal inflammation that might reduce the severity of IBD by inhibiting excessive immunoinflammatory responses (Dubuquoy et al., 2003, Annese et al., 2012). According to our study, we found that the anti-inflammatory effects of EVOO, PE or PE-enriched EVOO diets could be correlated with PPAR¿ activation in colon. 1.B. Extra Virgin Olive Oil polyphenolic fraction down-regulates inflammatory reponse in murine peritoneal macrophages suppressing NFKappa B and MAPKs signalling pathways Macrophages are dramatically involved in the pathogenesis of both acute and chronic inmuno-inflammatory disorders. Regarding IBD macrophages are pivotal mediators of innate immunity and appear to play a key role in stimulating the subsequent adaptative immunity (Mahida 2000). Thus, in order to discover the cellular implications in IBD prevention by PE, we evaluated the potential anti-inflammatory effect of this fraction on lipopolysaccharide (LPS)-stimulated peritoneal murine macrophages. Modulators of reactive oxygen species (ROS) production and ROS-induced signalling pathways, especially in macrophages, could represent potential targets for anti-inflammatory intervention (Li et al., 2012). Upon macrophages stimulation, the iNOS gene of macrophages is induced and large amounts of nitric oxide (NO) are generated. NO acts as an intracellular messenger which modulates the formation of endogenous ROS (reactive oxygen species) including hydrogen peroxide, peroxynitrite and other potentially oxidants, that orchestrate the inflammatory responses (Kang and Lee 2012). Our results revealed that PE exerted significant anti-inflammatory and anti-oxidant effects inhibiting LPS-induced intracellular ROS and NO production. Besides, the treatment decreased COX-2 and iNOS protein expressions via down-regulation of NFkB and MAPK signalling pathways. FUNCTIONALITY OF EXTRA VIRGIN OLIVE OIL AND ITS UNSAPONIFIABLE FRACTION ON IN VIVO AND IN VITRO INFLAMMATION MODELS 2.A. Dietary unsaponificable fraction from extra virgin olive oil supplementation attenuates acute ulcerative colitis in mice C57BL/6 mice were fed from weaning with sunflower oil (SD), EVOO diet and UF-enriched SD at 5% oil (SD+UF). After 30 days, mice were exposed to 3% DSS for 5 days developing acute colitis. After 4 days of DSS removal, animals were sacrificed and colons were histological and biochemically processed. Disease activity index and microscopic damage score were significantly improved in EVOO and SD+UF dietary groups versus SD group. In addition, both dietary treatments significantly decreased MCP-1 and TNF-¿ levels, iNOS and COX-2 over-expression as well as p38 MAPKs activation in colon mucosa. Moreover, an up-regulation of IKB expression was also observed after feeding the animals with both diets. However, no statistic differences were observed between data obtained from mice fed with EVOO or UF+SD diets. 2.B. Extra Virgin Olive Oil unsaponifiable fraction down-regulates inflammatory reponse in murine peritoneal macrophages suppressing NFKappa B and MAPKs signalling pathways Then, when we had evaluated the antiflammatory activity of UF in a murine model of acute colitis and in the same way that we preceded in the case of PE, we analyzed the effects of UF in murine peritoneal LPS-stimulated macrophages. Our results revealed that UF exerted significant anti-inflammatory and anti-oxidant effects inhibiting LPS-induced intracellular ROS and NO production. Besides, UF treatment decreased COX-2 and iNOS protein expressions via down-regulation of NFkB and MAPK signalling pathways. 2.C. The unsaponifiable fraction of extra virgin olive oil promotes apoptosis and attenuates activation of T cells in patients with inflammatory bowel disease Hitherto, the role of dietary supplementation of UF from EVOO in patients with IBD is unknown and the underlying mechanisms responsible for any possible beneficial effect remains to be elucidated. Our study demonstrated that UF induced apoptosis of blood and intestinal T cells isolated from IBD patients but not from healthy subjects. Because dysregulated T cell apoptosis is a feature of IBD patients, and not in healthy subjects (Mudter and Neurath 2007), our results might indicate that EVOO¿s UF exert a selective proapoptotic effect. Besides same therapeutic approaches, i.e. glucocorticoids, (Farrell and Kelleher 2003) to reduce inflammatory reactivity of IBD patients aim to decrease number of activated T cells (Wei and Feng 2010). EVOO¿s UF addition resulted in decreased frequencies of activated CD4+ T cells, identified by the expression of CD69 and CD25, in blood and intestinal mucosa, respectively. This effect was recorded in T cells of both patients and healthy subjects. Moreover, EVOO¿s UF decreased the levels of secreted IFN-¿, a cytokine known to promote inflammatory reactions, from stimulated T cells in cell cultures from both patients and healthy subjects (Breese et al., 1993). Together, these observations suggest that EVOO¿s UF attenuates T cell activation. In addition, the fact that EVOO¿s UF induced apoptosis of both activated (CD69+) and naïve (CD69-) T cells suggests that EVOO¿s UF exert downregulatory effects on activation of T cells independently of its apoptotic mechanisms. Moreover, we did not record any increased frequencies of Foxp3+ T regulatory cells in the presence of EVOO¿s UF, which indicates that induction of apoptosis and attenuation of T cell activation, and not suppression of effector T cells, are the anti-inflammatory mechanisms of action of importance. There is strong evidence that T cell migration to the gut initiates and perpetuates the intestinal inflammatory process and tissue destruction in IBD (Hart et al., 2010). The gut homing integrin ß7 regulates lymphocyte trafficking and contributes to lymphocyte recruitment into the intestinal tissue (Gorfu et al., 2009). Interestingly, our study demonstrates that the frequencies of integrin ß7 expressing blood T cells decreased in the presence of EVOO¿s UF. This effect was only seen in cell cultures of IBD patients, whereas the expression of integrin ß7+ T cells from healthy individuals was low irrespective of presence of EVOO¿s UF. Thus, in addition to inducing apoptosis and attenuating T cell activation, EVOO¿s UF modulates gut homing properties of T cells, altogether contributing to the anti-inflammatory effects of the olive oil fraction. CONCLUSION All together our results suggest that EVOO exerts preventive effects in the development of IBD, playing its multiple minor components a key role in these healthy benefits. Thus EVOO may be considered as supportive nutritional therapy for IBD patients as well as its PE and UF fractions might provide an attractive nutraceutical complement in management of IBD, mainly when the effects of inexpensive, side effect-free therapies based on dietary EVOO might suppose an improvement in public health on the prevention of chronic pathologies with high prevalence in the population. BIBLIOGRAFÍA Acin, S., M. A. Navarro, J. S. Perona, J. C. Surra, N. Guillen, C. Arnal, A. J. Sarria, J. M. Arbones-Mainar, R. Carnicer, V. Ruiz-Gutierrez and J. Osada (2007). 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