REVIEW ARTICLE

Mucosal Healing in Inflammatory Bowel Diseases: Is There a Place for Nutritional Supplementation? Annaïg Lan, PhD,* Franc¸ois Blachier, PhD,* Robert Benamouzig, MD, PhD,† Martin Beaumont, MS,* Christophe Barrat, MD,‡ Desire Coelho, PhD,§ Antonio Lancha, Jr, PhD,§ Xiangfeng Kong, PhD,k Yulong Yin, PhD,k Jean-Claude Marie, PhD,¶ and Daniel Tomé, PhD*

Abstract: Advanced mucosal healing (MH) after intestinal mucosal inflammation coincides with sustained clinical remission and reduced rates of hospitalization and surgical resection, explaining why MH is increasingly considered as a full therapeutic goal and as an endpoint for clinical trials. Intestinal MH is a complex phenomenon viewed as a succession of steps necessary to restore tissue structure and function. These steps include epithelial cell migration and proliferation, cell differentiation, restoration of epithelial barrier functions, and modulation of cell apoptosis. Few clinical studies have evaluated the needs for specific macronutrients and micronutrients and their effects on intestinal MH, most data having been obtained from animal and cell studies. These data suggest that supplementation with specific amino acids including arginine, glutamine, glutamate, threonine, methionine, serine, proline, and the amino acid-derived compounds, polyamines can favorably influence MH. Short-chain fatty acids, which are produced by the microbiota from undigested polysaccharides and protein-derived amino acids, also exert beneficial effects on the process of intestinal MH in experimental models. Regarding supplementation with lipids, although the effects of v-3 and v-6 fatty acids remain controversial, endogenous prostaglandin synthesis seems to be necessary for MH. Finally, among micronutrients, several vitamin and mineral deficiencies with different frequencies have been observed in patients with inflammatory bowel diseases and supplementation with some of them (vitamin A, vitamin D3, vitamin C, and zinc) are presumed to favor MH. Future work, including clinical studies, should evaluate the efficiency of supplementation with combination of dietary compounds as adjuvant nutritional intervention for MH of the inflamed intestinal mucosa. (Inflamm Bowel Dis 2015;21:198–207) Key Words: intestinal mucosal healing, inflammatory bowel diseases, nutritional supplementation

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nflammatory bowel diseases (IBD) are characterized by alterations of the intestinal mucosa associated with exacerbated immune functions of still unclear etiology.1 IBD, which include ulcerative colitis (UC) and Crohn’s disease (CD), are both characterized by chronic inflammation of the mucosa with alternating relapse and remission episodes. In CD remission, the inflamed mucosa has the possibility to heal progressively, leading to the total disappearance of all mucosal ulcerations; although in clinical practice, this endpoint is difficult to achieve2 (Fig. 1). For patients with UC, mucosal healing (MH) is described as the absence of friability, blood, erosions, and ulcers in all visualized segments of

Received for publication June 16, 2014; Accepted July 3, 2014. From the *UMR 914 INRA/AgroParisTech, Nutrition Physiology and Ingestive Behavior, Paris, France; †Department of Gastroenterology, Avicenne Hospital, Assistance Publique-Hôpitaux de Paris, Bobigny, France; ‡Department of Digestive Surgery, Jean Verdier Hospital, Assistance Publique-Hôpitaux de Paris, Bobigny, France; §Laboratory of Applied Nutrition and Metabolism, School of Physical Education and Sports, University of Sao Paulo, Sao Paulo, Brazil; kInstitute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China; ¶INSERM U773, Biomedical Research Center Bichat Beaujon/Paris7 University, Paris, France. The authors have no conflicts of interest to disclose. Reprints: Annaïg Lan, PhD, UMR 914 INRA/AgroParisTech, Nutrition Physiology and Ingestive Behavior, 16, rue Claude Bernard, F-75005, Paris, France (e-mail: [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000177 Published online 9 September 2014.

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the gut mucosa.3,4 According to recent evidence, it seems that an advanced MH coincides with a sustained clinical remission and improved clinical outcomes, as well as reduced rates of hospitalization and surgical resection. This explains why MH can be considered as a full therapeutic goal and an endpoint for clinical trials for both CD and UC,5–9 even if MH by itself obviously is not sufficient to cure IBD.10 Regarding CD more specifically, a recent consensus report by experts concluded that MH is both a relevant and achievable goal, as well as a predictive parameter for the long-term disease course.7 Although reports on the pharmacological treatment of IBD are numerous, there is still a paucity of information regarding the possible role of alimentary compounds and nutritional supplements, which would improve MH in IBD. Indeed, most studies have been related to the effects of dietary compounds on the process of mucosal inflammation itself, this topic being the subject of several review articles.11–13 The objective of this review is thus to focus on the beneficial effects of dietary supplementation with compounds presumably efficient for intestinal MH improvement. We present the process of MH in IBD with a brief description of the mechanisms involved, and then we examine the dietary compounds that seem beneficial in this process. For this purpose, we present and synthesize data obtained from experiments with Inflamm Bowel Dis  Volume 21, Number 1, January 2015

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as myofibroblasts, to the mucosa repair process.26–30 In fact, an increased number of myofibroblasts is observed in the lamina propria from colitis patients when compared with normal lamina propria.31 Wound healing can be viewed as the succession of steps necessary to restore tissue structure and function, whereas fibrosis can be viewed as the pathophysiological response after intestinal mucosa injury that leads to inappropriate tissue repair.32 The challenge for the scientific community is thus to find ways to accelerate MH without promoting tissue fibrosis.

Mechanisms Involved in Intestinal MH FIGURE 1. Schematic view of the intestinal MH after an active phase of inflammation. Advanced MH is likely to favor sustained clinical remission.

animal and cell models together with the limited data available from clinical studies.

Intestinal MH The intestinal epithelium is a single cellular monolayer, which represents the border between the endoluminal content and the “milieu intérieur” as defined by Claude Bernard more than 130 years ago. This structure that acts as a selective barrier against the compounds and microorganisms present in the intestinal lumen is renewed within a few days.14 This renewal is made possible through asymmetrical mitosis of pluripotent stem cells and daughter cell migration.15 This migration is concomitant with differentiation into cell lineages with associated physiological functions, which include absorption, mucus production, and enteroendocrine secretion.16 Fully mature cells are finally exfoliated into the lumen by a process named detachment-induced apoptosis (anoikis), which allows elimination of these cells.17–19 In IBD, due to a loss of epithelial cells, this barrier is disrupted, leading to the contact of the lamina propria compartment with compounds and microorganisms present in the intestinal lumen. When the aggression is of limited nature, restitution is made possible through migration of viable epithelial cells from the local subepithelial stem cell niche in the proximity of the wound edges, allowing the reestablishment of the intestinal epithelial continuity.20–23 A subsequent increase of nearby cell proliferation then intervenes to repopulate the damaged area.24 In the restitution process, cells in the immediate vicinity of the wound undergo a loss of classical absorptive epithelial cell polarization with reorganization of the cytoskeleton and loss of microvilli.21 These cells extend protrusions (lamellipodia) into the denuded area to allow wound closure.25 In the case of more severe mucosal injury, the regeneration capacity of local stem cells is believed to be insufficient to allow tissue healing. In such situations, bone marrow–derived mesenchymal stem cells migrate into the gastrointestinal wall where they likely contribute, as differentiated mesenchymal cells, such

The different factors and mechanisms allowing MH after active IBD seem to be numerous. Signaling events in intestinal epithelial cells (IEC) result mostly in proproliferative and promigratory effects as well as in antiapoptotic effects.7 Although the precise description of the growth factors and cytokines involved in MH and their mechanisms of action is out of the scope of this article (the readers being invited to refer to reviews on this topic33–38), an overview of the events required for MH is useful to determine by which mechanisms nutritional supplementation may improve this process. Recent articles devoted to intestinal MH reveal that numerous proteins seem to be involved in epithelial restitution and MH. Intestinal wound healing is dependent on the precise balance of several processes including migration, proliferation, differentiation, and apoptosis of the epithelial cells. Supplementation with dietary compounds (particularly those that are decreased in IBD) intervening in these processes, as energy substrates and/or as precursors for macromolecules in anabolic metabolism, as well as agents with signaling functions, represents a strategy to ameliorate intestinal MH (Fig. 2).

Remodeling of the Cytoskeleton and Cell Migration Cell migration, which is essential for epithelial restitution, is the process by which epithelial cells elongate and migrate to cover denuded surfaces. Disruption of the intestinal architecture is a common feature during chronic gut inflammation. Several

FIGURE 2. Schematic view of the effects of supplementation with dietary compounds on the process of intestinal MH. www.ibdjournal.org |

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transmembrane proteins are involved in the establishment and maintenance of the intercellular adhesion. Among them, E-cadherin, a cell–cell adhesion mediator,39 is linked to the actin cytoskeleton through interaction with catenins and other proteins to allow cell– cell adhesion. Loss of normal membrane E-cadherin and catenins has been detected at the mucosal edges around epithelial ulcerations in UC.40 The catenins and their complexes act not only as “intercellular glue” but also as integral components of intracellular signal transduction pathways, linking the membrane to downstream cytoplasmic and nuclear events.41 E-cadherin has been shown to be essential for migration during wound healing.42 Disruption of E-cadherin–mediated cell–cell contacts may lead to accumulation of cytosolic b-catenin41,43 and consequently to Wnt/Frizzled signaling initiation44 after nuclear translocation of b-catenin. Formation of a b-catenin/T-cell factor/lymphoid enhancer binding factor transcription factor complex eventually results in the expression of Wnt target genes, notably, genes involved in tissue repair and remodeling and in inflammatory responses.42,45 Intercellular adhesion proteins then likely play important roles in the intestinal epithelium not only for the formation of cell adhesions and migration, but also in the control of cell signaling during MH. Wnt signaling also regulates cell positioning in the crypt through regulation of the expression of the Ephrin B (EphB) family of receptors and their ligands. EphB signaling is involved in the ordered migration of epithelial cells along the crypt axis.46 Moreover, integrin-linked kinase performs crucial roles in the maintenance of the crypt-villus axis homeostasis.44 In addition, perturbation of the E-cadherin/catenin complex at intercellular junctions seems to be a way through which trefoil factor peptides (TFF) promote cell migration.47 Although there is a general consensus regarding the promitogenic activity of TFF peptides and their crucial role for epithelial protection and restitution, the cellular mechanisms mediating these processes are not yet completely understood.47,48 The TFFs offer an alternative promigratory pathway not affected by damage to the TGFb-dependent pathway.49 Interruption of cell–cell junctions through E-cadherin loss allows cells to migrate.50 IEC surrounding the injured surface lose their columnar polarization. The morphological changes observed are the result of actin cytoskeleton reorganization owing to small GTPase activities. For example, Ras-like GTPase Rho (such as RhoA) is required for epithelial growth factor-induced migration of intestinal crypt cells to restitute mucosal integrity.51 This transitory dedifferentiation in which epithelial cells lose their phenotypic and functional characteristics while acquiring mesenchymal features is called the epithelial to mesenchymal transition. This process, which is strongly dependent on the TGFb pathway, is involved in epithelial restitution.

Proliferation Wnt signaling promotes maintenance of epithelial stem cells and early progenitors by driving transcription of genes associated with proliferation. The expression of this key regulator of the intestinal stem cell niche is controlled by Hedgehog signaling.50 The deletion of Indian Hedgehog from the small intestinal epithelium initiates the intestinal wound repair response.52 Hedgehog

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signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo.53 The focal adhesion kinase (FAK, also called protein tyrosine kinase 2), a cytoplasmic tyrosine kinase protein, which is found concentrated in the focal adhesions (i.e., that form between cells growing in the presence of extracellular matrix constituents), has been shown to regulate cellular proliferation and to mediate cell survival. Indeed, in the absence of FAK, colonic epithelium repair is significantly impaired after inflammatory injury induced by acute dextran sodium sulfate treatment. This suggests that FAK functions in vivo as a regulator of adhesion-mediated survival and proliferation in response to inflammatory injury.54 Furthermore, a paramount role for IL-33 in epithelial restitution and repair55 through promotion of epithelial proliferation and mucus production has been evidenced. Conversely, other signaling pathways involved in the regulation of the intestinal epithelial homeostasis may limit wound repair after colitis, as shown for Dkk1, a secreted protein that inhibits the Wnt/b-catenin signaling. Indeed, Dkk1 depletion induced a strong proliferative response associated with wound repair after experimental colitis.56

Differentiation and Epithelial Barrier Function After IEC migration and proliferation, differentiation allows establishment of normal villus architecture and absorptive/secretory functions. Among factors involved in differentiation, Indian Hedgehog expressed by mature colonocytes regulates their differentiation in vitro and in vivo by negatively regulating the Wnt pathway.53 FAK (protein tyrosine kinase 2) is also involved in the differentiation57 and in the mediation of the promoting effect of the enteric glia on intestinal healing.58 Increased epithelial permeability is a common feature in IBD although the sequences of cell events involved in this phenomenon are not yet fully understood. Factors contributing to the regulation of tight junction complexes that seal the paracellular spaces between cells and also contribute to epithelial barrier function may thus promote intestinal repair. A recent study has shed light on a new role of the protein C system in controlling intestinal permeability by regulating tight junction protein expression and promoting MH.59 In addition, STAT5 signaling in enterocytes protects against tight junction barrier dysfunction and promotes intestinal mucosal wound healing.60 Also, TFF peptides may promote healing of mucosal lesions in vivo47 in addition to their promigrating and antiapoptotic properties. Indeed, TFF3 peptide stabilizes these junctions through upregulation of the tightening protein claudin-1 and redistribution of ZO-1 from the cytoplasm to the intercellular membrane with an increase in the binding to the protein occludin.48,61 Conversely, epithelial to mesenchymal transition contributes to the disassembly of tight junctions and loss of apical-basal polarity in IEC because of a repression of the transmembrane adhesion receptor E-cadherin.43

Apoptosis P53-mediated apoptosis under proinflammatory conditions is a common situation in IBD.61,62 Thus, it seems that antiapoptotic properties are crucial for epithelial restitution, in a situation where anchorage-dependent epithelial cells have to detach to

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migrate over a denuded area, making them susceptible to anoikis. TFF3 has an antianoikic effect on IEC through activation of NFkB.62 In addition to the serine/threonine kinase PI3K/Akt, which is a major signaling pathway to promote cell survival (as well as cell proliferation and/or differentiation), the serine/threonine kinase CK2 seems to be a key regulator of homeostatic properties of the intestinal epithelium that serves to promote wound healing, in part through protection of normal IEC from cytokine-induced apoptosis under conditions of inflammation.63 The key activity of CK2 in the regulation of Wnt and NFkB pathways in embryonic development and cancer reinforces the view that CK2 might be important for MH.64

Miscellaneous Factors Involved in MH Numerous other factors are involved in MH, including some proteins of the basement membranes such as laminins,65 the cell surface receptor Trem2 involved in colonic MH,66 or circulating factors, such as the blood coagulation factor XIII.67 In addition, some enzymatic activities (for instance, cyclooxygenase-268) and antimicrobial peptides secreted by the IEC (like the b-defensin 269) seem to be involved in MH. Finally, microRNAs (miRNAs) might also play an important role in these biological processes. For instance, a recent study showed that miR-200b expression was decreased in inflamed mucosa of patients with IBD (especially in UC), suggesting that this posttranscriptional gene regulator inhibits TGFb1-induced epithelial to mesenchymal transition and promotes proliferation of IEC.70 This latter point further illustrates the complexity of intestinal MH and the numerous actors implicated. The situation is further complicated by the fact that specific proteins involved in healing may paradoxically impair the MH when produced in excess. For instance, although an increase of the matrix metalloproteinase activity is necessary for intestinal wound healing,71 an overexpression of this latter activity after upregulation by cytokines delays epithelial healing.72

Which Roles Dietary Compounds Play in the Process of Intestinal MH? There is still no consensus about macronutrient and micronutrient needs in patients with IBD for MH. It has been reported that micronutrient deficiencies are more frequent than macronutrient deficiencies in patients with CD in remission.73 Unfortunately, there are only a few clinical studies evaluating the needs of specific macro- and micro-nutrients that may improve intestinal MH, most studies being performed with animal and cell models. Using these models, most studies have been devoted to the description of the efficiency of the alimentary compounds on MH with yet little investigation on the mechanisms involved.

Enteral Nutrition and Intestinal MH Poor nutritional status may characterize patients with IBD in the active phase of the disease. The parameters involved in such nutritional status include anorexia, intestinal malabsorption, increased intestinal losses, and increased catabolism.74 The poor

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intestinal MH may be attributed, at least in part, to such malnutrition. Protein-energy malnutrition and deficiencies of minerals and vitamins have been reported in patients with IBD.75–77 As reviewed by Pineton de Chambrun et al,4 an exclusive oral polymeric diet is efficient as an MH inducer in children with CD. Indeed, recent data have confirmed that exclusive enteral nutrition is effective for inducing clinical remission in pediatric CD patients.78–81 Elemental formulas with amino acids do not seem to be more efficient for clinical remission in adults and children than polymeric formulas containing whole proteins.82–85

Proteins, Amino Acids, and Related Compounds In patients with IBD, amino acids derived from proteins are presumably used as building blocks for macromolecule (DNA, RNA, and protein) synthesis, as energy substrates and as precursors of bioactive metabolites in the wounded mucosal area after an inflammatory episode. Consumption of protein hydrolysates may result in a more rapid uptake of amino acids when compared with whole alimentary proteins or amino acids in mixtures.86 To our knowledge, the effects of the amount of dietary protein ingestion in the course of intestinal MH have not been determined in clinical studies. The study by Jowett et al87 showed that the highest dietary protein consumption by patients with UC in remission was associated with a 3-fold increase of the risk of relapse when compared with patients with the lower protein intake. One possible explanation for such a result is related to the increased amounts of some deleterious amino acid–derived metabolites produced from undigested proteins in the large intestine luminal content by the microbiota,88 such as sulfide or ammonia, which inhibit colonocyte respiration89,90 when present at excessive concentrations. The study by Jowett et al87 illustrates the difficulty in establishing an optimal amount of dietary proteins for MH and suggests a maximal nondeleterious amount of dietary protein for patients with UC in remission. In infant rabbits with experimental bacterial enteritis, protein– calorie malnutrition is associated with delayed small intestine mucosa repair.91 Specific protein synthesis, in addition to the overall intestinal mucosa protein synthesis, may be necessary to facilitate epithelial cell migration and thus restitution after intestinal mucosa injury. For instance, collagen–epithelial cell interactions are clearly an important aspect of epithelial restitution.92,93 However, collagen deposition must be strongly regulated because excessive collagen deposition subsequent to downregulation of matrix degradation (notably through IL-13 action) leads to fibrosis.82 Experiments with animal models and with epithelial cells in culture have provided some information on compounds from dietary origin (and associated metabolites) involved in the process of intestinal wound healing. Compounds derived from amino acids, such as polyamines and their amino acid precursors (arginine and ornithine), have been implicated in the process of intestinal recovery after various experimental injuries in animal models. Recently, Iwashita et al94 have reported that glutamine can also be used as a substrate for polyamine synthesis in the rat small IEC-18 cell line. However, it is worth noting that glutamine is considered a less efficient substrate than arginine for ornithine and polyamine www.ibdjournal.org |

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production in enterocytes.95–97 In rats with intestinal mucosal injury induced by chemotherapy, it has been shown that increased activity of the enzyme ornithine decarboxylase (which converts ornithine to putrescine) and increased polyamine content plays a central role in mucosal regeneration.98 Studies using a model of stress-induced mucosal injury in rats have led to the suggestion that, in addition to the endogenous synthesis of polyamines in the intestinal mucosa, luminal polyamines are involved in the intestinal mucosal repair.88 This beneficial effect of polyamines seems to be partly related to the effects of these molecules on epithelial cell migration after wounding.99–103 In addition, polyamines are required for optimal epithelial cell growth and DNA synthesis104–107 (Fig. 3). Dietary supplementation with ornithine alpha-ketoglutarate (5 g$kg21$d21 for 4 d) has been shown to accelerate intestinal healing and to reduce bacterial translocation in the model of intestinal injury on rats receiving abdominal radiation.108 In the rat model, the administration of 1 g/kg ornithine alpha-ketoglutarate for 1 day after transient ischemia accelerated the repair of the small intestinal mucosa.99 Arginine supplementation (4% for 7 d) improves the intestinal mucosal recovery after experimental enteritis in rats.109,110 However, in this study, it was not determined if arginine is acting per se or through conversion into its metabolites. Among metabolites derived from arginine, although polyamines are considered beneficial for MH, nitric oxide, which combines with reactive oxygen species to form strong oxidant like peroxynitrite, may exert pathological roles when produced in excess as observed in colonic biopsies recovered from CD and UC patients.111 Among amino acids used in dietary supplements for improving MH, glutamine has shown some effects in different models of experimental enteritis. Indeed, oral glutamine supplementation (3%) is able to improve the healing of the small intestine mucosa after radiation-induced enteritis.112 In a model of anastomosis in rats, early enteral feeding with glutamine (12.5% given through an orogastric tube) was found to improve the microscopic aspect of the healing.113 The amino acid glutamate is highly metabolized in IEC.114,115 When given in the form of 5% monosodium glutamate for 5 days, this compound has been reported to promote small intestinal MH after chemically induced enteropathy in rats.116

In addition, mixtures of amino acids (threonine 15 g/kg, serine 10 g/kg, proline 15 g/kg, and cysteine 7.2 g/kg) have been shown to increase mucin synthesis after dextran sodium sulfate– induced colitis induction.117 The essential amino acid threonine is known to be highly metabolized by the gut,118 notably for the synthesis of mucins that requires high threonine availability.119 An intact mucus layer is known to be necessary for providing an “initial seal” after intestinal mucosa injury.7,120,121 Very recently, it has been shown that a mixture of glutamate (0.57 g/d in the form of monosodium glutamate), methionine (0.31 g/d), and threonine (0.50 g/d) given in the MH phase after chemical induction of colitis in rats improves colonic mucosal regeneration/ reepithelialization after 10-day supplementation, without affecting the spontaneous resolution of inflammation.122 In this experimental model, mucosal regeneration/reepithelialization was already visible 3 days after colitis induction, at a time when mucosal inflammation was still severe, suggesting that the effects of dietary supplementation on MH and inflammation can be partly dissociated.

Carbohydrates Very few studies have examined the effects of dietary carbohydrates on the process of intestinal MH. Supplementation with 1% pectin has been shown to improve the healing of experimental colonic anastomoses in rats.123

Short-chain Fatty Acids Short-chain fatty acids (SCFAs) are end products of the microbiota metabolism from dietary fibers, resistant starch, and protein-derived amino acids124,125 and represent a major source of energy for colonic epithelial cells.126 SCFAs, and more particularly butyrate, have been shown to help in the intestinal MH after chemically induced intestinal injury127 and colonic anastomoses.128,129 In addition, it has been shown that butyrate oxidation in colonocytes is a way to control its intracellular concentration and consequently its effects on gene expression in relation with cell growth.130,131 Plöger et al132 have discussed the potential effects of butyrate on colonic healing. The authors emphasize the capacity of this SCFA to directly contribute to MH through the restoration of tight junctions, notably by its effects on claudin-1, occludin, ZO-1, and ZO-2 expression. Butyrate, at concentrations between 0.1 and 1.0 mM, is also able to increase mucus synthesis in colonic biopsy specimens obtained from patients with UC.133 Studies using enemas containing high butyrate concentration (100 mM) showed an increased mucin-2 gene expression in the rat model.134 In a model of intestinal anastomoses in rats, butyrate enemas (60 mM) have been shown to increase the anastomotic bursting strength in association with collagen synthesis.135 Unfortunately, despite these encouraging effects in vitro and in animal models, enemas with butyrate used at high concentration and for long-term treatment (100 mM for 20 d) had no measurable effect in patients with UC.136

Lipids FIGURE 3. Schematic view of the dietary compounds presumably involved in intestinal MH.

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Using an in vitro assay model with confluent IEC-6 cell line wounded with a razor blade, it has been shown that

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eicosapentanoate, linoleate, a-linoleate, g-linoleate, and arachidonate (all used at 30 mM concentration) increase the cellular migration rates.137 The effects of v-3 and v-6 on cell migration are apparently explained by different mechanisms. Although v-6 fatty acids modulate mucosal restitution, at least in part, through eicosanoid production, v-3 seems to act through TGFb production. Conversely, in vitro experiments suggest that DHA and EPA may have a negative impact on the MH process. Turk et al138 showed that in a model of immortalized colonocytes, DHA (50 mM) reduced ligand-induced EGFR activation and that DHA and EPA (both 50 mM) reduced wound-induced EGFR transactivation. Supplementation for 10 days with arachidonic acid (5% of total fatty acids) was found to enhance ileal mucosa repair in the model of intestinal ischemia in suckling piglets.139 In the acetic acid–induced colitis model in rats, inhibition of endogenous colonic prostaglandin synthesis was shown to impair MH.140,141 Similar results were obtained using the model of indomethacininduced small intestinal lesions in rats.142 The effects of prostaglandin E2 and of synthetic prostaglandin analogs have been tested for their effects on the gastroduodenal mucosal lesions with some positive effects on the MH.143 All these results strongly suggest that endogenous prostaglandin synthesis play a major role in intestinal MH after injury. Also, lysophosphatidic acid, an intermediate in the phospholipids biosynthesis pathway, has been found to enhance MH by increasing IEC migration in a rat model of colitis provoked by trinitrobenzene.144 Regarding clinical studies, in a double-blind, randomized, placebo-controlled trial, Middleton et al145 studied the effects of supplementation with EPA (270 mg/d) and DHA (45 mg/d) on the maintenance of remission in patients with quiescent UC, but found no effect after 12 months of treatment. Feagan et al146 also found no effect of oral administration of v-3 fatty acids (4 g/d for up to 58 wk) on the prevention of relapse in patients with CD, calling into question the potential clinical utility of EPA and DHA supplementation in such situations.

Micronutrients Numerous vitamin and mineral deficiencies have been observed in patients with IBD with different degrees of putative causal relationships regarding the clinical significance of such deficiencies in terms of MH.13 Indeed, vitamin B1 (thiamine), vitamin B3 (niacin), vitamin B9 (folate), vitamin B6 (pyridoxine), vitamin B12 (cobalamines), vitamin D, vitamin E, vitamin A, vitamin C, vitamin K, b-carotene, calcium, magnesium, iron, selenium, copper, and zinc are often depleted in IBD with different frequencies.73,147–151 Among all these micronutrient deficiencies, some of which are presumed to play major roles in intestinal wound healing are discussed below.

Vitamin A The acidic form of vitamin A (retinoic acid) stimulates the production of collagen by the fibroblasts.152 Vitamin A deficiency is relatively common in patients with newly diagnosed IBD and is

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associated with zinc deficiency153; zinc and protein deficiencies154 being known to affect the transport of vitamin A. Vitamin A supplementation (between 2 and 10 mM) has been shown to accelerate the ileum wound healing ex vivo in the model of chemical induction of enterocolitis in the piglet.155 In the newborn rat model, vitamin A (between 5000 and 20,000 IU/kg) was able to ameliorate the healing of the chemically induced intestinal mucosal injury.156 Accordingly, vitamin A deficiency has been shown to impair colonic healing in the model of colonic anastomosis in rats.157 In the model of rabbits with intestinal anastomosis that received dexamethasone (0.1 mg/kg/d), high doses of vitamin A (10,000 IU$kg21$d21) reversed the inhibitory effect of dexamethasone on the healing.158 Finally, in the model of rats undergoing abdominal irradiation followed by intestinal anastomoses, vitamin A supplementation (150 IU/g of food) reduced the bursting strength at the colon anastomoses.159 Importantly, a clinical study showed that children recovering from diarrhea who received vitamin A supplementation regained normal mucosal integrity (measured by the indirect test of gut mucosal integrity using the lactulose/mannitol differential sugar absorption test) more rapidly than children receiving a placebo.160

Vitamin D Vitamin D deficiency is often observed in patients with IBD.161 1,25-dihydroxy-vitamin D3 (0.01 mM) has been shown to enhance tight junctions formed by the human colonic epithelial cells Caco-2 by increasing the expression of junction proteins. In addition, this compound stimulates IEC migration.162 An intervention study has shown that supplementation with vitamin D3 reduces the risk of relapse163 in patients with CD in remission. Importantly, Johnson et al164 recently demonstrated that a vitamin D analog (CARD-024) attenuates the profibrotic response of colonic myofibroblasts, suggesting that vitamin D analogs may reduce intestinal fibrosis.

Vitamin C Subnormal serum vitamin C levels are commonly measured in active and inactive IBD.148,150 Vitamin C (ascorbic acid) may play an important role in intestinal MH because this vitamin is involved in proline and lysine hydroxylation and collagen synthesis.165 Interestingly, this vitamin supports angiogenesis and regulates neutrophil activity during cutaneous wound healing.166 In a model of intestinal anastomoses in rats, high doses of vitamin C (100 or 200 mg/kg through the intramuscular route) ameliorated both the anastomotic strength and the histopathological score.167 Association of ascorbic acid with v-3 fatty acid has been shown to enhance the colonic wound healing in a model of ischemic colon anastomoses in rats.168

Zinc More than half of the patients with CD in remission are characterized by low zinc plasma concentrations.75 Zinc is involved in the catalytic activities of more than 200 enzymes169 including those involved in protein and DNA synthesis, gene expression, and www.ibdjournal.org |

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protection against deleterious effects of excessive reactive oxygen species.170–174 Several in vivo and in vitro studies have shown that zinc supplementation can improve intestinal wound healing. In IEC-6 in vitro wounding model, zinc was found to be able to increase cellular restitution.175 In the model of colonic anastomoses in rats, zinc deficiency was found to impair wound healing,176 whereas accordingly, balancing zinc deficiency in rats was able to improve the healing of colonic anastomoses.177

CONCLUSION AND PERSPECTIVES Most of the available data from the literature are, on one hand, related to the description of the process involved in intestinal MH and, on the other hand, related to the description of the effects of individual dietary compounds on the MH process. These studies have been done by using animal and intestinal cell models, with few clinical studies. These studies have enabled identification of dietary compounds that are active in the process of intestinal MH in different experimental situations. These compounds include several amino acids (arginine, glutamine, glutamate, threonine, serine, proline, and methionine), metabolites derived from amino acids (such as polyamines), SCFAs (particularly butyrate), prostaglandins, lysophosphatidic acid, vitamin A, vitamin D3, vitamin C, and zinc (Fig. 3). However, it is only fair to consider that the mechanisms of action by which these dietary compounds are active remain largely unknown. Particularly, it remains to be determined by which mechanisms the different dietary compounds act on the different signaling pathways involved in the restoration of intestinal tissue structure and functions, as well as the ways by which they act as energy substrates, as precursors for bioactive compounds and as regulators of the anabolic metabolism. In addition, the few clinical studies performed in an attempt to evaluate the efficiency of dietary compounds for intestinal MH do not yet allow for nutritional recommendations. Therefore, future studies should urgently test dietary compounds individually or in combination for their capacity to improve MH after an active phase of intestinal mucosal inflammation in clinical trials and with a mechanistic perspective (using animal and cell models). If successful, such an approach would be beneficial for patients by allowing better definition of the most effective adjuvant nutritional supplementations for intestinal MH.

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