Role of Ras in regulation of intestinal epithelial cell homeostasis and crosstalk with Wnt signaling.

Cross talk between different signaling pathways is thought to be important for regulation of homeostasis of, as well as oncogenesis of, the intestinal epithelium. Expression of an active form of K-Ras specifically in intestinal epithelial cells (IECs) of mice (IEC-RasDA mice) resulted in the development of hyperplasia in the small intestine and colon of mice. IEC-RasDA mice also manifested the increased proliferation of IECs. In addition, the number of goblet cells markedly increased, while that of Paneth cells decreased in IEC-RasDA mice. Development of intestinal organoids was markedly enhanced for IEC-RasDA mice compared with control mice. Whereas, the expression of Wnt target genes was significantly reduced in the in intestinal crypts from IEC-RasDA mice compared with that apparent for the control. Our results thus suggest that K-Ras promotes the proliferation of IECs as well as generation of goblet cells. By contrast, Ras counter-regulates the Wnt signaling and thereby contribute to the proper regulation of intestinal epithelial cell homeostasis.

RAB11A-mediated YAP localization to adherens and tight junctions is essential for colonic epithelial integrity.

Within the intestinal epithelium, regulation of intracellular protein and vesicular trafficking is of utmost importance for barrier maintenance, immune responses, and tissue polarity. RAB11A is a small GTPase that mediates the anterograde transport of protein cargos to the plasma membrane. Loss of RAB11A-dependent trafficking in mature intestinal epithelial cells results in increased epithelial proliferation and nuclear accumulation of Yes-associated protein (YAP), a key Hippo-signaling transducer that senses cell-cell contacts and regulates tissue growth. However, it is unclear how RAB11A regulates YAP intracellular localizations. In this report, we examined the relationship of RAB11A to epithelial junctional complexes, YAP, and the associated consequences on colonic epithelial tissue repair. We found that RAB11A controls the biochemical associations of YAP with multiple components of adherens and tight junctions, including α-catenin, ß-catenin, and Merlin, a tumor suppressor. In the absence of RAB11A and Merlin, we observed enhanced YAP-ß-catenin complex formation and nuclear translocation. Upon chemical injury to the intestine, mice deficient in RAB11A were found to have reduced epithelial integrity, decreased YAP localization to adherens and tight junctions, and increased nuclear YAP accumulation in the colon epithelium. Thus, RAB11A-regulated trafficking regulates the Hippo-YAP signaling pathway for rapid reparative response after tissue injury.

BMP Antagonists Secreted by Mesenchymal Stromal Cells Improve Colonic Organoid Formation: Application for the Treatment of Radiation-induced Injury.

Radiation therapy is crucial in the therapeutic arsenal to cure cancers; however, non-neoplastic tissues around an abdominopelvic tumor can be damaged by ionizing radiation. In particular, the radio-induced death of highly proliferative stem/progenitor cells of the colonic mucosa could induce severe ulcers. The importance of sequelae for patients with gastrointestinal complications after radiotherapy and the absence of satisfactory management has opened the field to the testing of innovative treatments. The aim of this study was to use adult epithelial cells from the colon, to reduce colonic injuries in an animal model reproducing radiation damage observed in patients. We demonstrated that transplanted in vitro-amplified epithelial cells from colonic organoids (ECO) of C57/Bl6 mice expressing green fluorescent protein implant, proliferate, and differentiate in irradiated mucosa and reduce ulcer size. To improve the therapeutic benefit of ECO-based treatment with clinical translatability, we performed co-injection of ECO with mesenchymal stromal cells (MSCs), cells involved in niche function and widely used in clinical trials. We observed in vivo an improvement of the therapeutic benefit and in vitro analysis highlighted that co-culture of MSCs with ECO increases the number, proliferation, and size of colonic organoids. We also demonstrated, using gene expression analysis and siRNA inhibition, the involvement of bone morphogenetic protein antagonists in MSC-induced organoid formation. This study provides evidence of the potential of ECO to limit late radiation effects on the colon and opens perspectives on combined strategies to improve their amplification abilities and therapeutic effects.

Intestinal immunity in hypopituitary dwarf mice: effects of age.

Hypopituitary dwarf mice demonstrate advantages of longevity, but little is known of their colon development and intestinal immunity. Herein we found that Ames dwarf mice have shorter colon and colonic crypts, but larger ratio of mesenteric lymph nodes (MLNs) over body weight than age-matched wild type (WT) mice. In the colonic lamina propria (cLP) of juvenile Ames mice, more inflammatory neutrophils (A: 0.15% vs. 0.03% in WT mice) and monocytes (A: 7.97% vs. 5.15%) infiltrated, and antigen presenting cells CD11c+ dendritic cells (A: 1.39% vs. 0.87%), CD11b+ macrophages (A: 3.22% vs. 0.81%) and gamma delta T (γδ T) cells (A: 5.56% vs. 1.35%) were increased. In adult Ames dwarf mice, adaptive immune cells, such as IL-17 producing CD4+ T helper (Th17) cells (A: 8.3% vs. 4.7%) were augmented. In the MLNs of Ames dwarf mice, the antigen presenting and adaptive immune cells also altered when compared to WT mice, such as a decrease of T-regulatory (Treg) cells in juvenile Ames mice (A: 7.7% vs.10.5%), but an increase of Th17 cells (A: 0.627% vs.0.093%). Taken together, these data suggest that somatotropic signaling deficiency influences colon development and intestinal immunity.

Corrupted Colonic Crypts Bordering Regenerating Mucosal Ulcers in Ulcerative Colitis.

BACKGROUND/AIM: Histology in protracted ulcerative colitis (UC) discloses high numbers of chronic inflammatory cells and crypts with architectural distortions. In severe cases, ulcerations are frequently found. The histogenesis of colonic crypts with architectural distortions in UC remains elusive. A recent review of colectomy specimens from patients with UC revealed crypts surrounding mucosal ulcerations exhibiting severe architectural distortions. They were called corrupted colonic crypts, CCCs. MATERIAL AND METHODS: Archival hematoxylin and eosin (H&E)-stained sections from three colectomies having several mucosal ulcers were selected for the study. The mucosa bordering mucosal ulcers was particularly scrutinized. RESULTS: The review of 49 sections (mean=16.3, range=14-20) in the three colectomies revealed 60 ulcers (mean=20, range=13-27). The following CCC phenotypes were found bordering mucosal ulcers: with asymmetric lateral fission (n=11), with dual or three-foiled corrupted fission (n=19), with cystic dilatations (n=3), L-shaped crypts (n=7), T-inverted crypts (n=6), shoe-shaped crypts (n=3), horizontal crypts (n=14), multi-lobate crypts (n=2), and/or inter-connecting crypts (n=5). CONCLUSION: The regeneration of ulcers in UC seems to proceed with neo-formation of corrupted crypts. In the same colectomies, none to occasional CCCs were found in large areas of the mucosa having severe chronic inflammation. Importantly, none of the occasional CCCs were found in other diseases of the colonic mucosa with chronic inflammation or in unspecific ulcers of the colon. Since neither chronic mucosal inflammation per se, nor unspecific ulcers of the colon are central for the formation of CCCs, it is suggested that crypt distortions of the non-ulcerated colonic mucosa in patients with UC might mirror formerly healed mucosal ulcerations.

Fish oil, lard and soybean oil differentially shape gut microbiota of middle-aged rats.

High-fat diets have been associated with overweight/obesity and increased mortality in middle-aged populations. However, it is still unclear how gut microbiota in middle-aged populations responds to dietary fats at a normal dose. In this study, we explored gut microbiota structure in middle-aged rats (aged 12 months) after feeding 4% (w/w) soybean oil, lard or fish oil for 3 months, respectively. The results showed that the gut microbiota structure in the fish oil group was substantially different from those of the soybean oil and lard groups in both in vitro and in vivo studies. The relative abundances of phylum Proteobacteria and genus Desulfovibrio in the caecal and colonic contents were the highest in the fish oil group (p < 0.05). The mRNA levels of biomarkers for inflammation in the colon, including IL-1ß, IL-6, IL-17, IL-18 and TNF-α, were also the highest in the fish oil group (p < 0.05). Meanwhile, the fish oil group had the highest microbial DNA abundance of a predicted lipid metabolism. Our results gave a new insight into the potentially negative impact of fish oil diet on health of middle-aged populations by changing gut microbiota and inducing inflammation as compared to soybean oil and lard diets.

The TNF-α antagonist etanercept reverses age-related decreases in colonic SERT expression and faecal output in mice.

Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age-related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24-month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg-1). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to "anti-TNFα" drugs as potential treatments for age-related chronic constipation.

Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon.

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4(+) DCS cells promote organoid formation of single Lgr5(+) colon stem cells. DCS cells can be massively produced from Lgr5(+) colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4(+) DCS cells serve as Paneth cell equivalents in the colon crypt niche.

The role of cell location and spatial gradients in the evolutionary dynamics of colon and intestinal crypts.

BACKGROUND: Colon and intestinal crypts serve as an important model system for adult stem cell proliferation and differentiation. We develop a spatial stochastic model to study the rate of somatic evolution in a normal crypt, focusing on the production of two-hit mutants that inactivate a tumor suppressor gene. We investigate the effect of cell division pattern along the crypt on mutant production, assuming that the division rate of each cell depends on its location. RESULTS: We find that higher probability of division at the bottom of the crypt, where the stem cells are located, leads to a higher rate of double-hit mutant production. The optimal case for delaying mutations occurs when most of the cell divisions happen at the top of the crypt. We further consider an optimization problem where the "evolutionary" penalty for double-hit mutant generation is complemented with a "functional" penalty that assures that fully differentiated cells at the top of the crypt cannot divide. CONCLUSION: The trade-off between the two types of objectives leads to the selection of an intermediate division pattern, where the cells in the middle of the crypt divide with the highest rate. This matches the pattern of cell divisions obtained experimentally in murine crypts. REVIEWERS: This article was reviewed by David Axelrod (nominated by an Editorial Board member, Marek Kimmel), Yang Kuang and Anna Marciniak-Czochra. For the full reviews, please go to the Reviewers' comments section.

Development and evaluation of a porcine in vitro colon organ culture technique.

The intestinal mucosa comprises a complex assemblage of specialized tissues that interact in numerous ways. In vitro cell culture models are generally focused on recreating a specific characteristic of this organ and do not account for the many interactions between the different tissues. In vitro organ culture (IVOC) methods offer a way to overcome these limitations, but prolonging cell viability is essential. This study aimed to determine the feasibility and optimal conditions for in vitro culture of swine colonic mucosa for use as an enteric pathogen infection model. Explants (n = 168) from commercial pigs (n = 12), aged 5 to 10 wk, were used to assess the impact of various culture protocols on explant viability. Explants were cultured for up to 5 d and formalin fixed at 24-h intervals. Following establishment of the culture protocol, explants (n = 208) from 13 pigs were evaluated at Day 0 and 5 of culture. Assessment of viability was based on histological changes (tissue architecture evaluated by H&E, immunostaining of cell proliferation marker Ki-67) and expression of genes encoding IL-1α, IL-8, TNF-α, IFN-γ, and e-cadherin. After 5 d in culture, 20% of explants displayed over 80% of epithelial coverage, whereas 31% of explants had more than 50% of their surface covered by columnar epithelium, and 81% had crypts but with a decreased number of Ki-67-positive cells when compared to Day 0. Notably, large variability in explant quality was observed between donor pigs. Best possible explants were obtained from the distal colon of pigs, processed immediately after euthanasia, cultured at the liquid-tissue-gas interface in media supplemented with a mixture of antibiotics and antifungals and an oxygen-rich gas mix.

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice.

Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid intraperitoneally and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study, we sought to determine whether the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 wk of age to wild-type, CD44(-/-), and TLR4(-/-) mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life, we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5(+) stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5(+) reporter mice from postnatal day 7 to day 14 decreased Lgr5(+) cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5(+) stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4.

Comparative in vitro fermentations of cranberry and grape seed polyphenols with colonic microbiota.

In this study, we have assessed the phenolic metabolism of a cranberry extract by microbiota obtained from the ascending colon and descending colon compartments of a dynamic gastrointestinal simulator (SHIME). For comparison, parallel fermentations with a grape seed extract were carried out. Extracts were used directly without previous intestinal digestion. Among the 60 phenolic compounds targeted, our results confirmed the formation of phenylacetic, phenylpropionic and benzoic acids as well as phenols such as catechol and its derivatives from the action of colonic microbiota on cranberry polyphenols. Benzoic acid (38.4µg/ml), 4-hydroxy-5-(3'-hydroxyphenyl)-valeric acid (26.2µg/ml) and phenylacetic acid (19.5µg/ml) reached the highest concentrations. Under the same conditions, microbial degradation of grape seed polyphenols took place to a lesser extent compared to cranberry polyphenols, which was consistent with the more pronounced antimicrobial effect observed for the grape seed polyphenols, particularly against Bacteroides, Prevotella and Blautia coccoides-Eubacterium rectale.

Apelin's effects on young rat gastrointestinal tract maturation.

Apelin is considered an important gut regulatory peptide with potential physiological roles in gastrointestinal cytoprotection and regulation of food intake and drinking behavior. The aim of this study was to determine the effects of intraperitoneal or intragastric apelin administration on gastric and intestinal epithelial apoptosis, mitosis and DNA repair enzyme 8-oxoguanine (OGG 1/2) expression in young Wistar rats (50±5 g b.wt.). Apelin-13 was intraperitoneally or intragastrically administered twice a day for 10 days (100 nmol/kg b.wt./2×day), and control groups received physiological saline as a placebo. The rats were sacrificed after treatment, and the gastric fundus, duodenum, middle jejunum and colon tissue samples were harvested for immunofluorescence studies. Intragastric administration of apelin-13 increased the apoptotic index in the stomach and colon tissues (P≤0.001) but decreased apoptosis in the duodenum and jejunum (P<0.001); this approach reduced the number of mitotic cells in the jejunum and colon but increased mitoses (P<0.001) in the duodenum. Finally, intragastric apelin-13 increased (P<0.001) OGG 1/2 enzyme expression in the stomach and jejunum and decreased its expression in the colon (P<0.01). However, intraperitoneal apelin-13 injection caused the opposite effect in the same regions of the gastrointestinal tract. In conclusion, apelin inhibits gastrointestinal tissue maturation in young rats, regardless of the administration route. However, further studies are required to clarify the mechanism of apelin action on gastrointestinal tract maturation in young rats.

Iron supplementation promotes gut microbiota metabolic activity but not colitis markers in human gut microbiota-associated rats.

The global prevalence of Fe deficiency is high and a common corrective strategy is oral Fe supplementation, which may affect the commensal gut microbiota and gastrointestinal health. The aim of the present study was to investigate the impact of different dietary Fe concentrations on the gut microbiota and gut health of rats inoculated with human faecal microbiota. Rats (8 weeks old, n 40) were divided into five (n 8 each) groups and fed diets differing only in Fe concentration during an Fe-depletion period (12 weeks) and an Fe-repletion period (4 weeks) as follows: (1) Fe-sufficient diet throughout the study period; (2) Fe-sufficient diet followed by 70 mg Fe/kg diet; (3) Fe-depleted diet throughout the study period; (4) Fe-depleted diet followed by 35 mg Fe/kg diet; (5) Fe-depleted diet followed by 70 mg Fe/kg diet. Faecal and caecal samples were analysed for gut microbiota composition (quantitative PCR and pyrosequencing) and bacterial metabolites (HPLC), and intestinal tissue samples were investigated histologically. Fe depletion did not significantly alter dominant populations of the gut microbiota and did not induce Fe-deficiency anaemia in the studied rats. Provision of the 35 mg Fe/kg diet after feeding an Fe-deficient diet significantly increased the abundance of dominant bacterial groups such as Bacteroides spp. and Clostridium cluster IV members compared with that of an Fe-deficient diet. Fe supplementation increased gut microbial butyrate concentration 6-fold compared with Fe depletion and did not affect histological colitis scores. The present results suggest that Fe supplementation enhances the concentration of beneficial gut microbiota metabolites and thus may contribute to gut health.

Hyaluronic acid regulates normal intestinal and colonic growth in mice.

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.

Aging-induced alterations in female rat colon smooth muscle: the protective effects of hormonal therapy.

Aging is associated to oxidative damage and alterations in inflammatory and apoptotic pathways. Aging impairs secretion of several hormones, including melatonin and estrogens. However, the mechanisms involved in aging of smooth muscle are poorly known. We have studied the changes induced by aging in the colonic smooth muscle layer of female rats and the protective effect of hormonal therapy. We used young, aged, and ovariectomized aged female rats. Two groups of ovariectomized rats (22 months old) were treated either with melatonin or with estrogen for 10 weeks before sacrifice. Aging induced oxidative imbalance, evidenced by H(2)O(2) accumulation, lipid peroxidation, and decreased catalase activity. The oxidative damage was enhanced by ovariectomy. In addition, aged colonic muscle showed enhanced expression of the pro-inflammatory enzyme cyclooxygenase 2. Expression of the activated forms of caspases 3 and 9 was also enhanced in aged colon. Melatonin and estrogen treatment prevented the oxidative damage and the activation of caspases. In conclusion, aging of colonic smooth muscle induces oxidative imbalance and activation of apoptotic and pro-inflammatory pathways. Hormonal therapy has beneficial effects on the oxidative and apoptotic changes associated to aging in this model.

Identity and ranking of colonic mesenchymal stromal cells.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.

High-performance inulin and oligofructose prebiotics increase the intestinal absorption of iron in rats with iron deficiency anaemia during the growth phase.

Considering the high frequency of anaemia due to Fe deficiency, it is important to evaluate the effects of prebiotics on the absorption of Fe. The aim of the present study was to evaluate the effects of high-performance (HP) inulin, oligofructose and synergy1 during recovery from anaemia in rats through the intestinal absorption of Fe, food intake, body growth, caecal pH and weight of the intestine. Wistar rats (n 47) were fed with rations of AIN93-G with no Fe to induce Fe deficiency anaemia. At 36 d of life, anaemic rats were divided into four groups: (1) the HP inulin group; (2) the synergy1 group; and (3) the oligofructose group, all with 100 g of the respective prebiotic per kg of ration; and (4) a control group, in which the prebiotic was replaced by maize starch. Then, 25 mg of elemental Fe/kg of ration was added to all rations to allow recovery from anaemia. The final values of Hb in the HP inulin, synergy1, oligofructose and control groups were, respectively: 98 (94-99); 83 (81-92); 100 (90-114); 77 (72-81) g/l, with a statistically significant difference (P ≤ 0·001) between the oligofructose and control groups and the HP inulin and control groups. The four groups had an increase in weight and body length and had similar consumption of rations. The intestinal weight and caecal pH were significantly different between the groups that consumed prebiotics and the control group. HP inulin and oligofructose increased the intestinal absorption of Fe in rats.

Influence of broccoli extract and various essential oils on performance and expression of xenobiotic- and antioxidant enzymes in broiler chickens.

The aim of our present study was to examine the regulation of xenobiotic- and antioxidant enzymes by phytogenic feed additives in the intestine and the liver of broilers. A total of 240 male Ross-308 broiler chickens (1 d old) were fed a commercial starter diet for 2 weeks. On day 15, the birds were assigned to six treatment groups of forty birds each. The control (Con) group was fed a diet without any additive for 3 weeks. The diet of group sulforaphane (SFN) contained broccoli extract providing 0.075 g/kg SFN, whereas the diets of the other four groups contained 0.15 g/kg essential oils from turmeric (Cuo), oregano (Oo), thyme and rosemary (Ro). Weight gain and feed conversion were slightly impaired by Cuo and Oo. In the jejunum SFN, Cuo and Ro increased the expression of xenobiotic enzymes (epoxide hydrolases 1 and 2 and aflatoxin B1 aldehyde reductase) and of the antioxidant enzyme haeme oxygenase regulated by an 'antioxidant response element' (ARE) compared to group Con. In contrast to our expectations in the liver, the expression of these enzymes was decreased by all the additives. Nevertheless, all the additives increased the Trolox equivalent antioxidant capacity of the jejunum and the liver and reduced Fe-induced lipid peroxidation in the liver. We conclude that the up-regulation of ARE genes in the small intestine reduces oxidative stress in the organism and represents a novel mechanism by which phytogenic feed additives improve the health of farm animals.

α-fetoprotein involvement during glucocorticoid-induced precocious maturation in rat colon.

AIM: To investigate the role of α-fetoprotein (AFP), a cancer-associated fetal glycoprotein, in glucocorticoid-induced precocious maturation in rat colon. METHODS: Colons from suckling Sprague-Dawley rats were used in this study. Corticosterone acetate at a dose of 100 µg/g body weight was given to normal pups on days 7, 9 and 11 after birth to induce hypercorticoidism. Control animals were injected with identical volumes of normal saline. Some rats receiving corticosterone 7 d after birth were also treated with mifepristone (RU38486), a glucocorticoid cytoplasm receptor antagonist to investigate the effects of glucocorticoids (GCs). The morphological changes of the crypt depth and villous height of the villous zone in colon were observed as indices of colon maturation. Expression levels of AFP in colons were detected by reverse transcriptase polymerase chain reaction and Western blotting. To identify the cellular localization of AFP in developing rat colons, double-immunofluorescent staining was performed using antibodies to specific mesenchymal cell marker and AFP. RESULTS: Corticosterone increased the crypt depth and villous height in the colon of 8- and 10-d-old rats with hypercorticoidism compared with that in the control animals (120% in 8-d-old rats and 118% in 10-d-old rats in villous height, P = 0.021; 145% in 8-d-old rats and 124% in 10-d-old rats in crypt depth, P = 0.017). These increases were accompanied by an increase of AFP expression in both mRNA and protein (2.5-folds in 8-d-old and 2.5-folds in 10-d-old rats higher than in control animals, P = 0.035; 1.8-folds in 8-d-old and 1.3-folds in 10-d-old rats higher than in control animals, P = 0.023). Increased crypt depth and villous height and increased expression of AFP in the colon of rats with hypercorticoidism were blocked by mifepristone. Both had positive staining for AFP or vimentin, and overlapped in mesenchymal cells at each tested colon. CONCLUSION: GCs promote the development of rat colon. AFP appears to be involved, in part, in mediating the effects of GCs in the developmental colon.