The influence of specific luminal factors on the colonic epithelium : high-dose butyrate and physical changes suppress early carcinogenic events in rats

INTRODUCTION Although luminal delivery of butyrate is one putative mechanism by which biology of the colonic epithelium might be influenced by changes in luminal contents, there is a paucity of supportive cause–effect evidence. This study aimed to directly establish whether distal colonic butyrate delivery is able to alter the response of the distal colonic epithelium to a carcinogen.
METHODS Groups of male Sprague-Dawley rats with chronically intubated colons received infusions of 80 mM butyrate or 0.9 percent saline into distal colon two or five times daily. Three weeks after exposure to azoxymethane (15 mg/kg subcutaneously), the density of aberrant crypts was quantified in distal colon.
RESULTS Infusions of 0.5 ml twice daily, whether containing saline or butyrate, decreased the number of aberrant crypt foci by 45 percent compared with rats receiving no infusions (P = 0.004, analysis of variance). Similar results were obtained when infusions were restricted to the postinitiation phase. When infusions were increased to 1 ml five times daily, saline infusions similarly suppressed aberrant crypt formation (38 percent), but butyrate infusions suppressed it to a greater degree (by 64 percent; P = 0.02 compared with saline infusion, t-test).
CONCLUSIONS High levels of butyrate delivery to the distal colonic lumen alter the epithelial response to a carcinogen in otherwise healthy rats. This finding directly supports the notion that the effects of butyrate on cells in vitro do occur in vivo provided a sufficient dose is delivered. The effect of infusion of liquid per se on the epithelial response highlights the potential impact physical changes alone can have on the colonic epithelium.

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short-chain fatty-acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short-chain fatty-acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post-operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p = 0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). CONCLUSION: Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Emprego dos ácidos graxos de cadeia curta na colite de derivação fecal : estudo em ratos Wistar

Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, after a colostomy. It is postulated that nutritional deficiency of the colonic epithelium by the absence of short-chain fatty acids (SCFA) is one of the factors responsible for the appearance of DC and that their employment could reverse the morphological changes of the mucosa. The treatment of choice for fecal diversion colitis (DC) is the reconstruction of the intestinal tract, although they suggested therapeutic options using enemas. This study evaluates the effect of SCFA in atrophy and inflammation in excluded colonic segments before and after the installation DC. Forty Wistar rats were divided into four groups (n = 10 for each group), submitted colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after postoperative day 40), for 07 days. Histological sections stained with HE were used for histological analysis of the thickness of the colonic mucosa excluded (t- Student p ≤0.05). Inflammatory reaction of the lamina propria and mucosa were measured with scores previously established (Mann Whitney p ≤ 0.05). There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 did not prevent the mucosal atrophy and significant increases in the numbers of lymphocytes (p=0.0006) and 50 eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Alterations in the intestinal wall due to protein malnutrition in rats. Evaluation of the rupture strength and the tissue's collagen

OBJETIVO: Avaliar o efeito da desnutrição protéica na parede intestinal do rato através da medida de força de ruptura e dosagem do colágeno tecidual no íleo e cólon distal. MÉTODOS: Foram utilizados 120 ratos, pesando em média 100g, que receberam durante 07 dias uma dieta padrão, contendo 20% de caseína para adaptação dos animais as condições do biotério. Após esse período os animais foram divididos em dois grupos de 60, o controle denominado grupo um que recebeu a dieta padrão, e o grupo teste denominado grupo dois, que recebeu dieta hipoprotéica contendo 2% de caseína. Os dois grupos receberam suas respectivas dietas por um período de 21 dias. Após esse período iniciou-se o sacrifício seqüencial dos animais em ambos os grupos, em número de 12 animais em cada momento, correspondendo ao dia Zero (MO), 4º dia (M1), 7º dia (M2), 14º dia (M3), e 21º dia (M4) sendo mantida a mesma dieta até o final do sacrifício. em cada momento foram avaliados o peso corpóreo, albumina sanguínea, hidroxiprolina tecidual, relação hidroxiprolina/proteína tecidual e a força de ruptura no segmento ileal e cólico dos animais. RESULTADOS: Observou-se que a força de ruptura do segmento ileal e do cólon distal foi menor nos animais desnutridos (Grupo 2). A perda da resistência mecânica foi maior no segmento do cólon distal do que no segmento ileal, provavelmente pela menor concentração do colágeno tecidual no cólon distal. CONCLUSÃO: A desnutrição protéica induz a diminuição da resistência mecânica no íleo e no cólon distal associado a diminuição do colágeno tecidual na parede intestinal.

Modifying effect of propolis on dimethylhydrazine-induced DNA damage but not colonic aberrant crypt foci in rats

Propolis is a honeybee product with several biological and therapeutic properties, including antimutagenic and anticarcinogenic activities. The effects of an aqueous extract of propolis (AEP) were evaluated on the formation of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and DNA damage in the colon of male Wistar rats by the ACF and Comet assays, respectively. AEP was administered orally at 0.01%, 0.03%, 0.1%, and 0.3% in the drinking water, which resulted in doses of approximately 12, 34, 108, and 336 mg/kg body weight/day. Animals were also given a single subcutaneous injection of 40 mg/kg DMH and sacrificed 4 hr later for evaluating DNA damage, or 4 doses of 40 mg/kg DMH, administered 2 doses/week for 2 weeks, and sacrificed 12 weeks after the last injection for evaluating ACF development in the distal colon. Administration of AEP either simultaneously with or after the DMH treatment resulted in no statistically significant reduction of ACF. In contrast, 0.01%, 0.03%, and 0.3% AEP, given simultaneously with DMH, reduced DNA damage induction in the mid and distal colon. However, 0.3% AEP alone increased DNA damage in the colon. In conclusion, AEP had no effect on the formation of DMH-induced ACF in rat colon, but it modulated DMH-induced DNA damage in colon cells. Further investigations are recommended in order to establish the conditions under which propolis produces either protective or deleterious effects. (C) 2004 Wiley-Liss, Inc.

Lack of chemoprevention of dietary Agaricus blazei against rat colonic aberrant crypt foci

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeitos da injeção de solução bicarbonatada de ácido acetilsalicílico em mucosa colorretal de coelhos, com vistas a aplicação no preparo pré-operatório do cólon

INTRODUÇÃO: A recidiva local no câncer colorretal tem como principal causa o implante de células tumorais nas anastomoses. 11-15 Dessa maneira, lavagem química do lúmen intestinal é preconizada para evitar tanto o implante quanto à recidiva local. 11-28 em estudos prévios constatamos que a solução bicarbonatada de ácido acetilsalicílico tem efeitos citolíticos e anti-tumorais in-vitro.31 OBJETIVOS: Avaliar a toxicidade da solução de aspirina na mucosa colônica de coelhos com o objetivo de usá-la no preparo intestinal de portadores de câncer colorretal. MATERIAIS E MÉTODOS: Foram utilizados 20 coelhos. Um clampe vascular foi colocado acima do cólon sigmóide. Os animais foram submetidos a um enema com 50 ml da solução de aspirina ou soro fisiológico de acordo com o grupo. Os animais foram sacrificados ao término do procedimento ou tardiamente de acordo com o grupo. RESULTADOS: A solução de aspirina não altera a mucosa colônica de coelhos. CONCLUSÃO: O uso da solução bicarbonatada de ácido acetilsalicílico no preparo intestinal de portadores de câncer colorretal é clinicamente possível.

Microscopic, morphometric and ultrastructural analysis of anastomotic healing in the intestine of normal and diabetic rats

The purpose of this study was to investigate if experimental alloxanic diabetes could cause qualitative changes in intestinal anastomoses of the terminal ileum and distal colon in rats, as compared to controls. 192 male Wistar rats, weighing ± 300g were split into four experimental groups of 48 animals each, after 3 months of follow-up: a control group with ileum anastomoses (G1), a control group with colon anastomoses (G2), a diabetic group with ileum anastomoses (G3) and a diabetic group with colon anastomoses (G4). Animals were evaluated and sacrificed on days 4, 14, 21 and 30 after surgery, and fragments of the small and large intestine where the anastomoses were performed were removed. Samples from 6 animals from each sacrifice moment were submitted to ultrastructural analysis of the collagen fibers using a scanning electron microscope and samples from another 6 animals were submitted to histopathology and optical microscopy studies using picrosirius red-staining. Histopathological analysis of picrosirius red-stained anastomosis slides using an optical microscope at 40x magnification showed that the distribution of collagen fibers was disarranged and also revealed a delay in scar tissue retraction. The morphometric study revealed differences in the collagen filled area for the ileum anastomoses 14 days post surgery whereas, in the case of colon anastomoses, differences were observed at days 4 and 30 post surgery, with higher values in the diabetic animals. Ultrastructure analysis of the ileum and colon anastomoses using a scanning electron microscope revealed fewer wide collagen fibers, the presence of narrower fibers and a disarranged distribution of the collagen fibers. We conclude that diabetes caused qualitative changes in scar tissue as well as in the structural arrangement of collagen fibers, what could explain the reduced wound strength in the anastomosis of diabetic animals. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart.

Displasia neuronal intestinal: análise de critérios morfológicos e comparação de métodos diagnósticos

Pós-graduação em Patologia - FMB

Oral treatment with Hev b 13 ameliorates experimental colitis in mice

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human mononuclear cells to release interleukin (IL)-10 in vitro. This immunoregulatory cytokine appears to play an important role in preventing inflammation and mucosal damage in animal models of colitis and in Crohn's disease patients. The aim of this study was to evaluate the therapeutic effect of Hev b 13 in mice with 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. Two hours following colonic instillation of the haptenizing agent, and daily thereafter for 5 days, Hev b 13 was administered by oral gavage. In mice treated with daily doses of either 0.5 mg/kg or 5.0 mg/kg of Hev b 13, the clinical signs of diarrhoea, rectal prolapse and body weight loss and also histological damage of the distal colon, were reduced significantly, in comparison with water-treated diseased mice. These findings suggest a potent anti-inflammatory activity of Hev b 13; this activity is speculated to be related to its interaction with cells from the immune system.

Oral treatment with Hev b 13 prevents experimental arthritis in mice

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human monocytes to release interleukin (IL)-10 in vitro, and to exert a potent anti-inflammatory effect in vivo. Moreover, Hev b 13 has been shown to reduce clinical signs of inflammation and also histological damage to the distal colon of mice with 2,4,6-trinitrobenze sulphonic acid (TNBS)-induced colitis after its oral administration. The aim of this study was to investigate the effect of Hev b 13 on human mononuclear cells, as well as its therapeutic use in the methylated bovine serum albumin (mBSA) model of antigen-induced arthritis. Five days before the intra-articular challenge, and daily thereafter for 8 days, Hev b 13 was administered by oral gavage. In mice treated with a dose of 0.5 mg/kg of Hev b 13, the severity of oedema, leucocyte infiltration, pannus formation and cartilage erosion were reduced significantly. These findings underscore the anti-inflammatory activity suggested previously for Hev b 13, an activity speculated to be related to its interaction with monocytes/macrophages and the consequent stimulation of IL-10 release and reduction of tumour necrosis factor (TNF) release. The study also opens a wide range of possible applications in the field of immune-mediated inflammatory diseases.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

Intestinal atresia and ectopia in a bovine fetus

A 2-year-old Red Holstein cow was presented with uterine torsion at 235 days of pregnancy. The fetus extracted by cesarean section had weak vital signs and marked abdominal distention. An edematous pouch that contained tubular structures with peristaltic activity was associated with the umbilical cord. Because of poor prognosis, both dam and fetus were euthanized. At necropsy, the fetus had severe distention of the forestomachs, abomasum, and proximal small intestine; absence of distal small intestine, cecum, and proximal colon; atresia of the 2 blind ends of the intestine; and atrophy of distal colon and rectum. The tubular structures associated with the umbilical cord were identified as the segments of intestine that were absent in the fetus. Intestinal atresia combined with ectopia may be caused by local ischemia during temporary herniation and rotation of the fetal gut into the extraembryonic coelom. The close connection between ectopic intestine and amniotic sheath of the umbilical cord in this case may have facilitated vascularization and allowed development and viability of the ectopic intestine.

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.

Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.

The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro.