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and Nitric Oxide Production in Trinitrobenzenesulfonic Acid-Induced Colitic Rats1 ,2
*
Department of Pharmacology, School of Pharmacy, and
Department of Parasitology, School of Sciences, University of Granada, Spain,
**
Department of Pathology, Hospital Universitario "Virgen de las Nieves," Granada, Spain and
Madaus Laboratories S.A., Barcelona, Spain
4To whom correspondence should be addressed. E-mail: jgalvez{at}ugr.es.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(TNF) and nitric oxide (NO). Rats were fed the fiber-supplemented diet for 2 wk before TNBS colitis induction and thereafter until colonic evaluation 1 wk later. The results obtained showed that dietary fiber supplementation facilitated recovery from intestinal insult as evidenced both histologically, by a preservation of intestinal cytoarchitecture, and biochemically, by a significant reduction in colonic myeloperoxidase activity and by restoration of colonic glutathione levels. This intestinal anti-inflammatory effect was associated with lower TNF levels and lower NO synthase activity in the inflamed colon, showing significant differences when compared with nontreated colitic rats. Moreover, the intestinal contents from fiber-treated colitic rats showed a significantly higher production of SCFA, mainly butyrate and propionate. We conclude that the increased production of these SCFA may contribute to recovery of damaged colonic mucosa because they constitute substrates for the colonocyte and, additionally, that they can inhibit the production of proinflammatory mediators, such as TNF and NO.
KEY WORDS: • dietary fiber • rat colitis • tumor necrosis factor • nitric oxide synthase • short chain fatty acids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). As a consequence, the up-regulation in the synthesis and release of different proinflammatory mediators, including reactive oxygen and nitrogen metabolites, eicosanoids, platelet-activating factor and cytokines, takes place (2
). All these mediators actively contribute to the pathogenic cascade that initiates and perpetuates the inflammatory response of the gut. In fact, the intestinal epithelium acts as a defense against invasion by luminal toxins and bacteria but, as a consequence of the inflammation, the barrier function of the epithelium is impaired and the subsequent translocation of endotoxins and antigens up-regulates the immune response even more (3
). Therefore, we speculate that an improvement in mucosal integrity of the intestinal barrier function may contribute to a reduction of intestinal inflammation (4
,5
).
An important role in the maintenance of colonic homeostasis has been attributed to SCFA, mainly acetate, propionate and butyrate. They are produced in the large bowel by the anaerobic bacterial fermentation of undigested dietary carbohydrates and fiber polysaccharides, with butyrate being considered the major fuel source for colonocytes (6
). It has been suggested that a diminished ß-oxidation of luminal butyrate to CO2 and ketones, resulting in energy deficiency within colonic epithelial cells, could contribute to the pathogenesis of ulcerative colitis (7
). In addition, several authors have reported decreased fecal concentrations of SCFA in patients with ulcerative colitis and in the cotton-top tamarin model of idiopathic colitis (8
,9
). All these facts suggest that butyrate enemas may alleviate the symptoms of ulcerative colitis by restoring luminal levels of butyrate and thus facilitating the mucosal recovery from inflammation, as previously reported (10
,11
). However, other studies have described the lack of effectiveness of treatment with SCFA enemas (or only butyrate enemas), most probably because of poor compliance with the protocols (12
,13
). To avoid this, Fernandez-Bañares et al. (14
) carried out a controlled investigation of oral administration of a SCFA substrate (fermentable dietary fiber from Plantago ovata seeds) in patients with ulcerative colitis. This study reported the efficacy and safety of this kind of fermentable dietary fiber versus mesalamine to maintain remission in these patients. Furthermore, the beneficial effect exerted by long-term P. ovata oral administration was associated with increased butyrate concentrations in the distal colon.
The aim of the present study was to elucidate the probable mechanisms involved in the beneficial effects of P. ovata seeds in the treatment of IBD. We tested the preventive effects of a dietary fiber-supplemented diet (5% P. ovata seeds) in the trinitrobenzenesulfonic acid (TNBS)-induced rat model of colitis, a well established model of intestinal inflammation with some resemblance to human IBD (15
). Special attention was paid to its effects on the production of some of the mediators involved in the inflammatory response, such as tumor necrosis factor (TNF) and nitric oxide (NO).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Reagents.
All chemicals, including lipopolysaccharide (LPS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were obtained from Sigma Chemical (Madrid, Spain) unless otherwise stated. Glutathione reductase was provided by Boehringer Mannheim (Barcelona, Spain); L-[2,3,4,5-3H]arginine monohydrochloride was purchased from Amersham Iberica (Barcelona, Spain); 2-methylvaleric acid was obtained from Fluka (Madrid, Spain); 2', 7'-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was supplied by Molecular Probes (Leiden, The Netherlands). P. ovata seeds were provided by Madaus S.A. (Barcelona, Spain). The fiber-supplemented diet was prepared by adding 5 g of P. ovata seeds to 95 g of pulverized standard diet for rats (Panlab A04; Panlab, Barcelona, Spain). The composition of the standard diet was as follows: 17.2% protein, 2.7% fat, 59.7% carbohydrates, 3.9% fiber, 4.4% minerals and 12% humidity.
Experimental design.
Female Wistar rats (180–200 g) obtained from the Laboratory Animal Service of the University of Granada (Granada, Spain) were housed individually in makrolon cages and maintained in an air-conditioned atmosphere with a 12-h light-dark cycle, and they had free access to tap water and food. The rats were randomly assigned to three groups (n = 15); two (noncolitic and control groups) received the standard diet and the other (treated group) was fed the fiber-supplemented diet for 3 wk. Two weeks after starting the experiment, the rats were starved overnight and those from the control and treated groups were rendered colitic by the method originally described by Morris et al. (16
). Briefly, they were anesthetized with halothane and given 10 mg of TNBS dissolved in 0.25 mL of 50% ethanol (v/v) by means of a Teflon cannula inserted 8 cm through the anus. During and after TNBS administration, the rats were kept in a head-down position until they recovered from the anesthetic and were then returned to their cages. Rats from the noncolitic group were administered intracolonically 0.25 mL of phosphate-buffered saline (PBS) instead of TNBS. All rats were killed with an overdose of halothane 1 wk after induction of colitis.
Assessment of colonic damage.
The body weight and food intake were recorded daily throughout the experiment. Once the rats were killed, the colon was removed and placed on an ice-cold plate, the luminal contents were collected for SCFA quantification (see below) and the colonic segment was cleaned of fat and mesentery and blotted on filter paper. Each specimen was weighed and its length was measured under a constant load (2 g). The colon was longitudinally opened and scored for macroscopically visible damage on a 0–10 scale by two observers unaware of the treatment, according to the criteria described by Bell et al. (17
). (Table 1
), which take into account the extent as well as the severity of colonic damage. The colon was subsequently divided into four segments for biochemical determinations. Two fragments were frozen at -80°C for myeloperoxidase (MPO) and NO synthase (NOS) activities and another sample was weighed and frozen in 1 mL of 50 g/L trichloroacetic acid for total glutathione content determination. The remaining sample was immediately processed for the measurement of TNF and leukotriene B4 (LTB4) levels. All biochemical measurements were completed within 1 wk from the time of sample collection and were performed in duplicate.
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). The results were expressed as MPO units per gram of wet tissue; one unit of MPO activity was defined as that degrading 1 µmol hydrogen peroxide/min at 25°C. Total glutathione content was quantified with the recycling assay described by Anderson (19
) and the results were expressed as nmol/g wet tissue.
Colonic samples for TNF and LTB4 determinations were immediately weighed, minced on an ice-cold plate and suspended in a tube with 10 mmol/L sodium phosphate buffer (pH 7.4) (1:5 wt/v). The tubes were placed in a shaking water bath (37°C) for 20 min and centrifuged at 9000 x g for 30 s at 4°C; the supernatants were frozen at -80°C until assay. TNF was quantified by enzyme-linked immunoabsorbent assay (Amersham Pharmacia Biotech, Little Chalfont, UK) and the results were expressed as picograms per gram of wet tissue. LTB4 was determined by enzyme immunoassay (Amersham Pharmacia Biotech) and the results were expressed as nanograms per gram of wet tissue.
Colonic NOS activity was determined by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline (20
). For this purpose, the intestinal samples were homogenized (1:5 wt/v) for 60 s in 10 mmol/L N-2-ethanesulphonic acid (pH 7.4) containing sucrose (0.32 mol/L), EDTA (100 µmol/L), phenylmethylsulfonyl fluoride (1 g/L) and leupeptin (10 mg/L); the resulting homogenate was centrifuged at 10,000 x g for 10 min at 4°C, and the supernatants were assayed for protein content according to the method proposed by Bradford (21
). Samples (40 µg of protein) were incubated at room temperature for 30 min in the presence of nicotinamide adenine dinucleotide phosphate (1 mmol/L), calmodulin (0.3 µmol/L), tetrahydrobiopterin (10 µmol/L), CaCl2 (2 mmol/L), L-valine (10 mmol/L; to inhibit nonspecific arginase activity) and L-[3H]arginine (100 µmol/L, 10 mCi/L). Incubations were terminated by the addition of 1 mL N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] (20 mmol/L, pH 5.5) containing 1 mmol/L EGTA and 1 mmol/L EDTA. L-[3H]citrulline was separated from arginine by adding 1.5 mL of a 1:1 suspension of Dowex (50W) in water. Formation of citrulline in the presence of excess of L-NAME (10 mmol/L) was also determined to be able to subtract background citrulline formation. Radioactivity was measured in supernatants by liquid scintillation counting and the results were expressed as pmol L-citrulline/(mg protein x 30 min).
SCFA quantification in colonic contents.
Colonic contents from 10 different rats from each group were obtained and homogenized with NaHCO3 (150 mmol/L, pH 7.8) (1:5 wt/v) in a nitrogen atmosphere. Aliquots of 10 mL from each fecal sample were then incubated in duplicate for 24 h at 37°C either in the presence of P. ovata seeds (at a final concentration of 10 g/L) or in the absence of substrate (control incubations). Incubation was terminated by freezing and specimens were stored at -80°C until gas-liquid chromatographic analysis of the SCFA. Briefly, sulfuric acid was added to fecal homogenates and they were centrifuged at 10,000 x g for 5 min at 4°C and SCFA from supernatants were extracted in ethylether. After this, 2 µl of the sample was injected splitless into a CarloErba 8060 gas chromatograph equipped with a 250-µm (internal diameter), 30 m INNOWAX capillary column with a film thickness of 0.25 µm, associated with a Mass Detector Platform II (Micromass, UK). Carrier and make-up gas were both helium, each with a flow rate of 1 mL/min. Injection temperature was 200°C. The initial oven temperature was 80°C; this was maintained for 2 min before the temperature was raised 5°C/min to 200°C. Acetate, propionate and butyrate concentrations were calculated from areas of peaks (internal standard was 2-methylvaleric acid) automatically calculated using Masslynx program version 2.1 from Fisons connected on-line to the mass detector.
Histological analysis.
Five additional rats from each group were used for colonic histological studies. Representative whole gut specimens were taken from a region of the inflamed colon corresponding to the gross macroscopic damage in the distal colon and were fixed in 4% buffered formaldehyde. Cross-sections were selected and embedded in paraffin. Equivalent colonic segments were also obtained from the noncolitic group. Sections (5 µm thick) were obtained at different levels and stained with hematoxylin and eosin. The histological damage was scored on a scale from 0 to 20 according to criteria adapted from those reported by Millar et al. (22
) (Table 2
). Two pathologists (A.C. and M.D.L.) who were unaware of the treatment conditions recorded the histological damage.
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Five additional rats from each group were used. The colonic segment adjacent to gross macroscopic damage (2 cm in length) was gently flushed and the serosa and outer muscle layers were separated by blunt dissection. Each sample was cut into two longitudinal segments, which were placed on cover glasses and incubated in the dark in Dulbecco’s modified Eagle’s medium (DMEM) containing BCECF-AM (10 µmol/L) for 45 min at 37°C. Samples were washed three times in Earle’s balanced salts solution to rid them of any surplus BCECF-AM. They were then incubated in Hanks’ solution (without glucose) supplemented with either glucose (5.5 mmol/L) or butyrate sodium salt (4 mmol/L). Intracellular pH was measured immediately after substrate supplementation (t = 0) and after 15 min of incubation at 37°C in a Hitachi F2000 spectrofluorometer with an excitation source at an angle of 45 degrees. The pHi was calculated on the basis of the ratio 505:439 nm as an excitation source, these being recorded for 2 min by computer using F.2000.U.1.02 software. The linear regression of the data were calculated according to the pH. To calibrate the pHi, we made a series of high K+ buffers composed of NaCl (20 mmol/L), KH2PO4 (135 mmol/L) and different volumes of KH2PO4 (110 mmol/L) in NaCl (20 mmol/L) to adjust the different solutions to final pH values of 6, 6.7 and 7.67. We then added nigericin (10 µmol/L) to each solution, as described by Musgrove and Hedley (23
), and several samples from the noncolitic group labeled with BCECF-AM were incubated in these solutions for 10 min. After measuring the pHi values of these samples, the linear regression of the data were calculated according to the pH.
Effects of SCFA on IL-8 production and NOS activity in HT-29 cells.
The human colon adenocarcinoma cell line HT-29, obtained from the Cell Culture Unit of the University of Granada (Granada, Spain) (European Collection of Cell Cultures reference number: 91072201), was used as a model intestinal epithelium. In these assays we tested the ability of butyrate or propionate to inhibit the production and/or release of IL-8 as well as their effects on NOS activity. Cells were grown in DMEM supplemented with 10% fetal bovine serum (Boehringer Mannheim, Mannheim, Germany), 2 mmol/L L-glutamine, in a humidified 5% CO2 atmosphere at 37°C. Cells were seeded into 12-well plates and grown to confluence. Monolayers were preincubated for 30 min with SCFA, butyrate or propionate, at concentrations ranging from 0.1 to 8 mmol/L, and then stimulated with 10 mg/L LPS. After 20 h, supernatants were collected, centrifuged to remove debris at 7000 x g for 10 min and stored at -80°C for the determination of IL-8 levels by enzyme-linked immunosorbent assay (BioSource International, Nivilles, Belgium), and the percentage inhibition of cytokine production was calculated for every SCFA concentration. Cells were washed twice with PBS and lysated in the same buffer used for NOS activity assay in colonic samples (see above). NOS activity was assayed as described. At least four different experiments were performed in these assays.
In another set of experiments, the spectrofluorometric determination of NO released by HT-29 cells was determined, according to the method proposed by Leikert et al. (24
). For this purpose, after incubation with 10 mg/L LPS for 20 h in presence of different concentrations of SCFA (0.1–8 mmol/L), cells were washed with PBS and then preincubated with L-arginine (100 µmol/L for 5 min at 37°C). Subsequently, 1 µmol/L A23187 and 0.1 µmol/L 4,5-diaminofluorescein were added and cells were incubated in the dark (at 37°C for 5 min). Then the fluorescence of the supernatants was measured at room temperature using a spectrofluorometer (F2500; Hitachi) with excitation wavelength set at 495 nm and emission wavelength at 515 nm. The band width was 10 nm for both excitation and emission. Percentage inhibition of NO production was calculated for every SCFA concentration. Five different experiments were performed.
No cytotoxicity was detected in either of the SCFA studied, at any concentration assayed, as evidenced by the colorimetric MTT (tetrazolium) assay (25
), which revealed a viability >95% in all cases.
Statistics.
All results are expressed as the mean ± SEM. Data were analyzed by one-way analysis of variance and post hoc least significance tests. Nonparametric data (score) are expressed as the median (range) and were analyzed using the Mann-Whitney U-test. Differences between proportions were analyzed with the chi-squared test. All statistical analyses were carried out with the Statgraphics 5.0 software package with differences considered significant set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Rats subjected to dietary fiber supplementation showed an overall lower impact of TNBS-induced colonic damage compared with the TNBS control group. This was observed in the course of the experiment, because treated rats showed a faster weight recovery than control rats, regaining normal values from the 4th d after colitis induction (P > 0.05 versus noncolitic rats). However, rats from the TNBS control group showed a lower weight gain throughout the experiment in comparison with the rats from the noncolitic group (P < 0.01; data not shown). This effect was accompanied by a normalization of food intake and a decreased incidence of diarrhea (22 versus 70% in TNBS control group after 7 days of TNBS administration, P < 0.01). The anti-inflammatory effect was evidenced macroscopically by a lower colonic damage score than that of control rats (P < 0.01), with a significant reduction in the extent of colonic necrosis and/or inflammation induced by TNBS/ethanol. Treated rats showed a lower colonic weight:length ratio compared with the TNBS control group, which increased significantly as a consequence of the inflammatory process (Table 3
). The histological studies confirmed the intestinal anti-inflammatory effect exerted by dietary fiber (Fig. 1
). Histological assessment of colonic samples from the TNBS control group revealed severe transmural disruption of the normal architecture of the colon, with extensive ulceration and inflammation involving all the intestinal layers of the colon, with a median score (range) of 18 (14
–20
) (Fig. 2
). Colonic samples were characterized by severe edema, interstitial microhemorrhages and diffuse leukocyte infiltration, mainly composed of neutrophils and, to a lesser extent, lymphocytes and histiocytes. However, histological analysis of the colonic specimens from rats treated with fiber revealed a more pronounced recovery in the intestinal cytoarchitecture than controls, with a score of 11 (10
–18
) (P < 0.05 versus TNBS control group) (Fig. 2)
. Thus, most of the samples (four of five) showed almost complete restoration of the epithelial cell layer, in contrast to the extensive ulceration observed in nontreated rats; moreover, the transmural involvement of the lesions was reduced, affecting only the mucosal layer. The goblet cells appeared replenished with their mucin content, together with the absence of dilated crypts. The improvement in colonic histology was accompanied by a reduction in the inflammatory infiltrate, revealing a patchy distribution, although neutrophils were the predominant cell type. Fibrosis was not conspicuous in these samples.
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). In addition, colonic glutathione concentration returned to normal values in fiber-treated colitic rats (Table 4)
, in contrast with the glutathione depletion that occurred in colitic control rats as a consequence of the colonic oxidative stress induced by the inflammatory process, as previously reported in this model of experimental colitis (26
).
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and LTB4 (Table 4)
as well as by a greater colonic NOS activity (Fig. 3
) in comparison with noncolitic rats. Treatment of colitic rats with fiber resulted in a significant reduction of colonic TNF levels and in lower colonic NOS activity compared with TNBS control rats; no statistical differences with normal rats were observed (Table 4
and Fig. 3
). However, no significant effect on colonic LTB4 levels in comparison with nontreated rats was observed (P > 0.1; Table 4
).
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,28
), we determined the metabolic capacity of the inflamed colonic mucosa in both control and treated colitic rats. For this purpose, we determined the modification in the pHi of the colonic mucosal layers from the different groups of rats studied after incubation with glucose or sodium butyrate. Incubation of the colonic tissue from noncolitic rats with both substrates resulted in a significant decrease of pHi values, most probably due to the oxidation of these energetic products, as has been suggested in other in vitro studies (29
,30
). When mucosal layers from TNBS control rats were incubated in the presence of either glucose or butyrate, a significantly smaller decrease of pHi was observed compared with the noncolitic rats (Fig. 4
). However, colonic mucosa from fiber-treated colitic rats evidenced a restoration of their metabolic function, as shown by a similar decrease in the pHi of noncolitic rats after incubation of the samples with both substrates (Fig. 4)
.
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When the colonic contents from TNBS control rats were incubated for 24 h either in the presence or in the absence of fiber, reductions in the levels of SCFA were observed compared with noncolitic rats (P < 0.05, Table 5
). However, the intestinal contents obtained from the colitic treated rats showed greater butyrate and propionate production under both experimental conditions than those from TNBS control rats. No significant modification in acetate levels was observed in any of the groups studied (Table 5)
.
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Incubation of HT-29 cells in the presence of LPS increased IL-8 production in comparison with the basal release of this cytokine by the cells (15.9 ± 0.3 versus 0.06 ± 0.05 µg/L, P < 0.01). When these cells were cultured in the presence of both SCFA, IL-8 basal production was not affected (data not shown). However, both propionate and butyrate strongly inhibited LPS-induced IL-8 production in HT-29 cells in a concentration-dependent manner, with IC50 values of 2.69 ± 1.11 and 1.49 ± 0.26 mmol/L, respectively.
LPS increased NOS activity in HT-29 cells compared with nonstimulated cells (1718.5 ± 208.3 versus 612.7 ± 87.3 pmol L-citrulline/(mg protein x 30 min), P < 0.05). Butyrate dose-dependently inhibited LPS-stimulated NOS activity (IC50 = 1.87 ± 0.49 mmol/L), whereas propionate did not inhibit NOS activity at any of the concentrations assayed. Accordingly, butyrate reduced LPS-stimulated NO production in these cells with a maximum inhibitory effect of 84.5 ± 5.5% at 8 mmol/L (IC50 = 0.86 ± 2.68 mmol/L), whereas propionate showed a weak inhibitory effect (24.7 ± 3.1% inhibition at the concentration of 8 mmol/L).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,31
,32
) and in experimental models of colitis (33
,34
). Thus, dietary supplementation of P. ovata seeds (5%) facilitated recovery from TNBS-induced colonic damage. This was evidenced both histologically, with a significant reduction in the extent and severity of involved tissue, and biochemically, by a decrease in colonic MPO activity, a marker of neutrophil infiltration, and by a restoration of colonic glutathione content. There is ample experimental and clinical evidence to suggest that the inflamed colon undergoes substantial oxidative stress by neutrophil-derived oxidants and MPO, which contribute markedly to tissue damage during chronic intestinal inflammation (35
,36
). The present study shows that oxidative damage results in glutathione depletion, as has also been reported to occur both in human IBD and in experimental models of colitis (26
,37
,38
). As a consequence, a reduction in the activity of MPO together with a restoration of glutathione levels can be interpreted as a manifestation of the intestinal anti-inflammatory effect exerted by fiber in this model of experimental colitis. This was confirmed microscopically by amelioration of the histological lesions that characterize this experimental model of rat colitis. Thus, most of the samples from colitic treated rats (four of five) showed partial restoration of the intestinal epithelium, evidenced by the presence of mature goblet cells with their mucin content replenished and a lower edema and granulocyte infiltration in comparison with nontreated colitic rats. It is important to note that the amelioration in colonic histology obtained after fiber treatment given to colitic rats was not due to a "shield"-like effect of the fiber, which would prevent the damage induced by TNBS. On the contrary, two observations from the histological analysis reveal that fiber supplementation resulted in a faster recovery of the damaged colonic mucosa (Fig. 1)
. First, there was an evident presence of phenomena of epithelial regeneration at the margins of the mucosal ulcers, together with a "covering" process of the granulomatous tissue by enterocytes coming from these margins. The second factor was the existence of postinflammatory healing tissue in the submucosa, which was covered by a normal mucosa on the surface of the colonic tissue.
Although much progress has been made in understanding the pathogenesis of human IBD, its precise etiology remains unknown and probably involves a number of factors, including genetics and mucosal immune mechanisms. The inflammatory process results in alteration of intestinal epithelial cell function, including an interference with colonic SCFA use, especially of butyrate, which is considered the most important SCFA in colonocyte metabolism (6
). This has also been observed in the present study, because butyrate use by colonic epithelial cells was impaired in control colitic rats, because no modification in the pHi values was observed when mucosal layers from these rats were incubated in the presence of this substrate. However, the beneficial effect exerted by dietary fiber supplementation was associated with a restoration of the metabolic function of the colonic epithelial cells, this being indicative of a faster regeneration of the colonic tissue in comparison with nontreated rats. It is important to note that the tissue selected to perform these studies was obtained from the zone adjacent to the grossest colonic damage. This was done in an attempt to compare tissues from both treated and nontreated colitic rats, which would have been in the process of healing for the same period of time and thus would present similar characteristics.
The results obtained in the present study revealed that intestinal inflammation in colitic rats is associated with a lower production of SCFA than in noncolitic rats. This may be indicative of a defect in the colonic bacterial flora in rats with inflammation in the metabolism of the fiber and the production of different SCFA. As previously reported, a failure of homeostasis between the bacteria and the colonocyte may play a role in the inflammatory process (39
). When dietary fiber was administered to colitic rats, there was a significant increase in fecal butyrate and propionate levels, thus providing the substrates to the colonocytes and resulting in a fast recovery from inflammation. These results are in accordance with previous studies, of both humans suffering ulcerative colitis and those using experimental models of rat colitis, which showed that the intestinal anti-inflammatory effect of dietary fiber supplementation was associated with an increase in SCFA production (14
,40
).
Different mechanisms collaborate in the intestinal anti-inflammatory activity shown by dietary fiber supplementation in this model of colitis. One such mechanism could be the reduction in colonic neutrophil infiltration, as evidenced both histologically and biochemically. Margination and extravasation of circulating granulocytes markedly contribute to chronic injury in this model of IBD. Different mediators participate in the recruitment and activation of granulocytes, including LTB4, platelet-activating factor and chemokines like IL-8 (41
); inhibition in the synthesis and/or release of these mediators can result in a lower leukocyte infiltrate. The results obtained in the present study revealed that fiber treatment of colitic rats was not associated with a decrease in colonic LTB4 levels, and thus a direct inhibitory effect on LTB4 synthesis and/or release can be ruled out as a mechanism involved in the anti-inflammatory effect. For this reason, in vitro studies were performed to establish whether butyrate or propionate, the colonic levels of which rose after dietary fiber supplementation, inhibited the production of the chemokine IL-8 in HT-29 cells when stimulated by LPS. This activity can not be attributed to any alteration of the general physiology of the cells as evidenced by the MTT assay because MTT is only cleaved by living, metabolically active cells (25
). The results obtained showed that both SCFA effectively inhibited this chemokine production, which could contribute to the lower leukocyte infiltration observed in colitic rats after fiber supplementation. The HT-29 cells were used as an in vitro model of intestinal epithelium because intestinal epithelial cells have been described as playing a central role in IBD, synthesizing this proinflammatory cytokine (42
). As a consequence, in addition to the role as substrates for the colonic epithelial cells attributed to the SCFA, their effect on cytokine synthesis and/or release can directly contribute to the intestinal anti-inflammatory activity ascribed to dietary fiber. This activity was associated with a significant decrease in colonic TNF levels compared with nontreated rats. The inhibitory effect on TNF production is relevant, as shown by the development of anti-TNF agents, such as infliximab, in the treatment of human IBD (43
,44
). In addition, although the normalization of the glutathione levels observed in the fiber-treated colitic rats could be attributed to the antioxidant properties exerted by SCFA, a direct antioxidant effect can be ruled out because in vitro experiments have shown that both butyrate and propionate, at concentrations up to 8 mmol/L, did not inhibit the induced lipid peroxidation in liver membrane-enriched fraction P2 (unpublished data).
During the last decade it has become increasingly clear that chronic colonic inflammation is associated with enhanced NO production, mainly via inducible NOS (iNOS) activity, in both humans and experimental rats (45
–47
). NO overproduction by iNOS has been suggested to be deleterious to intestinal function (45
,46
), thus contributing to gastrointestinal immunopathology during the chronic inflammatory events that take place in IBD. The important role attributed to NO in these intestinal conditions prompted us to study whether the beneficial effects of dietary fiber on chronic TNBS-induced colitis could be related to an effect on colonic NO production. The results obtained in the present study reveal that colonic inflammation is associated with a higher NOS activity in comparison with noncolitic rats. This is in agreement with previous observations reported both for the same experimental model of rat colitis (46
) and for human IBD (47
), which described the enhanced NO production in the inflamed mucosa by colonic iNOS. The intestinal anti-inflammatory effect exerted by fiber was associated with a significant inhibition of colonic NOS activity. This can be attributed to the inhibitory effect exerted by butyrate on the induction of NOS, and subsequently in NO production, as has been demonstrated in HT-29 cells stimulated with LPS. As a consequence, an inhibition in NO production may also contribute to the beneficial effect, thus preventing, at least partially, the deleterious activity ascribed to NO when it is produced in high amounts by iNOS. Similar beneficial effects have been reported after NOS inhibition in different experimental models of intestinal inflammation (45
,46
,48
).
The inhibitory effect of dietary fiber supplementation on cytokine production and NOS activity can be related to the ability of SCFA to interfere with transcription factors. Nuclear factor-
B (NF-B) is a transcription factor that, in combination with others, plays a central role in regulating the expression of genes encoding iNOS as well as numerous cytokines in immune and inflammatory responses (49
). Thus, it has been previously reported that butyrate decreases TNF production by intestinal biopsies and by isolated lamina propria mononuclear cells via inhibition of NF-B activation and IB degradation (50
). The inhibitory effect of butyrate on NF-B activation in HT-29 cells, probably derived from its ability to inhibit deacetylases, has also been reported (51
).
In conclusion, dietary fiber supplementation facilitates the recovery of the inflamed tissue in the TNBS model of rat colitis, an effect associated with an amelioration in the production of some of the mediators involved in the inflammatory response of the intestine, such as cytokines, including TNF, and NO. This beneficial effect could be ascribed to the enhanced production of propionate and butyrate, which may act through the combination of different mechanisms. First, both are considered substrates for the colonocyte, thus facilitating the restoration of the intestinal barrier and avoiding the entry of luminal agents that contribute to maintaining the exacerbated immune response. Second, these SCFA are able to modulate the immune response through down-regulation of different mediators, such as proinflammatory cytokines, and of NOS activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by a grant from Instituto de Salud "Carlos III" (FIS 01/0936).
3 J.G. and A.Z. contributed equally to the supervision of this work.
5 Abbreviations used: BCECF-AM, 2',7'-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester; DMEM, Dulbecco’s modified Eagle’s medium; IBD, inflammatory bowel disease; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; LTB4, leukotriene B4; MPO, myeloperoxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-B, nuclear factor-B; NO, nitric oxide; NOS, NO synthase; PBS, phosphate-buffered saline; pHi, intracellular pH; TNBS, trinitrobenzenesulfonic acid; TNF, tumor necrosis factor.
Manuscript received 1 March 2002. Initial review completed 11 April 2002. Revision accepted 7 August 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Fiocchi, C. (1998) Inflammatory bowel disease: aetiology and pathogenesis. Gastroenterology 115:182-205.[Medline]
2. Sartor, R. B. (1997) Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am. J. Gastroenterol. 92((Suppl.)):5S-11S.[Medline]
3. MacDermott, R. P. (1996) Alterations of the mucosal immune system in inflammatory bowel disease. J. Gastroenterol. 31:907-916.[Medline]
4. Yamada, T., Fujimoto, K., Tso, P., Fujimoto, T., Gaginella, T. S. & Grisham, M. B. (1992) Misoprostol accelerates colonic mucosal repair in acetic acid-induced colitis. J. Pharmacol. Exp. Ther. 260:313-318.
5. Higa, A., McKnight, W. & Wallace, J. L. (1993) Attenuation of epithelial injury in acute experimental colitis by immunomodulators. Eur. J. Pharmacol. 239:171-176.[Medline]
6. Mortensen, P. B. & Clausen, M. R. (1996) Short-chain fatty acids in the human colon: relation to gastrointestinal health and disease. Scand. J. Gastroenterol. 31:132S-148S.
7. Roediger, W. E. & Nance, S. (1986) Metabolic induction of experimental ulcerative colitis by inhibition of fatty acid oxidation. Br. J. Exp. Pathol. 67:773-782.[Medline]
8. Vernia, P., Gnaedinger, A., Hauck, W. & Breuer, R. I. (1988) Organic anions and the diarrhoea of inflammatory bowel disease. Dig. Dis. Sci. 33:1353-1358.[Medline]
9. Stonerook, M. J., Tefend, K. S., Sharma, U. M., Peck, O. C. & Wood, J. D. (1996) Faecal short-chain fatty acids associated with inflammation in cotton-top tamarin model for idiopathic colitis. Dig. Dis. Sci. 41:618-624.
10. Patz, J., Jacobsohn, W. Z., Gottschalk-Sabag, S., Zeides, S. & Braverman, D. Z. (1996) Treatment of refractory distal ulcerative colitis with short chain fatty acid enemas. Am. J. Gastroenterol. 91:731-734.[Medline]
11. Steihart, A. H., Brzezinski, A. & Baker, J. P. (1994) Treatment of refractory ulcerative proctosigmoiditis with butyrate enemas. Am. J. Gastroenterol. 89:179-183.[Medline]
12. Breuer, R. I., Soergel, K. H., Lashner, B. A., Christ, M. L., Hanauer, S. B. & Vanaguas, A. (1997) Short chain fatty acid rectal irrigation for left-sided ulcerative colitis: a randomized, placebo controlled trial. Gut 40:485-491.
13. Scheppach, W. (1996) Treatment of distal ulcerative colitis with short-chain fatty acid enemas: a placebo controlled trial. Dig. Dis. Sci. 41:2254-2259.[Medline]
14. Fernandez-Bañares, F., Hinojosa, J. L., Sanchez-Lombraña, J. L., Navarro, E., Martinez-Salmeron, J. F., Garcia-Puges, A., Gonzalez-Huix, F., Riera, J., Gonzalez-Lara, V., Dominguez-Abascal, F., Gine, J. J., Moles, J., Gomollon, F. & Gassull, M. A. (1999) Randomized clinical trial of Plantago ovata seeds (dietary fiber) as compared with mesalamine in maintaining remission in ulcerative colitis. Am. J. Gastroenterol. 94:427-433.[Medline]
15. Elson, C. O., Sartor, R. B., Tennyson, G. S. & Riddell, R. H. (1995) Experimental models of inflammatory bowel disease. Gastroenterology 109:1344-1367.[Medline]
16. Morris, G. P., Beck, P. L., Herridge, W., Depew, W., Szewczuk, M. R. & Wallace, J. L. (1989) Hapten induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96:795-803.[Medline]
17. Bell, C. J., Gall, D. G. & Wallace, J. L. (1995) Disruption of colonic electrolyte transport in experimental colitis. Am. J. Physiol. 268:G622-G630.
18. Krawisz, J. E., Sharon, P. & Stenson, W. F. (1984) Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity: assessment of inflammation in rat and hamster models. Gastroenterology 87:1344-1350.[Medline]
19. Anderson, M. E. (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113:548-555.[Medline]
20. Chiesi, M. & Schwaller, R. (1995) Inhibition of constitutive endothelial NO-synthase activity by tannin and quercetin. Biochem. Pharmacol. 49:195-501.
21. Bradford, M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[Medline]
22. Millar, A. D., Rampton, D. S., Chander, C. L., Claxson, A. W. D., Blades, A., Coumbe, A., Panetta, J., Morris, C. J. & Blake, D. R. (1996) Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis. Gut 39:407-415.
23. Musgrove, E. & Hedley, D. (1990) Flow cytometry. Dar
zynkiewicz, Z. Crissman, H. A. eds. Methods in Cell Biology 1990:59-69 Academic Press San Diego, CA. .
24. Leikert, J. F., Räthel, T. R., Müller, C., Vollmar, A. M. & Dirsch, V. M. (2001) Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein. FEBS Lett. 506:131-134.[Medline]
25. Mosmann, T. (1983) Rapid colorimetric assay for cellular-growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63.[Medline]
26. Galvez, J., Garrrido, M., Merlos, M., Torres, M. I. & Zarzuelo, A. (2000) Intestinal anti-inflammatory activity of UR-12746, a novel 5-ASA conjugate, on acute and chronic experimental colitis in the rat. Br. J. Pharmacol. 130:1949-1959.[Medline]
27. Roediger, W. E. (1980) The colonic epithelium in ulcerative colitis: an energy-deficiency disease?. Lancet 2:712-715.[Medline]
28. Chapman, M. A., Grahn, M. F., Boyle, M. A., Hutton, M., Rogers, J. & Williams, N. S. (1994) Butyrate oxidation is impaired in the colonic mucosa of sufferers of quiescent ulcerative colitis. Gut 35:73-76.
29. Zoran, D. L., Barhoumi, R., Burghardt, R. C., Chapkin, R. S. & Lupton, J. R. (1997) Diet and carcinogen alter luminal butyrate concentration and intracellular pH in isolated rat colonocytes. Nutr. Cancer 27:220-230.
30. Akagi, K., Aoki, Y., Nasu, R., Nagata, K., Itagaki, Y. & Sawada, S. (1999) Enhancement of antitumor effect of hyperthermia with glucose administration in murine mammary carcinoma. Oncol. Rep. 6:593-596.[Medline]
31. Hallert, C., Kaldma, M. & Petersson, B. G. (1991) Ispaghula husk may relieve gastrointestinal symptoms in ulcerative colitis in remission. Scand. J. Gastroenterol. 26:747-750.[Medline]
32. Mitsuyama, K., Saiki, T., Kanauchi, O., Iwanaga, T., Tomiyasu, N., Nishiyama, T., Tateishi, H., Shirachi, A., Ide, M., Suzuki, A., Noguchi, K., Ikeda, H., Toyonaga, A. & Sata, M. (1998) Treatment of ulcerative colitis with germinated barley foodstuff: a pilot study. Aliment. Pharmacol. Ther. 12:1225-1230.[Medline]
33. Kanauchi, O., Andoh, A., Iwanaga, T., Fujiyama, Y., Mitsuyama, K., Toyonaga, A. & Bamba, T. (1999) Germinated barley foodstuffs attenuate colonic mucosal damage and mucosal nuclear factor B activity in a spontaneous colitis model. J. Gastroenterol. Hepatol. 14:1173-1179.[Medline]
34. Araki, Y., Fujiyama, Y., Andoh, A., Koyama, S., Kanauchi, O. & Bamba, T. (2000) The dietary combination of germinated barley foodstuff plus Clostridium butyricum suppresses the dextran sodium-induced experimental colitis in rats. Scand. J. Gastroenterol. 35:1060-1067.[Medline]
35. Harris, M. L., Schiller, H. J., Reilly, P. M., Donowitz, M., Grisham, M. B. & Bulkley, G. B. (1992) Free radicals and other reactive oxygen metabolites in inflammatory bowel disease: cause, consequence or epiphenomenon?. Pharmacol. Ther. 53:375-408.[Medline]
36. Yamada, T. & Grisham, M. B. (1994) Pathogenesis of tissue injury: role of reactive metabolites of oxygen and nitrogen. Targan, S. R. Shanahan, F. eds. Inflammatory Bowel Disease: From Bench to Bedside 1994:133-150 Williams & Wilkins Baltimore, MD. .
37. Lih-Brody, L., Powell, S. R., Collier, K. P., Reddy, G. M., Cerchia, R., Kahn, E., Weissman, G. S., Katz, S., Floyd, R. A., McKinley, M. J., Fisher, S. E. & Mullin, G. E. (1996) Increased oxidative stress and decreased antioxidant defences in mucosa of inflammatory bowel disease. Dig. Dis. Sci. 41:2078-2086.[Medline]
38. Loguercio, C., D’Argenio, G., Delle Cave, M., Cosenza, V., Della Valle, N., Mazzacca, G. & del Vecchio Blanco, C. (1996) Direct evidence of oxidative damage in acute and chronic phases of experimental colitis in rats. Dig. Dis. Sci. 41:1204-1211.[Medline]
39. Simpson, E. J., Chapman, M. A. S., Dawson, J., Berry, D., Macdonald, I. A. & Cole, A. (2000) In vivo measurement of colonic butyrate metabolism in patients with quiescent ulcerative colitis. Gut 46:73-77.
40. Kanauchi, O., Iwanaga, T. & Mitsuyama, K. (2001) Germinated barley foodstuff feeding. Digestion 63:60-67.
41. Podolsky, D. K. & Fiocchi, C. (2000) Cytokines, chemokines, growth factors, eicosanoids and other bioactive molecules in inflammatory bowel disease. Kirsner, J. B. eds. Inflammatory Bowel Disease 5th edn. 2000:191-207 W. B. Saunders Philadelphia, PA. .
42. Van Deventer, S. J. (1997) Review article: chemokine production by intestinal epithelial cells: a therapeutic target in inflammatory bowel disease?. Aliment. Pharmacol. Ther. 11:116-120.
43. Rutgeerts, P., D’Haens, G., Targan, S., Vasiliauskas, E., Hanauer, S. B., Present, D. H., Mayer, L., Van Hogezand, R. A., Braakman, T., DeWoody, K. L., Schaible, T. F. & Van Deventer, S. J. (1999) Efficacy and safety of retreatment with anti-tumor necrosis factor antibody (infliximab) to maintain remission in Crohn’s disease. Gastroenterology 117:761-779.[Medline]
44. Chey, W. Y. (2001) Infliximab for patients with refractory ulcerative colitis. Inflamm. Bowel. Dis. 7((Suppl.)):30S-33S.
45. Rachmilewitz, D., Karmeli, F., Okon, E. & Bursztyn, M. (1995) Experimental colitis is ameliorated by inhibition of nitric oxide synthase activity. Gut 37:247-255.
46. Hogaboam, C. M., Jacobson, K., Collins, S. M. & Blennerhassett, M. G. (1995) The selective beneficial effects of nitric oxide inhibition in experimental colitis. Am. J. Physiol. 268:G673-G684.
47. Kimura, H., Miura, S., Shigematsu, T., Ohkubo, N., Tsuzuki, Y., Kurose, I., Higuchi, H., Akiba, Y., Hokari, R., Hirokawa, M., Serizawa, H. & Ishii, H. (1997) Increased nitric oxide production and inducible nitric oxide synthase activity in colonic mucosa of patients with active ulcerative colitis and Crohn’s disease. Dig. Dis. Sci. 42:1047-1054.[Medline]
48. Miller, M. J., Sadowska-Krowicka, S. H., Chotinaruemol, S., Kakkis, J. L. & Clark, D. A. (1993) Amelioration of chronic ileitis by nitric oxide synthase inhibition. J. Pharm. Exp. Ther. 264:11-16.
49. Choi, C. Y., You, H. J. & Jeong, H. G. (2001) Nitric oxide and tumor necrosis factor- production by oleanolic acid via nuclear factor-B activation in macrophages. Biochem. Biophys. Res. Commun. 288:49-55.[Medline]
50. Segain, J. P., Raingeard de la Bletiere, D., Bourreille, A., Leray, V., Gervois, N., Rosales, C., Ferrier, L., Bonnet, C., Blottiere, H. M. & Galmiche, J. P. (2000) Butyrate inhibits inflammatory responses through NFB inhibition: implications for Crohn’s disease. Gut 47:397-403.
51. Inan, M. S., Rasoulpour, R. J., Yin, L., Hubbard, A. K., Rosenberg, D. W. & Giardina, C. (2000) The luminal short-chain fatty acid butyrate modulates NF-B activity in a human colonic epithelial cell line. Gastroenterology 118:724-734.[Medline]
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