Impact of basal diet on dextran sodium sulphate (DSS)-induced colitis in rats

(1)

DOI 10.1007/s00394-014-0800-2 ORIGINAL CONTRIBUTION

Impact of basal diet on dextran sodium sulphate (DSS)-induced colitis in rats

Ahlem Boussenna · Nicolas Goncalves-Mendes · Juliette Joubert-Zakeyh · Bruno Pereira · Didier Fraisse · Marie-Paule Vasson · Odile Texier · Catherine Felgines

Received: 15 September 2014 / Accepted: 6 November 2014

in half of the rats by administration of DSS in drinking water (4 % w/v) during the last 7 days of experimentation.

At the end of the experimental period, colon sections were taken for histopathological examination, determination of various markers of inﬂammation (myeloperoxidase: MPO, cytokines) and oxidative stress (superoxide dismutase:

SOD, catalase: CAT, glutathione peroxidase: GPx and glutathione reductase: GRed activities), and evaluation of the expression of various genes implicated in this disorder.

Results DSS ingestion induced a more marked colitis in animals receiving the purified diet, as reflected by higher histological score and increased MPO activity. A significant decrease in SOD and CAT activities was also observed in rats fed the purified diet. Also, in these animals, administration of DSS induced a significant increase in interleukin (IL)-1α, IL-1β and IL-6. In addition, various genes implicated in inflammation were over-expressed after ingestion of DSS by rats fed the purified diet.

Conclusions These results show that a purified diet pro- motes the onset of a more severe induced colitis than a non- purified one, highlighting the influence of basal diet in colitis development.

Keywords Puriﬁed diet · Dextran sodium sulphate · Colitis · Oxidative stress · Rat

Introduction

Inﬂammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic pathology consisting of an uncontrolled inﬂammation that ultimately leads to mucosal disruption and ulceration [1].

It appears to result from a dysregulated immune response, associated with genetic and environmental factors [1].

Abstract

Purpose Dextran sodium sulphate (DSS)-induced colitis is a widely used model for inﬂammatory bowel disease.

However, various factors including nutrition may affect the development of this colitis. This study aimed to compare and characterize the impact of puriﬁed and non-puriﬁed basal diets on the development of DSS-induced colitis in the rat.

Methods Wistar rats were fed a non-puriﬁed or a semi- synthetic puriﬁed diet for 21 days. Colitis was then induced

A. Boussenna · D. Fraisse · O. Texier · C. Felgines Unité de Nutrition Humaine, Equipe ECREIN, Laboratoire de Pharmacognosie et Phytothérapie, Clermont Université, Université d’Auvergne, 63000 Clermont-Ferrand, France A. Boussenna

3inature Biosphère, Parc Naturopôle, 03800 Saint-Bonnet-de-Rochefort, France N. Goncalves-Mendes · M.-P. Vasson

Unité de Nutrition Humaine, Equipe ECREIN, Laboratoire de Biochimie Biologie Moléculaire et Nutrition, Clermont Université, Université d’Auvergne, 63000 Clermont-Ferrand, France

J. Joubert-Zakeyh

Service d’Anatomie et de Cytologie Pathologiques, CHU de Clermont-Ferrand, 63003 Clermont-Ferrand, France B. Pereira

Biostatistics Unit (DRCI), CHU de Clermont-Ferrand, 63003 Clermont-Ferrand, France

C. Felgines (*)

Laboratoire de Pharmacognosie et Phytothérapie, Faculté de Pharmacie, Clermont Université, Université d’Auvergne, 28 Place Henri-Dunant, BP 38, 63001 Clermont-Ferrand Cedex 1, France

e-mail: catherine.felgines@udamail.fr

(2)

During the inflammatory process, a large number of immu- nological cells, including activated macrophages, polymorphonuclear neutrophils (PMNs), and eosinophils, infiltrate the lamina propria of the gut, where they produce large amounts of reactive oxygen species (ROS) [2]. Oxidative damage is exacerbated by the depletion of enzymatic and non-enzymatic antioxidant defences accompanying the inflammatory process [2].

Over the past two decades, several experimental models, with a variable range of clinical and histopathologic fea- tures similar to those observed in human IBD, have been developed in an attempt to understand the pathophysiol- ogy of this disease and evaluate various pharmacological or nutritional interventions [3]. Dextran sodium sulphate (DSS)-induced acute colitis is one of the most widely used models for IBD because of its similarities to human IBD, especially UC, in aetiology, pathogenesis and therapeutic response [4]. Furthermore, several therapeutic agents for IBD have shown efﬁcacy in this disease model, indicating that DSS-induced colitis can be used as a relevant model for the translation of animal data to human disease [5].

Although the exact mechanism by which DSS induces colitis is not yet fully understood, it is generally thought that DSS exerts toxic effects on intestinal epithelial cells and increases exposure to luminal antigens by disrupting the mucosal barrier [6]. DSS-induced colitis is highly dependent on increased leucocyte recruitment responses and on production of inflammatory mediators such as cytokines, which contribute to tissue damage [6]. DSS has also been shown to affect the expression of various genes implicated in inflammation, immune response, and oxidative damage such as genes of pro-inflammatory cytokines, intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) [7, 8].

Several authors have reported that DSS-induced colitis depends on various factors, such as molecular weight and concentration of DSS and duration of administration, but also on genetic, microbiological and nutritional factors [4, 6]. Previous studies in our laboratory using DSS administration (4 % for 7 days) to rats fed a non-purified commercial rodent diet showed that such conditions induced only a mild-to-moderate colitis and a somewhat high variability in its intensity [9]. On the other hand, we observed that identical administration of DSS (4 % for 7 days) to rats fed a purified diet made of refined ingredients, and widely used for nutritional studies, induced more exacerbated symptoms of colitis [10]. Also, a recent study showed that feed- ing a purified diet instead of a non-purified one increased the severity of the DSS-induced colitis in mice [11]. It is thus important to know the impact of the basal diet on the development of DSS colitis when evaluating any potential beneficial role of macro- or micronutrients. Accordingly, the aim of this study was to compare and characterize the

impact of two basal diets (purified and non-purified) on DSS-induced colitis in the rat. The influence of the diet on colitis development was assessed by histological analysis, determination of various markers of inflammation (myeloperoxidase: MPO, cytokines) and oxidative stress (antioxi- dative enzyme activities), and evaluation of the expression of various genes implicated in this disorder.

Materials and methods

Animals and experimental design

Five-week-old male Wistar rats weighing 160–180 g (n = 32) were purchased from Janvier (Le Genest-Saint- Isle, France) and housed in a temperature-controlled room (22 °C) with a controlled reverse lighting cycle (12-h dark/

light cycle). They were allowed to adapt to the Auvergne University Experimental Animal Laboratory for 1 week before beginning the experiment. This animal study was approved by the local Ethics Committee (Registration No. CE4-08). The rats were randomly divided into two groups (n = 16 per group) and received ad libitum either a non-puriﬁed (NP) commercial rodent pelleted diet (2016 Teklad Global standard diet, Harlan, Gannat, France;

12.4 kJ/g) or a puriﬁed (P) pelleted diet based on the AIN- 93G recommendations (UPAE INRA, Jouy-en-Josas, France; 15.6 kJ/g) (Table 1) for 3 weeks. In the last week, one half of the rats in each group received 4 % (w/v) DSS (molecular weight 36–50 kDa; MP Biomedicals, Illkirch, France) dissolved in drinking water (NP-DSS and P-DSS groups), whereas the other rats kept on drinking untreated water (NP and P groups). During this week, DSS-induced colitis severity was assessed daily using the disease activity index (DAI) calculated on the basis of weight loss, stool consistency, and rectal bleeding, as previously described [12].

Animal weight and food consumption were measured daily. Body weight gain was determined between day

Table 1 Composition of the puriﬁed diet

Ingredients g/kg of diet

Wheat starch 629.36

Casein 200

Corn oil 70

Cellulose 50

AIN 93 G-Mx (mineral mix) 35

AIN 93-Vx (vitamin mix) 10

l-Cystine 3

Choline bitartrate 2.5

t-Butyl-hydroquinone 0.14

(3)

15 and day 22 (i.e. period of DSS administration) and expressed as a percentage of day 15 body weight. Food intake was expressed as the sum of food consumed during the 7 days of colitis induction. At day 22, rats were anaes- thetized by intraperitoneal administration of sodium pento- barbital (40 mg/kg body weight) and killed by decapita- tion. The colon was excised from the caecum to the rectum, freed of adherent adipose tissue, and its length measured under a constant load (2 g). The last distal centimetre was sampled for histological analysis, and the remaining por- tion was divided longitudinally into four sections, rapidly frozen in liquid nitrogen, and stored at −80 °C until further analysis.

Histopathological examination

The section for histological examination was treated as previously described [9]. Rats were scored individually.

For each rat, the score was the mean of three colonic sections analysed blindly by two operators. Nine parameters were considered and scored as described [9]: epithelium and crypt destruction, cryptic dilation, PMN infiltration, mononuclear infiltration, oedema, dystrophic epithelium detachment, erosion, vascular congestion, and depth of inflammation.

Determination of MPO and colonic antioxidant enzyme activities

MPO activity in colon samples was determined by evaluating guaiacol oxidation by MPO-produced hydroxyl radicals in the presence of hydrogen peroxide, as previously described [9]. Protein concentration was determined using a Pierce BCA protein assay kit (Perbio, Brebières, France). MPO activity was deﬁned as the quantity of enzyme degrading 1 μmol of H₂O₂ per min and expressed as units per mg of proteins. The assay was performed in triplicate.

For colonic antioxidant enzyme assay, a colon sample section was mechanically homogenized in ice in a 100 mM Trizma buffer pH 7.4 with 1 mM EDTA and 1 mM phe- nylmethanesulphonyl ﬂuoride (PMSF). The homogenate was sonicated for 30 s in ice and centrifuged for 10 min at 1,000×g at 4 °C. Supernatants were aliquoted and stored at

−80 °C until further analysis. Antioxidant enzyme activities were determined in triplicate using superoxide dismutase (SOD), glutathione reductase (GRed), glutathione peroxidase (GPx), and catalase (CAT) kits (Sigma, Saint- Quentin-Fallavier, France). Protein concentration of the supernatants was determined as previously stated. Activi- ties were deﬁned as the quantity of enzyme degrading 1 μmol of substrate per minute and expressed in units per mg of proteins.

Colonic cytokines

Before cytokine analysis, colon tissue samples (≈50 mg) were prepared using a Cell Lysis kit (Biorad, Marnes- La-Coquette, France). Brieﬂy, samples were rinsed in the wash buffer and then thawed in the lysis buffer containing a protease inhibitor cocktail and PMSF (500 mM). Colon samples were then homogenized in ice. Homogenates were treated with a freeze and thaw cycle, then sonicated in ice, and centrifuged for 4 min at 4,500×g. Supernatants were aliquoted and frozen at −80 °C until analysis. Total protein concentration of supernatants was determined as previously stated. All tissue samples were diluted with cell lysis buffer to a ﬁnal protein concentration of ≈1 mg/ml before cytokine analysis. Cytokines were analysed using the xMAP technology developed by Luminex (Austin, TX).

The Bio-Plex assay of 12 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, GM-CSF, IFN-γ, and TNF-α) was performed according to the manufactur- er’s protocol (Biorad). The detection limit of the multiplex cytokine assay was <10 pg/ml for each cytokine.

Quantitative reverse transcriptase (qRT)-PCR analysis A colon sample section was mechanically ground in liquid nitrogen using a BioPulverizer (BioSpec Products, Bartles- ville, USA). Total RNAs were then extracted from these samples (≈30 mg) using a NucleoSpin RNA II kit (Mach- erey-Nagel, Hoerdt, France). RNA quantity and purity were assessed by the ratio of absorbance at 260 and 280 nm (Nanodrop ND-8000, Labtech, Uckﬁeld, United King- dom). RNA purity is considered as correct when A260/

A280 ratio is in the range 2.0–2.1. Total RNAs (0.5 μg) were reverse-transcribed using SuperScript II reverse transcriptase (Fisher Scientiﬁc, Illkirch, France) and a combi- nation of random hexamers (Eurogenetec, Angers, France).

The list of primers used for real-time PCR ampliﬁcation is presented in Table 2. Gene of 18S RNA was used as ref- erence gene for normalizing mRNA levels in colon tissue.

The relative expression of the mRNAs corresponding to the genes of interest was measured by real-time PCR using a Rotor-Gene Q system (Qiagen, Courtabœuf, France). The RT-PCR mixture contained 5 μl of diluted cDNA template, 10 μl of Rotor-Gene sybrgreen PCR master mix (Qia- gen), and 0.5 μl of forward and reverse primers. Samples were heated for 5 min at 95 °C and then subjected to 40 cycles of denaturing at 95 °C for 5 s and annealing/elonga- tion at 60 °C for 10 s. Relative expression values between groups were analysed using Rotor-Gene software and calculated using the CT method: for each sample, the difference between the threshold cycle (CT) values for the gene of interest and housekeeping gene (18S) was calculated (ΔCT). The ΔΔCT, which represents the difference

(4)

between the ΔCT of each tissue sample compared with the ΔCT of samples of the NP group, was then calculated using the ΔΔCT method [13]. Finally, the fold induction of target gene expression in each sample was calculated as fold change = 2^−ΔΔ^CT. Real-time PCR efﬁciency was determined by analysis of serial dilutions from a mix of colon cDNA for each target gene. When the gene was undetectable, we assigned to these samples a CT value corresponding to the value of the lower range point of the standard curve.

Statistical analysis

The results are expressed as means along with their standard errors. Data analysis was performed using the Stat- View^® 5.0 statistical software (SAS Institute Inc., USA).

Considering the 2 × 2 factorial design, two-way analysis of variance (ANOVA) was carried out in order to assess

the effects of DSS (DSS), diet (D), and their interaction (DSS × D). The normality of the variables was assessed by the Shapiro–Wilk test and their homoscedasticity by Bartlett’s test. When variables presented a normal distribu- tion, comparison between groups was made by two-way ANOVA followed by the Tukey–Kramer test. Otherwise, it was made by the Kruskal–Wallis test followed by Dunn’s test. Concerning the analysis of repeated measures, a random-effects model (mixed model) was considered, as usually proposed, to study the ﬁxed effects group, time-points, and interaction group × time taking into account between- and within-subject variability. Differences were considered signiﬁcant at the level of P < 0.05.

Results

Food and energy intake and body weight

Despite a lower food intake in P-fed versus NP-fed rats throughout the experiment, body weight was not signiﬁ- cantly different between groups from the beginning of the experiment until the ﬁrst day of DSS administration (data not shown). Food and energy intake and body weight gain during the last week of the experiment are reported in Fig. 1. Food consumption depended on the diet: during the last week of experimentation, healthy rats ingested less P than NP diet. However, since P diet was more energy-rich than NP diet, healthy rats had similar energy intake and their body weight gain was similar. DSS consumption lowered food intake for both diets. This resulted in a decrease in body weight gain, more marked for rats fed the P diet.

The two-way ANOVA revealed an interaction between DSS and diet (P < 0.05), reﬂecting a diet-dependent effect of DSS on body weight gain.

Disease activity index, colon length, and histopathological examinations

After DSS administration, DAI was statistically higher on days 5, 6, and 7 in rats fed P versus NP diet (Fig. 2a).

In non-DSS-treated rats, P diet induced colon shorten- ing (Fig. 2b), and DSS administration led to a significant reduction of colon length (P < 0.05), with both diets (NP: −17.4 %, P: −24.4 %). Colon of rats receiving DSS (Fig. 3c, d) presented histological alterations compared with non-DSS-treated rats (Fig. 3a, b). In P-fed rats, DSS affected all histological parameters except vascular congestion, whereas only inflammation extent, PMN infiltration, and crypt dilation were affected in NP-fed rats (Table 3). The histological score (Fig. 3e) was markedly increased after DSS administration, more especially in P-fed rats.

Table 2 Primer information

NFκB1 nuclear factor kappa B p50; NFκBp65 Nuclear factor kappa B p65, TNFα tumour necrosis factor alpha, IL1β interleukin-1 beta, IL6 interleukin-6, IL4 Interleukin-4, iNOS inducible nitric oxide synthase, COX2 cyclooxygenase 2, SOD2 superoxide dismutase 2, MMP9 matrix metalloproteinase 9, ICAM1 intercellular adhesion molecule-1, ARN18S ribosomal RNA 18S, and F forward, R reverse Gene Primer sequence (5′–3′)

NFκB1-F CAG-CTC-TTC-TCA-AAG-CAG-CA

NFκB1-R TCC-AGG-TCA-TAG-AGA-GGC-TCA

NFκBp65-F TGT-ATT-TCA-CGG-GAC-CTG-GC

NFκBp65-R CAG-GCT-AGG-GTC-AGC-GTA-TG

TNFα-F GCC-TCT-TCT-CAT-TCC-TGC-TC

TNFα-R GAG-CCC-ATT-TGG-GAA-CTT-CT

IL1β-F TGC-TGA-TGT-ACC-AGT-TGG-GG

IL1β-R CTC-CAT-GAG-CTT-TGT-ACA-AG

IL6-F TAG-TCC-TTC-CTA-CCC-CAA-CTT-CC

IL6-R TTG-GTC-CTT-AGC-CAC-TCC-TTC

IL4-F GTA-CCG-GGA-ACG-GTA-TCC-AC

IL4-R TGG-TGT-TCC-TTG-TTG-CCG-TA

iNOS-F GGC-TGG-AAG-CCC-CGC-TAT-GG

iNOS-R GGC-AGG-CAG-CGC-ATA-CCA-CT

COX2-F GAC-CCG-CAG-CCT-ACC-AAG

COX2-R ACT-GTA-GGG-TTA-ATG-TCA-TCT-AGT-C

SOD2-F ATT-AAC-GCG-CAG-ATC-ATG-CA

SOD2-R CCT-CGG-TGA-CGT-TCA-GAT-TGT

MMP9-F GAC-ACC-GCT-CAC-CTT-CAC-C

MMP9-R CCG-CGA-CAC-CAA-ACT-GG

ICAM1-F CTG-GAG-AGC-ACA-AAC-AGC-AGA-G

ICAM1-R AAG-GGC-GCA-GAG-CAA-AAG-AAG-C

RNA18S-F CAA-CTT-CTT-AGA-GGG-ACA-AGT-GG

RNA18S-R ACG-CTG-AGC-CAG-TCA-GTG-TA

(5)

Colonic MPO and antioxidant enzyme activities

MPO and antioxidant enzyme activities are reported in Table 4. In non-colitic rats, diet did not affect MPO activity. However, this activity was markedly increased (167 %) in P-fed rats following DSS administration (P < 0.05).

There were no signiﬁcant differences for SOD, CAT, and GRed activities between control groups (NP vs. P). How- ever, GPx activity was increased in control animals fed P

versus NP diet. On the other hand, DSS administration did not affect antioxidant enzyme activities in NP-fed rats, but induced a signiﬁcant decrease in SOD and CAT activities in P-fed rats.

Colonic cytokine levels

Healthy rats fed P diet presented lower colonic level of IFN- γ than animals fed NP diet (Table 5). DSS administration to Fig. 1 Cumulative intake (a) of food (bars) and energy (black line)

and body weight gain (b) following colitis induction by DSS (4 % for 7 days). These parameters were determined during the DSS administration period. Body weight gain is expressed as a percentage of ﬁrst day DSS treatment. Values are means (n = 8 for each group) with their standard errors. Statistical signiﬁcance was analysed by two-

way ANOVA followed by the Tukey–Kramer test. Significant effects induced by DSS for each diet are indicated with *P < 0.05. Signifi- cant differences between diets (P vs. NP or P-DSS vs. NP-DSS) are indicated by ^#P < 0.05. NP rats fed the non-purified diet, NP-DSS DSS-treated rats fed the non-purified diet, P rats fed the purified diet, P-DSS DSS-treated rats fed the purified diet

Fig. 2 Time course of the disease activity index (DAI) (a) and colon length (b) of rats following DSS administration (4 % for 7 days).

Values are expressed as means (n = 8 for each group) with their standard errors. a DSS induced earlier and more pronounced signs of disease in rats fed P diet. Statistical analysis was performed by random-effects model. *P < 0.05, ***P < 0.001: signiﬁcant difference between NP-DSS and P-DSS rats for the same day. b Statisti-

cal significance was analysed by two-way ANOVA followed by the Tukey–Kramer test. Significant effects induced by DSS for each diet are indicated with *P < 0.05. Significant differences between diets (P vs. NP or P-DSS vs. NP-DSS) are indicated by ^#P < 0.05. NP rats fed the non-purified diet, NP-DSS DSS-treated rats fed the non-purified diet, P rats fed the purified diet, P-DSS DSS-treated rats fed the purified diet

(6)

NP rats induced a decrease only in IL-13 level. However, in P-fed rats, induction of colitis by DSS induced a marked increase in IL-1α, IL-1β, and IL-6 levels and a decrease in TNF-α, GM-CSF, IL-4, IL-5, and IL-13 levels.

Gene expression analysis

In healthy rats, gene expression was not signiﬁcantly affected by diet (Fig. 4). The effect of DSS was more pronounced in P-fed than in NP-fed rats. In P-fed rats,

DSS administration led to over-expression of TNF-α, IL-1β, iNOS, MMP-9, and ICAM1 genes. In NP-fed rats, only IL-6 gene was signiﬁcantly up-regulated after DSS administration.

Discussion

The DSS-induced colitis model is widely used to evaluate the impact of various pharmacological or nutritional Fig. 3 Histological analysis of colonic mucosa from rats follow-

ing colitis induction by DSS administration (4 % for 7 days). Colon sections from rats fed the non-purified diet (a) and from rats fed the purified diet (b) showed normal mucosa. Colon from DSS-induced colitis rats fed the non-purified diet (c) presented moderate cryptic dilation and moderate inflammatory cell infiltration, whereas colon from DSS-treated rats fed the purified diet (d) presented epithelium destruction. Hematoxylin–phloxin–saffron staining; origi-

nal magniﬁcation ×10. Histological score (e) is expressed as means (n = 8 for each group) with their standard errors. Differences among means were analysed by Kruskal–Wallis followed by Dunn’s test.

Signiﬁcant effects induced by DSS for each diet are indicated with

*P < 0.05. Significant differences between diets (P vs. NP or P-DSS vs. NP-DSS) are indicated by ^#P < 0.05. NP rats fed the non-purified diet, NP-DSS DSS-treated rats fed the non-purified diet, P rats fed the purified diet, P-DSS DSS-treated rats fed the purified diet

Table 3 Histological analysis of colonic sections in rats following colitis induction by DSS administration (4 % for 7 days)

Values are expressed as mean ± SEM (n = 8)

NP rats fed the non-purified diet, NP-DSS rats fed the non-purified diet then treated by DSS, P rats fed the purified diet, P-DSS rats fed the puri- fied diet then treated by DSS

Significant effects induced by DSS for each diet are indicated with * P < 0.05. Significant differences between diets (P vs. NP or P-DSS vs. NP- DSS) are indicated by ^#P < 0.05. Statistical significance was analysed by Kruskal–Wallis test followed by Dunn’s test

Non-puriﬁed diet Puriﬁed diet

NP (arbitrary units) NP-DSS (arbitrary units) P (arbitrary units) P-DSS (arbitrary units) Epithelium and glandular destruction 0.21 ± 0.14 1.21 ± 0.23 0.17 ± 0.09 6.79 ± 2.12*,^#

Cryptic dilation 0.00 ± 0.00 1.33 ± 0.14* 0.08 ± 0.05 1.21 ± 0.29*

Polymorphonuclear inﬁltration 0.00 ± 0.00 1.67 ± 0.14* 0.04 ± 0.04 2.96 ± 0.04*,^# Mononuclear inﬁltration 0.00 ± 0.00 0.13 ± 0.13 0.00 ± 0.00 1.38 ± 0.53*,^#

Oedema 0.00 ± 0.00 0.62 ± 0.21 0.13 ± 0.13 1.37 ± 0.28*,^#

Dystrophic epithelium detachment 0.00 ± 0.00 0.37 ± 0.16 0.00 ± 0.00 0.92 ± 0.29*

Erosion 0.00 ± 0.00 0.08 ± 0.08 0.00 ± 0.00 1.29 ± 0.34*,^#

Vascular congestion 0.00 ± 0.00 0.08 ± 0.05 0.00 ± 0.00 0.04 ± 0.04

Depth of inﬂammation 0.00 ± 0.00 2.00 ± 0.19* 0.04 ± 0.04 2.96 ± 0.04*,^#

(7)

interventions on intestinal inflammation. A great number of studies using the DSS model have been carried out in rodents fed commercial standard chow, whereas only a few have used semisynthetic purified diets based on refined ingredients [14, 15]. Purified diets are widely used in various studies evaluating nutritional interventions, since they allow precise control of diet composition, including

modulation by adding speciﬁc macro- or micronutrients.

However, to date, there are only scant data on the impact of the basal diet on DSS-induced colitis development [11].

Goto et al. [11] thus reported that a puriﬁed diet increased disease severity and MPO activity and lowered caecal short-chain fatty acid (SCFA) concentrations in DSS colitic mice. In this study, we evaluated and compared for the Table 4 Colonic myeloperoxidase and antioxidant enzyme activities in rats following colitis induction by DSS administration (4 % for 7 days)

NP rats fed the non-purified diet, NP-DSS rats fed the non-purified diet then treated by DSS, P rats fed the purified diet, P-DSS rats fed the puri- fied diet then treated by DSS, MPO myeloperoxidase, SOD superoxide dismutase, CAT catalase, GPx glutathione peroxidase, and GRed glutathione reductase

Signiﬁcant effects induced by DSS for each diet are indicated with * P < 0.05

Signiﬁcant differences between diets (P vs. NP or P-DSS vs. NP-DSS) are indicated by ^#P < 0.05

a Two-way ANOVA followed by Tukey–Kramer test

b Kruskal–Wallis test followed by Dunn’s test

NP (U/mg proteins) NP-DSS (U/mg proteins) P (U/mg proteins) P-DSS (U/mg proteins)

MPO^b 1.84 ± 0.21 2.09 ± 0.23 1.69 ± 0.13 2.82 ± 0.31*

SOD^a 26.7 ± 1.3 24.3 ± 0.7 24.5 ± 1.51 18.9 ± 1.2*,^#

CAT^a 61.6 ± 2.9 49.0 ± 3.5 53.4 ± 4.3 37.3 ± 3.6*

GPx^b 1.42 ± 0.12 1.58 ± 0.13 2.85 ± 0.39^# 2.49 ± 0.30

GRed^a 0.101 ± 0.003 0.102 ± 0.003 0.105 ± 0.002 0.092 ± 0.005

Table 5 Colonic tissue cytokine levels in rats following colitis induction by DSS administration (4 % for 7 days)

NP rats fed the non-purified diet, NP-DSS rats fed the non-purified diet then treated by DSS, P rats fed the purified diet, P-DSS rats fed the puri- fied diet then treated by DSS, TNF-α tumour necrosis factor alpha, IL-1α interleukin-1 alpha, IL-1β interleukin-1 beta, IL-6 interleukin-6, IFN-γ interferon gamma, GM-CSF granulocyte–macrophage colony-stimulating factor, IL-2 inteleukin-2, IL-4 inteleukin-4, IL-5 inteleukin-5, IL-10 inteleukin-10, IL-12 p70 interleukin-12 p70, and IL-13 interleukin-13

Signiﬁcant effects induced by DSS for each diet are indicated with *P < 0.05

Signiﬁcant differences between diets (P vs. NP or P-DSS vs. NP-DSS) are indicated by ^#P < 0.05

a Two-way ANOVA followed by Tukey–Kramer test

b Kruskal–Wallis test followed by Dunn’s test

NP (pg/mg proteins) NP-DSS (pg/mg proteins) P (pg/mg proteins) P-DSS (pg/mg proteins)

TNF-α^a 82.8 ± 4.2 70.3 ± 1.9 78.5 ± 3.8 60.1 ± 4.0*

IL-1α^b 23.9 ± 2.0 41.1 ± 4.2 22.0 ± 2.4 134 ± 31.2*,^#

IL-1β^b 1,230 ± 155 2,242 ± 185 1,542 ± 265 4,131 ± 617*,^#

IL-6^b 132 ± 8 120 ± 9 112 ± 5 562 ± 215*,^#

IFN-γ^a 41.9 ± 3.1 35.6 ± 3.4 29.4 ± 1.6^# 22.5 ± 3.0^#

GM-CSF^a 22.9 ± 1.0 19.3 ± 1.8 18.4 ± 1.3 12.2 ± 1.3*,^#

IL-2^a 399 ± 17 357 ± 22 345 ± 13 292 ± 29

IL-4^a 51.8 ± 3.7 38.7 ± 4.6 43.7 ± 2.8 30.2 ± 2.4*

IL-5^a 45.8 ± 1.6 38.7 ± 3.4 38.2 ± 1.4 29.2 ± 1.9*,^#

IL-10^a 192 ± 8 160 ± 14 167 ± 9 139 ± 13

IL-12 p70^a 16.3 ± 0.8 12.5 ± 1.0 14.2 ± 0.6 13.0 ± 2.5

IL-13^a 27.8 ± 1.9 21.6 ± 2.2* 22.7 ± 0.7 13.7 ± 0.9*,^#

(8)

first time the effects of two commonly used basal diets, a NP standard chow and a semisynthetic P diet, on the development of DSS-induced colitis in rats by evaluating histological alterations, markers of inflammation and oxidative stress, and expression of various genes implicated in this disorder. We showed that the purified diet exacerbated the symptoms of colitis and the alteration of various markers of inflammation and oxidative stress. The NP diet consisted mainly of ingredients from natural sources such as wheat, corn, soybean oil, and brewer’s yeast, and thus contained various non-digestible ingredients. By contrast, the P diet was made of refined ingredients and contained cellulose as the sole source of non-digestible material. As previously suggested [11], these two diets may have different effects on the composition and metabolic activity of intestinal microflora. Such differences may be at least partly responsible for the difference in DSS colitis severity. Goto et al.

[11] have hypothesized that abundant ﬁbre in the NP diet might adsorb DSS itself, thereby reducing its cytotoxic effect.

Whichever the diet, control rats presented similar body weight gain despite a lower food intake in P-fed rats. The energy density of the two diets was different (12.4 vs.

15.6 kJ/g for NP and P diets, respectively), but energy intake was identical. As commonly shown [4], DSS significantly lowered food intake, thereby decreasing body weight gain. This decrease was more marked in P-fed rats, and growth of P-DSS rats was near zero. Moreover, the clinical score (DAI) increased more and earlier in P-fed rats, reflecting the earlier onset of colitis symptoms such as diarrhoea and bloody stools in these animals. As previously reported [12], higher DAI coincides with smaller colon length and higher histopathological score and MPO activity. Histological score, which reflects alteration of colonic mucosa, was increased by DSS. This increase was much more pronounced in P-fed rats. In these rats, colonic alterations were mainly related to epithelium and crypt destruction, massive inflammatory cell (PMN and mononuclear cells) infiltration, erosion, and oedema. Difference in the fibre content of these two diets is probably involved in the observed differences: certain high-fibre diets or diets rich in digestion-resistant carbohydrates have been shown to attenuate experimental colitis in animals [16]. Fibre increases the formation by intestinal microflora of SCFAs such as butyrate that may exert a beneficial effect against colitis development [16]. This is in line with the work of Fig. 4 Changes in gene expression in colon tissue of rats follow-

ing colitis induction by DSS administration (4 % for 7 days). Gene expression was determined by quantitative real-time PCR. Data were normalized to the housekeeping gene 18S and gene expression was represented compared with the NP group, which was set at 1. Results are expressed as the mean relative values (n = 7–8) with their standard errors. Statistical analysis was based on a ΔΔCT method for comparing relative fold-expression differences. Except for SOD2 expression, statistical signiﬁcance was analysed by Kruskal–Wallis followed by Dunn’s test. Signiﬁcant effects induced by DSS for each

diet are indicated with *P < 0.05. Signiﬁcant differences between diets (P vs. NP or P-DSS vs. NP-DSS) are indicated by ^#P < 0.05.

NP rats fed the non-purified diet, NP-DSS DSS-treated rats fed the non-purified diet, P rats fed the purified diet, P-DSS DSS-treated rats fed the purified diet. NFκB-p65 nuclear factor kappa B p65, NFκB1 nuclear factor kappa B p50, TNF-α tumour necrosis factor alpha, IL- 1β interleukin-1 beta, IL-6 interleukin-6, IL-4 interleukin-4, SOD2 superoxide dismutase 2, COX2 cyclooxygenase 2, iNOS inducible nitric oxide synthase, MMP9 matrix metalloproteinase 9, and ICAM1 intercellular adhesion molecule-1

(9)

Goto et al. [11], which showed higher SCFA concentrations in caecal contents from mice fed a non-purified diet rather than a purified one. Moreover, we detected some polyphenols that may have been bound to cereal materials in the NP diet (unpublished results). Polyphenols are antioxidant micronutrients and have been shown to exert beneficial effects in various intestinal inflammation models [17].

Hence, they could contribute to the lowered sensitivity to DSS of rats fed the NP diet.

MPO activity is used as a marker of inflammatory response in the colonic mucosa, and its activity is lin- early related to neutrophil infiltration [18]. On the other hand, DSS is known to induce neutrophil infiltration, thus increasing colonic MPO activity [18]. In this study, MPO activity significantly increased only in DSS-treated rats fed the P diet, in relation to the marked infiltration of the mucosa by PMNs. On the contrary, PMN infiltration was only moderately increased in NP-DSS rats, in relation to the slight, non-significant increase of MPO. Histologi- cal lesions with no significant increase in MPO activity as observed in NP-DSS rats have been previously reported [19, 20]. MPO increase is not the only factor implicated in the development of colonic lesions. One possible mechanism of action of DSS is a direct cytotoxicity on the colonic mucosa [4], an effect that may be reduced by fibre present in the NP diet. In addition, various radical species and inflammatory mediators may also be implicated in colonic damage [2]. Susceptibility to these various factors may thus be modulated by dietary factors such as the basal diet.

Inﬂammation is accompanied by recruitment and activation of phagocytic leucocytes in the mucosa. These cells will release high amounts of ROS such as superoxide anion [2]. This uncontrolled overproduction of ROS could over- whelm protective mechanisms, thereby resulting in cellular oxidative damage [21]. Usually, ROS may be neutralized by endogenous antioxidant enzymes: SOD converts O₂·⁻ to H₂O₂, which is subsequently neutralized to water by CAT or GPx. However, high local concentrations of ROS can attack antioxidant enzymes (mainly SOD) and inactivate them [22]. Thus, in P-fed rats, DSS administration sig- niﬁcantly decreased SOD and CAT activities as previously reported [23, 24]. This impairment of antioxidant enzymes may contribute to tissue damage in P-fed rats as reported in IBD patients [25]. Surprisingly, GPx activity was higher in P- than in NP-fed rats. The mechanism of this effect is unclear: the impact of various nutrients on GPx activity has only been scantily investigated.

Not only does oxidative stress induce direct damage to intestinal cells, it also causes dysregulated redox signalling, leading to activation of many signalling pathways involved in inflammation, including that of NF-κB [2]. NF-κB can activate the transcription of numerous pro-inflammatory genes, including pro-inflammatory cytokines, chemokines,

adhesion molecules, COX2, and iNOS. Many studies sug- gest that ICAM1 is involved in leucocyte migration to the site of inﬂammation, leading to severe intestinal damage.

Enhanced colonic mucosal endothelial cell ICAM1 expression and cell infiltration are early events in the inflammatory cascade of acute colitis [26]. Thus, the dramatic increase in ICAM1 gene expression observed in P-DSS rats was probably involved in the large leukocyte infiltration and the subsequent development of marked histological alterations. In addition, the massive leukocyte infiltration may also result in the increased iNOS mRNA levels observed in DSS-treated P-fed rats, since major sources of iNOS in the colon include recruited cells such as neutrophils and other leucocytes [8].

Cytokines are reported to play a major role in the immu- nopathogenesis of IBD [27]. In this study, we showed for the first time that the purified diet exacerbated the effect of DSS on colonic cytokine levels. Conversely, an increased production of SCFAs induced by dietary fibre present in NP diet [11] could limit the production of inflammatory mediators such as cytokines [28] and so offer protection against DSS-induced colitis. Increased levels of pro-inflammatory cytokines, such as TNF-α, IL1-β, and IL-6, are reported in IBD patients with active disease [29], as well as in experimental IBD models [6]. In this study, elevated colonic levels and/or gene expression of pro-inflammatory cytokines IL-1β and IL-6 were observed after colitis induction in P-fed rats. The enhanced monocyte/macrophage infiltration into the colonic mucosa in P-DSS rats may contribute to this effect [30]. It has been suggested that IL1-β is involved in the development of DSS-induced colitis and stimulates itself and IL-6 gene expression, thus inducing progression of disease [31]. DSS significantly induced TNF-α gene expression in P-fed rats, whereas a slight but significant reduction in colonic TNF-α level was observed. As reported [32], kinetics of TNF-α production may lag behind gene expression. In addition, as previously suggested [33], our data do not argue for a major role of this cytokine in DSS- induced colitis. As previously observed [34], DSS did not affect IL-4 gene expression. However, colonic levels of this cytokine, as well as IL-5 and IL-13, were lowered by DSS in P-fed rats. A decreased mucosal IL-4 production has been reported in some IBD patients [35]. GM-CSF colonic level was lowered by DSS in P-fed rats. This effect could participate in colitis development, since GM-CSF therapy has been shown to protect against DSS-induced colitis [36].

MMP-9 can be released by various cells in response to pro-inﬂammatory cytokines and may be a key enzyme responsible for regulation of accelerated extracellular matrix breakdown and remodelling in IBD [37]. It has been demonstrated that this enzyme is abundantly expressed in colon tissues of patients with UC [38]. A signiﬁcant up- regulation of MMP-9 gene expression and activity has been

(10)

observed in mice after DSS administration [39]. It has also been shown that DSS-induced colitis is markedly attenuated in mice lacking MMP-9 [40]. Puriﬁed diet exacerbated DSS-induced pro-inﬂammatory cytokine production that may consecutively stimulate induction of MMP-9 gene expression. The high expression of MMP-9 could thus contribute to the marked histological lesions reported in P-DSS rats.

In summary, this study found that a semisynthetic purified diet in rats promoted the onset of a more marked DSS-induced colitis than that observed with a non-purified standard chow. This effect was accompanied by modifi- cations of the pro-inflammatory gene expression pattern.

Fibre and micronutrients such as polyphenols present in commercial standard chow may protect against DSS- induced acute colitis. These findings underline the impor- tance of taking into account the composition of the basal diet in designing DSS experiments and argue for the use of a purified diet devoid of protective components in future studies designed to evaluate the potential beneficial effects of various micronutrients against DSS colitis.

Acknowledgments The authors thank the GenoToul Anexplo- Phenotypage platform (IFR150, Toulouse, France) for the assay of colonic cytokines with Luminex technology, and Sophie Garcin for her technical assistance in handling rats. This work was funded by 3inature Biosphère.

Conflict of interest The authors have no conﬂict of interest.

References

1. Sands BE (2007) Inﬂammatory bowel disease: past, present, and future. J Gastroenterol 42:16–25

2. Zhu H, Li YR (2012) Oxidative stress and redox signaling mechanisms of inﬂammatory bowel disease: updated experimental and clinical evidence. Exp Biol Med (Maywood) 237:474–480 3. Jurjus AR, Khoury NN, Reimund JM (2004) Animal models

of inﬂammatory bowel disease. J Pharmacol Toxicol Methods 50:81–92

4. Solomon L, Mansor S, Mallon P, Donnelly E, Hoper M, Loughrey M, Kirk S, Gardiner K (2010) The dextran sulphate sodium (DSS) model of colitis: an overview. Comp Clin Pathol 19:235–239

5. Melgar S, Karlsson L, Rehnstrom E, Karlsson A, Utkovic H, Jansson L, Michaelsson E (2008) Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inﬂammatory bowel disease. Int Immunopharmacol 8:836–844

6. Perse M, Cerar A (2012) Dextran sodium sulphate colitis mouse model: traps and tricks. J Biomed Biotechnol 2012:718617 7. Okayama M, Hayashi S, Aoi Y, Nishio H, Kato S, Takeuchi K

(2007) Aggravation by selective COX-1 and COX-2 inhibitors of dextran sulfate sodium (DSS)-induced colon lesions in rats. Dig Dis Sci 52:2095–2103

8. Reed KL, Fruin AB, Gower AC, Gonzales KD, Stucchi AF, Andry CD, O’Brien M, Becker JM (2005) NF-kappaB activation precedes increases in mRNA encoding neurokinin-1 receptor,

proinﬂammatory cytokines, and adhesion molecules in dextran sulfate sodium-induced colitis in rats. Dig Dis Sci 50:2366–2378 9. Lenoir L, Rossary A, Joubert-Zakeyh J, Vergnaud-Gauduchon J,

Farges MC, Fraisse D, Texier O, Lamaison JL, Vasson MP, Fel- gines C (2011) Lemon verbena infusion consumption attenuates oxidative stress in dextran sulfate sodium-induced colitis in the rat. Dig Dis Sci 56:3534–3545

10. Felgines C, Fraisse D, Besson C, Vasson MP, Texier O (2014) Bioavailability of lemon verbena (Aloysia triphylla) polyphenols in rats: impact of colonic inﬂammation. Br J Nutr 111:1773–1781 11. Goto H, Takemura N, Ogasawara T, Sasajima N, Watanabe J, Ito

H, Morita T, Sonoyama K (2010) Effects of fructo-oligosaccha- ride on DSS-induced colitis differ in mice fed nonpuriﬁed and puriﬁed diets. J Nutr 140:2121–2127

12. Cooper HS, Murthy SN, Shah RS, Sedergran DJ (1993) Clinico- pathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69:238–249

13. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔ C(T)) method. Methods 25:402–408

14. Larrosa M, Luceri C, Vivoli E, Pagliuca C, Lodovici M, Moneti G, Dolara P (2009) Polyphenol metabolites from colonic micro- biota exert anti-inﬂammatory activity on different inﬂammation models. Mol Nutr Food Res 53:1044–1054

15. Sanchez-Fidalgo S, Sanchez de Ibarguen L, Cardeno A, Alar- con de la Lastra C (2012) Inﬂuence of extra virgin olive oil diet enriched with hydroxytyrosol in a chronic DSS colitis model. Eur J Nutr 51:497–506

16. Schrenk D (2009) Dietary ﬁber, low-molecular-weight food constituents and colo-rectal inﬂammation in animal models—a review. Mol Nutr Food Res 53:1281–1288

17. Romier B, Schneider YJ, Larondelle Y, During A (2009) Dietary polyphenols can modulate the intestinal inﬂammatory response.

Nutr Rev 67:363–378

18. Iba Y, Sugimoto Y, Kamei C, Masukawa T (2003) Possible role of mucosal mast cells in the recovery process of colitis induced by dextran sulfate sodium in rats. Int Immunopharmacol 3:485–491 19. Kruidenier L, van Meeteren ME, Kuiper I, Jaarsma D, Lamers

CB, Zijlstra FJ, Verspaget HW (2003) Attenuated mild colonic inﬂammation and improved survival from severe DSS-colitis of transgenic Cu/Zn-SOD mice. Free Radic Biol Med 34:753–765 20. Sann H, Erichsen J, Hessmann M, Pahl A, Hoffmeyer A (2013)

Efﬁcacy of drugs used in the treatment of IBD and combina- tions thereof in acute DSS-induced colitis in mice. Life Sci 92:708–718

21. Pavlick KP, Laroux FS, Fuseler J, Wolf RE, Gray L, Hoffman J, Grisham MB (2002) Role of reactive metabolites of oxygen and nitrogen in inﬂammatory bowel disease. Free Radic Biol Med 33:311–322

22. Kruidenier L, Verspaget HW (2002) Review article: oxidative stress as a pathogenic factor in inﬂammatory bowel disease—

radicals or ridiculous? Aliment Pharmacol Ther 16:1997–2015 23. Korkina L, Suprun M, Petrova A, Mikhal’chik E, Luci A, De

Luca C (2003) The protective and healing effects of a natural antioxidant formulation based on ubiquinol and Aloe vera against dextran sulfate-induced ulcerative colitis in rats. BioFactors 18:255–264

24. Oh PS, Lim KT (2006) Plant originated glycoprotein has anti- oxidative and anti-inﬂammatory effects on dextran sulfate sodium-induced colitis in mouse. J Biomed Sci 13:549–560 25. Dagli U, Balk M, Yucel D, Ulker A, Over H, Saydam G, Sahin B

(1997) The role of reactive oxygen metabolites in ulcerative colitis. Inﬂamm Bowel Dis 3:260–264

26. Breider MA, Eppinger M, Gough A (1997) Intercellular adhesion molecule-1 expression in dextran sodium sulfate-induced colitis in rats. Vet Pathol 34:598–604

(11)

27. Sanchez-Munoz F, Dominguez-Lopez A, Yamamoto-Furusho JK (2008) Role of cytokines in inﬂammatory bowel disease. World J Gastroenterol 14:4280–4288

28. Galvez J, Rodriguez-Cabezas ME, Zarzuelo A (2005) Effects of dietary ﬁber on inﬂammatory bowel disease. Mol Nutr Food Res 49:601–608

29. Reimund JM, Wittersheim C, Dumont S, Muller CD, Baumann R, Poindron P, Duclos B (1996) Mucosal inﬂammatory cytokine production by intestinal biopsies in patients with ulcerative colitis and Crohn’s disease. J Clin Immunol 16:144–150

30. Tokuyama H, Ueha S, Kurachi M, Matsushima K, Moriyasu F, Blumberg RS, Kakimi K (2005) The simultaneous blockade of chemokine receptors CCR2, CCR5 and CXCR3 by a non-pep- tide chemokine receptor antagonist protects mice from dextran sodium sulfate-mediated colitis. Int Immunol 17:1023–1034 31. Kwon KH, Murakami A, Hayashi R, Ohigashi H (2005) Inter-

leukin-1beta targets interleukin-6 in progressing dextran sulfate sodium-induced experimental colitis. Biochem Biophys Res Commun 337:647–654

32. Dharmani P, Leung P, Chadee K (2011) Tumor necrosis factor- alpha and Muc2 mucin play major roles in disease onset and progression in dextran sodium sulphate-induced colitis. PLoS ONE 6:e25058

33. Lopez-Posadas R, Requena P, Gonzalez R, Suarez MD, Zarzuelo A, Sanchez de Medina F, Martinez-Augustin O (2010) Bovine glycomacropeptide has intestinal antiinﬂammatory effects in rats with dextran sulfate-induced colitis. J Nutr 140:2014–2019 34. Kostadinova FI, Baba T, Ishida Y, Kondo T, Popivanova BK,

Mukaida N (2010) Crucial involvement of the CX3CR1–CX3CL1

axis in dextran sulfate sodium-mediated acute colitis in mice. J Leukoc Biol 88:133–143

35. Karttunnen R, Breese EJ, Walker-Smith JA, MacDonald TT (1994) Decreased mucosal interleukin-4 (IL-4) production in gut inﬂammation. J Clin Pathol 47:1015–1018

36. Sainathan SK, Hanna EM, Gong Q, Bishnupuri KS, Luo Q, Col- onna M, White FV, Croze E, Houchen C, Anant S, Dieckgraefe BK (2008) Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis. Inﬂamm Bowel Dis 14:88–99

37. Medina C, Videla S, Radomski A, Radomski MW, Antolin M, Guarner F, Vilaseca J, Salas A, Malagelada JR (2003) Increased activity and expression of matrix metalloproteinase-9 in a rat model of distal colitis. Am J Physiol Gastrointest Liver Physiol 284:G116–G122

38. Baugh MD, Perry MJ, Hollander AP, Davies DR, Cross SS, Lobo AJ, Taylor CJ, Evans GS (1999) Matrix metalloproteinase levels are elevated in inﬂammatory bowel disease. Gastroenterology 117:814–822

39. Suzuki K, Sun X, Nagata M, Kawase T, Yamaguchi H, Suku- maran V, Kawauchi Y, Kawachi H, Nishino T, Watanabe K, Yoneyama H, Asakura H (2011) Analysis of intestinal ﬁbrosis in chronic colitis in mice induced by dextran sulfate sodium. Pathol Int 61:228–238

40. Santana A, Medina C, Paz-Cabrera MC, Diaz-Gonzalez F, Farre E, Salas A, Radomski MW, Quintero E (2006) Attenuation of dextran sodium sulphate induced colitis in matrix metalloproteinase-9 deﬁcient mice. World J Gastroenterol 12:6464–6472