Inflamm. Res. (2014) 63:873–883 DOI 10.1007/s00011-014-0761-1

Inflammation Research

ORIGINAL RESEARCH PAPER

Preventive effects of Escherichia coli strain Nissle 1917 with different courses and different doses on intestinal inflammation in murine model of colitis Sumei Sha • Bin Xu • Xiangyun Kong • Ni Wei • Jian Liu • Kaichun Wu

Received: 23 January 2014 / Accepted: 23 July 2014 / Published online: 14 August 2014 Ó Springer Basel 2014

Abstract Objective To analyze the in vivo effect of Escherichia coli Nissle 1917 (EcN) with different courses and different doses to Sprague–Dawley rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Methods The probiotic was orally administered with different courses of treatment (with or without preadministration) and different doses (107–109 CFU/day) to Sprague–Dawley rats with TNBS-induced colitis.

Responsible Editor: Liwu Li. Sumei Sha, Bin Xu, and Xiangyun Kong contributed equally to this work. S. Sha  B. Xu  N. Wei  J. Liu  K. Wu (&) State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 17 Changle Western Road, Xi’an, Shaanxi 710032, People’s Republic of China e-mail: [email protected] S. Sha e-mail: [email protected]

Therapeutic effects, levels of cytokine in serum, mRNA and protein expression were analyzed. Results Oral EcN administration after TNBS-induced improved colitis dose dependently. In parallel, a reduction of disease activity index and colonic MPO activity together with a decreased level of TNF-a and a trend of increased IL-10 expression was detected. Pre-administration of 107 CFU/day EcN to TNBS-treated rats resulted in a significant protection against inflammatory response and colons isolated from these rats exhibited a more pronounced expression of ZO-1 than the other groups. In the group of pre-administration of 109 CFU/day, the condition was not improved but deteriorated. Conclusions This study convincingly demonstrates that pre-administration of probiotic EcN with low dose is able to protect colitis of rats and mediate up-regulation of ZO-1 expression, but long-term of high-dose EcN may do harm to colitis. Keywords Probiotic  Trinitrobenzene sulfonic acid  Myeloperoxidase  Tumor necrosis factor-alpha  Interleukin-10

N. Wei e-mail: [email protected]

Introduction

J. Liu e-mail: [email protected]

Inflammatory bowel disease (IBD) is a group of chronic relapsing inflammatory disorders of the gastrointestinal tract and primarily comprises ulcerative colitis (UC) and Crohn’s disease (CD). In the US, approximately 1.5 million people suffer from IBD. The incidences vary between 9 and 12 per 100,000 for UC and between 6 and 8 per 100,000 for CD [1]. Chinese IBD patients were less than in Western countries in the past. But the number of patients with IBD is increasing in China in the last decade [2]. The

B. Xu No. 174 Hospital of People’s Liberation Army, Xiamen, Fujian 361000, People’s Republic of China e-mail: [email protected] X. Kong Department of Gastroenterology, Xi’an No.1 Hospital, Xi’an, Shaanxi 710002, People’s Republic of China e-mail: [email protected]

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exact etiology of chronic IBD is still unknown but seems complex and multifactorial. Mounting evidence suggests an important role for the intestinal microbiota in the chronic mucosal inflammation that occurs in IBD [3], and novel molecular approaches have further identified a microbial dysbiosis in IBD patients [4]. Since several probiotic products are commercially available and marketed as beneficial for a wide variety of gastrointestinal diseases, the usefulness of probiotics is also a frequently asked question in the consultation room of IBD. Several mechanisms of action of probiotic products have been postulated, which may interfere with aetiological factors in IBD. Therefore, the interest of physicians and researchers in the application of probiotics in IBD is increasing. Probiotic bacteria are live microorganisms which contribute health benefits to their host [5]. They create an unfavorable environment for pathogens by various mechanisms, which include promoting the integrity of the gut defense barrier by normalizing intestinal permeability, modulating intestinal secretory Ig function, controlling intestinal inflammatory responses, and balancing the release of cytokines. In addition, probiotics maintain the normal microecology of the gastrointestinal flora and antimicrobial effects mediated by nutrient competition, alteration of local pH, production of bacteriocins, modification of pathogen-derived toxins, and stimulation of epithelial mucin production [6]. Escherichia coli Nissle 1917 (Mutaflor, also called EcN) is one of the most investigated probiotic bacteria. It is a gram-negative bacterium which belongs to the enterobacteriaceae family and is classified and identified according to O and H serogroups. The number of reports discussing the underlying mechanisms of EcN has increased rapidly in recent years. It has been shown that EcN induces human b-defensin-2 expression in the cell culture in a time- and densitydependent manner [7]. Studies demonstrate EcN with potent immunomodulatory properties that reducing in the secretion of proinflammatory cytokines and other markers of intestinal inflammation, it may also alter the intestinal microflora [8], other effects including local and systemic immunomodulation [9–11] and barrier enhancement [12, 13]. However, the exact mechanism or mechanisms by which EcN exerts its beneficial effects are even today still not completely understood. Over the last decade or so, the clinical use of probiotic bacteria is growing and besides the initial successful administration of EcN in the management of infectious gastroenteritis [14]. EcN is now successfully used in several European countries for the treatment of various diseases of the digestive tract, including diarrhea [15], diverticulitis [16] and IBD [17]. In the latter, it is particularly used for maintenance therapy in patients with UC in remission, showing an equivalent efficacy to the standard

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drug mesalazine, used in this condition [18]. In 2004, EcN was recommended as an alternative to standard mesalazine treatment to maintain remission based on its clinical efficacy and low side-effect profile in the guidelines for diagnosis and treatment of UC issued by the German Society of Gastroenterology and Digestive Diseases (DGVS) [19]. Beneficial effects have also been reported for CD [20]. But in elegant experiments, [21] showed that it is not completely safe. Their results suggest that if both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of EcN may occur and have potentially severe adverse effects. Differences in dosages used further hamper a comparison between studies, and dose–effect studies are limited. Although at least 108–10 colony-forming units per day are generally recommended, clear information on minimal efficacious (viable) dosages is not available [22]. The aim of this study was thus to describe the in vivo effects of EcN, given orally, with different courses and different doses in experimental trinitrobenzene sulfonic acid (TNBS) model of rat colitis, in which there is an pathological change similar with CD localized in the large intestine.

Materials and methods Experimental animals and bacteria Sixty female adult Sprague–Dawley rats weighing 195 ± 20 g were purchased from the experimental animal research center. All rats used in this study were 6–8 weeks old. All animals were held in standard caging conditions and received standard rodent chow and drinking water ad libitum. The research was approved by the local institutional review board, and the treatment of animals was according to the principles of laboratory animal care. The non-pathogenic E. coli strain Nissle 1917, also known as EcN or Mutaflor, was provided by Ardeypharm (Herdecke, Germany). EcN was grown in LB media and harvested at mid-logarithmic phase. Bacteria were washed and collected by centrifugation (3 min at 1,0009g), and the resulting pellet was diluted in 2 ml sterile PBS prior to injection. Induction of colitis The rats were randomly divided into six groups (n = 10 each, Fig. 1): (1) low dose with pre-administration EcN group, (2) high dose with pre-administration EcN group, (3) low dose without pre-administration EcN group, (4) high-dose without pre-administration EcN group, (5) untreated control group, (6) blank control. Before induction of colitis, rats were starved for 24 h but had free access to

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Fig. 1 Experimental design. Animals were separated into six groups as following: Group 1: TNBS-induced colitis treated with 2 9 107 CFU EcN intragastrically for a week both before and after the induction of colitis. Group 2: TNBS-induced colitis treated with 2 9 109 CFU EcN intragastrically for a week both before and after the induction of colitis. Group 3: TNBS-induced colitis treated with 2 9 107 CFU EcN intragastrically for a week. Group 4: TNBS-

induced colitis treated with 2 9 109 CFU EcN intragastrically for a week. Group 5: untreated TNBS-induced colitis control group, receiving intragastrically PBS at a dose of 2.0 ml/rat/day for two weeks. Group 6: healthy control group, receiving 25 % aqueous ethanol once at a dose of 1.0 ml/rat into the colon and intragastric PBS at a dose of 2.0 ml/rat/day for a week

Table 1 Scoring of disease activity index (DAI)

Table 3 Scoring criteria of full-thickness distal colon sections

Score

Weight loss (%)

Stool consistency

Rectal bleeding

Mucosal epithelium

None (0); mild surface (1); moderate (2); extensive full thickness (3)

0

None

Normal

Normal

Crypts

Ulceration

1

1–5

2

5–10

3

10–20

4

[20

Lower third (0); mild mid-third (1); moderate mid-third (2); upper third (3)

Loose stools

Mitotic activity: Diarrhea

Gross bleeding

DAI value is the combined scores of weight loss, stool consistency, and bleeding divided by 3

Neutrophilic infiltrate Mucus depletion Lamina propria

Plasmacytoid infiltrate Neutrophilic infiltrate

Table 2 Criteria for scoring ‘‘macroscopic mucosal damage’’ component of combined damage score Score Appearance 0

Normal

1

Localized hyperemia, no ulcers

2

Ulceration without hyperemia or bowel wall thickening

3

Ulceration with inflammation at one site

4

Two or more sites of ulceration and inflammation

5 Major sites of damage extending [1 cm along length of colon 6–10 Major sites of damage extending [2 cm along length of colon, with score increasing by 1 for each additional cm

water. The rats were lightly anesthetized with 5 % chloralic hydras. A rubber catheter (OD, 2 mm) was inserted rectally into the colon with the tip approximately 8 cm proximal to the anus. TNBS (dissolved in 1.0 ml 50 % ethanol) was instilled into the colon of rats in 1–5 groups at a dose of 25 mg/rat, while rats in group 6 received only 1.0 ml 25 % ethanol instead of TNBS. After

Fibrin deposition Submucosal

Vascularity None (0); mucosal (1); submucosal (2); transmural (3) Neutrophilic infiltrate Oedema

The colonic mucosa damage index (CMDI) was used to assess the severity of colonic damage Scoring scale: 0, none; 1, mild; 2, moderate; 3, severe. Maximum score: 30

the induction of colitis, different courses of treatment (with or without pre-administration) with different doses (107– 109 CFU/day) were administrated to experimental animal. Characteristics of stool and body weight of each rat were observed closely and recorded to assess the disease activity index (DAI) as described previously [23] (Table 1). All rats were killed 1 week after induction of colitis; the colon was transected in the distal rectum and just distal to the cecum at the time of killing. The colon was then measured and the colon length was corrected for the mouse’s pretreatment weight and

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calculated as: colon length (cm)/pretreatment weight (g). And the colon was removed for the assessment of colonic damage and scored for macroscopically visible damage on a 0–10 scale by two observers unaware of the treatment, according to the criteria described by [24] (Table 2), which takes into account the extent as well as the severity of colonic damage. In addition, approximately 3 cm of proximal colon (1–2 cm from cecum) and 3 cm of distal colon (1–2 cm from anus) samples sections were stained with haematoxylin and eosin, the colonic mucosa damage index (CMDI) was assessed by two histopathologists independently without knowing the experimental information. The two pathologists reached the same conclusion in all cases, according to the criteria previously described [25] (Table 3). Derivation of colonic MPO activity and serum levels of TNF-a and IL-10 The activity of MPO, which is found in neutrophils, can be used for evaluating the degree of colonic inflammation. Following the instruction of the commercial reagent kit, 100 mg of the frozen colon was used and assayed spectrophotometrically for MPO activity; the results were expressed as MPO units g-1 wet tissue; one unit of MPO activity was defined as that degrading 1 lmol H2O2 min-1 at 25 °C. Serum levels of TNF-a and IL-10 were also measured according to the instruction of corresponding ELISA kits for rat. Data were calculated as the mean cytokine response (in pg/ml of serum) of each treatment from triplicate wells, plus or minus the standard error of the mean. Western blot analysis Total protein was extracted from 2.0 cm of each colon, and 35 lg of protein were separated on an 8 % SDS-PAGE and transferred onto nitrocellulose (NC) membrane (0.45 mm, Millipore, USA). After a 2 hour block by 5 % fat-free milk in TBS (0.5 % Tween 20), the NC membrane was incubated with anti-ZO-1 antibody (1:200 dilution, Zymed) and anti-bactin (1:2,000 dilution, Sigma, USA) at 4 °C overnight. After three 5 min washes by TBST, the NC membrane was incubated with horseradish peroxidase (HRP) coupled with goat anti-Rabbit (1:2,000 dilution, BIOS) at room temperature for 1 h, respectively. Then, reaction products were visualized by enhanced chemiluminescence (ECL-kit, Thermo Fisher Scientific, USA). Representatives of three independent experiments were presented. Immunofluorescence and immunohistochemistry stain Fresh frozen tissue sections were fixed with 4 % paraformaldehyde, washed extensively and blocked with goat

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serum. Subsequently, the sections were incubated with the primary antibody rabbit anti-ZO-1 (1:200 dilution, Zymed) followed by incubation with a Cy3-labeled secondary antiRabbit IgG antibody (Jackson Immuno Research, West grove, PA, USA). Cell nucleus was counterstained using DAPI. The sections were then dried, covered with gelatine and visualized by fluorescence microscopy. A slide maintained in 0.01 mol/L PBS (pH 7.4), instead of the primary antibody solution, served as a negative control. As for the immunohistochemistry stain, the tissue paraffin blocks were cut into 4 lm sections. The sections were heated at 70 °C for 30 min, dewaxed in xylene and dehydrated through a gradient concentration of alcohol. Antigens were retrieved in 0.01 mol/L citrate buffer (pH 6.0) by a microwave for 25 min. Then, sections were washed thrice for 3 min with PBS. After blocking the endogenous peroxidase and non-specific staining with 3 % (v/v) H2O2 and normal goat serum, the sections were incubated with anti-ZO-1 antibody (1:200 dilution, Zymed) overnight at 4 °C. The slides were then washed with PBS three times for 3 min and incubated with HRP-conjugated goat anti-Rabbit IgG secondary antibody for 1 h. Finally, the sections were visualized by DAB solution (Zhongshan Golden Bridge Biotechnology Co, Ltd), and counterstained with hematoxylin (Zhongshan Golden Bridge Biotechnology Co, Ltd). A slide incubated with anti-actin primary antibodies served as the positive control, and a slide maintained in 0.01 mol/L PBS (pH 7.4), instead of the primary antibody solution, served as a negative control. Statistical analysis All the statistical analyses were performed with Statistical Program of Social Sciences (SPSS) for windows, version 16.0. For analysis of numeric values, the one-tailed analysis of variance and the student’s t test were used. A pvalue of \0.05 was considered significant. Error bars represent the standard error of the mean.

Results Change in DAI of all rats during treatment 10–24 h after administration of TNBS, rats in groups 1–5 began to show such symptoms as obvious diarrhea, inactivity and anorexia, while there were only four rats with minor diarrhea in group 6. DAI of all rats differed significantly at different times (multiple comparison by ‘‘time’’ factor, p \ 0.05). Following treatment, DAI began to decrease in the treated groups (1–4) except group 2 on the second day. DAI was also significantly different among the 6 groups (multiple comparison by ‘‘group’’ factor,

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Fig. 2 Disease activity indexes of all rats were analyzed by DAIs (a), colon length (cm)/pretreatment weight (g) (b), macroscopically visible damage (c) and CMDI (d). Colon length was calculated as

colon length (cm)/pretreatment weight (g) at the time of animal killing. aP \ 0.05 vs. group 6, bP \ 0.05 vs. group 5, and cP \ 0.05 vs. group 2

p \ 0.05), with group 6 the lowest and group 2 the highest (p \ 0.05). DAIs of treated groups 1, 3 and 4 were significantly lower (p \ 0.05) but higher in group 2 in comparison to the untreated group 6 (Fig. 2a).

increased (p \ 0.05). However, the damage in the treated group 1 and 4 decreased significantly (p \ 0.05), as compared to untreated group 5. In contrast, these lesions were greatly aggravated in groups 2. The colonic mucosa damage was not significantly different between treated groups 1 and 4 (p [ 0.05), or between treated groups 3 and untreated control group 6 (p = 0.9001) (Fig. 2c).

Colon length and macroscopic rating of colonic mucosa damage Shortening of the colon has been associated with TNBSinduced colitis and has also been shown to correlate with disease severity. In our study, a significant decrease in colon length was seen after TNBS induced. But the colon length was shorter in group 5 than EcN treated animals group 1 and 4 except group 3. While the colon length in group 2 was the shortest, all of the five groups were shorter than healthy control group 6 (Fig. 2b). Obvious hyperemia, swelling, edema, and ulceration could be seen on the colonic mucosal surface of rats in group 5, in which there were two rats with colonic ulcers as long as 4.0 cm. In comparison to group 6, the colonic mucosa damage of group 5 was significantly

Histopathologic changes of colonic mucosa damage index (CMDI) in all rats Ulceration, submucosal edema, cytoplasmic mucin depletion (Fig. 3e), infiltration of mucosa, submucosal granulomatosis, and thickening of intestinal wall (Fig. 3e) were common in untreated group 5. When compared to the healthy control group 6 (Fig. 3f), histopathologic score of untreated group 5 was pronouncedly elevated (p \ 0.05). Such lesions typical of CD, however, were greatly relieved in treated groups 1 and 4 (Fig. 3a, d). CMDIs of treated groups 1 and 4 were significantly inhibited (p \ 0.05,

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878 Fig. 3 Hematoxylin and eosin staining of the colon and ileum samples sections from rats treated with EcN and healthy control. Two representative pictures from each group were shown. a group 1 (a1, colon; a2, ileum); b group 2 (b1, colon; b2, ileum); c group 3 (c1, colon; c2, ileum); d group 4 (d1, colon; d2, ileum); e group 5 (e1, colon; e2, ileum) and f group 6 (f1, colon; f2, ileum). Scale bars 50 lm

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Fig. 4 Colonic MPO (a), serum TNF-a (b) and IL-10 (c) from rats treated with EcN and healthy control were analyzed. a P \ 0.05 vs. group 6, b P \ 0.05 vs. group 5, and c P \ 0.05 vs. group 2

respectively, Fig. 2d), when compared to untreated group 5. There was a non-significant difference between treated groups 1 and 4 (p [ 0.05, Fig. 2d). In comparison to either treated group 1 or group 4, CMDI of pre-treated with 109 CFU/day group 2 was significantly inhibited (p \ 0.05, respectively) (Figs. 2d, 3b). When compared to the untreated group 5 (Fig. 3e), histopathologic score of 107 CFU/day treated group 3 (Fig. 3c) was not significantly changed (p [ 0.05, Fig. 2d). Colonic MPO activity assay Colonic MPO activity of untreated group 5 was escalated significantly compared to that of healthy control group 6 (Fig. 4a; p \ 0.05). Treatment with 109 CFU/day or pretreated with 107 CFU/day (p \ 0.05) significantly suppressed colonic MPO activity compared to untreated group 5. The difference in colonic MPO activities between treatment with group 1 and 4 did not reach significance (p [ 0.05). Meanwhile, 107 CFU/day treated group 3 did not differ from untreated group 5 (p [ 0.05). And pre-treated with 109 CFU/ day (p \ 0.05) did not activate colonic MPO activity compared to untreated group 5 (p [ 0.05). Serum levels of TNF-a and IL-10 In untreated group 5, serum levels of both TNF-a and IL10 were significantly increased when compared to the

healthy control group 6 (p \ 0.05). When pre-treated with 107 CFU/day EcN group 1 (p \ 0.05) or treated with 109/ day EcN group 4 (p \ 0.05), serum TNF-a levels were significantly reduced, as compared to untreated group 5. No significant difference in serum TNF-a was observed between these two treatment groups (p [ 0.05). However, in treated with 107 CFU/day EcN group 3, the change of serum TNF-a level was not significantly reduced (p [ 0.05). And in group 2 serum TNF-a level was significantly increased compared to untreated group 5 (p \ 0.05) (Fig. 4b). The pattern of serum IL-10 was distinct from that of TNF-a. Following treatment with EcN, serum IL-10 rose significantly in group 1 and 4, compared to untreated group 5 (p \ 0.05). There was a non-significant difference between these two groups (p [ 0.05). But in group 2, serum IL-10 level was significantly decreased compared to untreated group 5 (p \ 0.05). And in group 3, the change of serum IL-10 level was not significant (p [ 0.05) (Fig. 4c). Colonic expression of tight junction protein ZO-1 Colonic mucosa was assayed for ZO-1 by western blot. A representative western blot probed for ZO-1 is shown in Fig. 5a. There was a loss of expression of ZO-1 in the colonic mucosa of rats in untreated group 5 compared to EcN treated animals, especially in group 2. Among the EcN-treated groups, expression of ZO-1 in group 1 and 4

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Fig. 5 Western blot (a) and immunofluorescence staining of frozen tissue sections (b1–b6) from rats treated with EcN and healthy control were stained (magnification, 9200) with an anti-ZO-1 antibody (red). Nuclei were counterstained with DAPI (blue). Merged images are shown. b1, group 1; b2, group 2; b3, group 3; b4, group 4; b5, group 5; b6, group 6. There was an increase of expression of ZO-1 in the colonic mucosa of rats pre-treated with 107 CFU/day EcN group 1 or treated with 109 CFU/day EcN group 4 compared to untreated control group 5, but a loss of expression of ZO-1 in group 2 and no change in group 3 compared with group 5 was observed. Anti-b-actin antibody was used as internal control, binding to the corresponding protein with a molecular weight of 42 kDa

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Fig. 6 ZO-1 protein expressions in ileum and colon of group 1–6 were detected by immunohistochemistry stain. a group 1 (a1, colon; a2, ileum); b group 2 (b1, colon; b2, ileum); C group 3 (c1, colon; c2, ileum); d group 4 (d1, colon; d2, ileum); e group 5 (e1, colon; e2, ileum) and f group 6 (f1, colon; f2, ileum). Scale bars 50 lm. There was an increase of expression of ZO-1 by both immunohistochemistry stain and western blot in the colonic mucosa of rats pre-treated with 107 CFU/day EcN group 1 or treated with 109 CFU/day EcN group 4 compared to untreated control group 5, but a loss of expression of ZO1 in group 2 and no change in group 3 compared with group 5 was observed

was the highest, followed by group 3 and the lowest in group 2. To morphologically confirm our western blot findings, immunofluorescence and immunohistochemistry stain for ZO-1 was performed on colonic sections from control animals. In control animals (Fig. 5, B6), ZO-1 (red)

Preventive effects of Escherichia coli strain Nissle 1917 in murine model of colitis

can be seen between the epithelial cells lining the epithelial surface of the colon and its crypts. In untreated group 5, there was remarkable loss of colonic ZO-1 expression as can be seen by decrease in ZO-1 staining (Fig. 5, B5). There was an increase of expression of ZO-1 in the colonic mucosa of rats pre-treated with 107 CFU/day EcN group 1 (Fig. 5, B1) or treated with 109 CFU/day EcN group 4 (Fig. 5, B4) compared to untreated control group 5, but a loss of expression of ZO-1 in group 2 (Fig. 5, B2). And no apparent change in group 3 (Fig. 5, B3) compared with group 5 was observed. The nuclear stain indicates the presence of epithelial cells even in the untreated animals, demonstrating that the loss of ZO-1 was not because of loss of epithelial cells. Similar tendency was observed by immunohistochemistry stain (Fig. 6).

Discussion IBD such as CD and UC are chronic recurrent diseases characterized by inflammation, ulceration, and stricturing of the gastrointestinal tract. The etiology and pathogenesis of IBD still remain unclear. Thus, medical treatment so far has merely a palliative and symptomatic character. However, the pathogenesis has been thought to relate to a dysregulated immune response to an environmental trigger in a genetically susceptible host. From studies of human and animal models for experimental colitis, increasing evidence is generated that the resident intestinal flora plays a critical role in the development of the intestinal inflammation. Hence, probiotics appear to be therapeutically effective. The use of probiotics as novel therapeutic agents and as an alternative to standard medication in gastrointestinal diseases is promising, although their mechanism of action is still under investigation. EcN has evolved into one of the best characterized probiotics, and its therapeutic efficacy and safety have convincingly been proven. It contains the Escherichia coli strain Nissle 1917 (EcN) that belongs to the normal, physiological intestinal microflora. Escherichia coli Nissle 1917 was isolated in 1917 from the feces of a German soldier who seemed to be protected from infectious diarrheal diseases. Since then, EcN has been studied intensively and demonstrated with significant probiotic properties such as antagonistic effects toward other members of the intestinal microbiota, immunomodulation and reinforcement of the intestinal barrier function, which make this strain an attractive candidate for probiotic therapy in IBD. This bacterial strain does not express any virulence factor. In vitro, E. coli strain Nissle 1917 shows an antagonistic effect against pathogenic bacterial strains such as enteropathogenic and uropathogenic E. coli strains, Salmonella enteritis, Shigella dysenteriae and Yersinia

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enterocolitica [26]. Furthermore, clinical observations demonstrate beneficial effects of EcN in the treatment of IBD. It especially was shown to be as effective as mesalazine in the maintenance of remission in patients with UC [27]. A potential mechanism by which probiotics may exhibit their beneficial activities is modulation of the epithelial barrier function. So far, however, no animal model of experimental colitis has been tested for further investigation of the optimized dose and course by which EcN may exert its beneficial effects. To solve this problem, 60 rats intrarectally given TNBS dissolved in ethanol at the dose of 25 mg/rat developed characteristics similar as clinic symptoms, mucosal lesions and colonic histopathologic changes with CD in humans were studied in our research. Different effects of EcN on particular courses and doses were shown in our research. The key findings of this study can be summarized as follows: A low dose of preadministration EcN was as effective as high dose of administration in remission of TNBS-induced colitis in rats, but a high dose of pre-administration EcN would exacerbate the intestinal inflammation. Different results were seen when EcN was administered in a model of intestinal colitis. In contrast to the effects of the probiotics on clinical and histopathological scores in low dose of pre-administration group, application of EcN did not ameliorate the histological appearance in high dose of pre-administration group of the TNBS-induced colitis in rats. Moreover, high dose of EcN is considered to be effective and safe when administered after TNBS-induced in rats. Our results indicate a marked clinical response to the oral administration of EcN with a dose of 2 9 107 CFU/day for one week both before and after the induction of colitis; same results were observed in TNBSinduced colitis treated with 2 9 109 CFU EcN intragastrically for a week, but not in TNBS-induced colitis treated with 2 9 109 CFU EcN intragastrically for one week both before and after the induction of colitis. Administration of EcN ameliorated experimental colitis by reducing disease activity index and modifying proinflammatory cytokine secretion, as well as restoring the impaired epithelial function. The intestinal epithelial barrier function is the result of a complex interaction of luminal factors (e.g., microflora), physical properties (e.g., mucus, epithelial cell layer, tight junctions, etc.), and the intestinal immune system. The intestinal barrier formed by the epithelial cells and the junctional complex, consisting of tight junctions (TJ), adherens junctions, gap junctions and desmosomes, excludes the majority of these microbes and their metabolites from access to the subepithelial cells. A breakdown of this tightly regulated homeostasis is seen in enteric infections but also in chronic diseases such as IBD [28]. It

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has been suggested that EcN also exhibits strengthening effects on the intestinal epithelial barrier. In our study, linear expression of ZO-1 is seen at the luminal surface in healthy intestinal in rats. However, in TNBS-induced colitis in rats, dot-like expression of ZO-1 is observed on the cell surface. From the results obtained, we have concluded that this probiotic E. coli ameliorated TNBS-induced colitis both in pre-administration of 2 9 107 CFU/day and administration of 2 9 109 CFU/day after TNBS-induced in rats. In consequence, the anti-inflammatory effects of this probiotic treatment were restricted to different courses and different doses. Thus, this study provides evidence that optimizes the use of this probiotic. Nevertheless, the transfer from bench to bedside is still incomplete. Novel and innovative clinical trials are lacking, despite the fact that EcN has been proven to be beneficial. Acknowledgments This study was supported by grants from the National Scientific Support Project (nos. 20100202, 02012BAI06B03 and BSW11J013), National Natural Science Foundation of China (Nos. 81322037 and 81370504) and National Excellent Doctoral Dissertation of PR China (No. 201182). Conflict of interest this paper.

There are no conflicts of interest to disclose in

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Preventive effects of Escherichia coli strain Nissle 1917 with different courses and different doses on intestinal inflammation in murine model of colitis.

To analyze the in vivo effect of Escherichia coli Nissle 1917 (EcN) with different courses and different doses to Sprague-Dawley rats with trinitroben...
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