Chitosan oligosaccharide as potential therapy of inflammatory bowel disease: Therapeutic efficacy and possible mechanisms of action

Pharmacological Research 66 (2012) 66–79

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Chitosan oligosaccharide as potential therapy of inflammatory bowel disease: Therapeutic efficacy and possible mechanisms of action Mohammad Yousef a , Rath Pichyangkura c , Sunhapas Soodvilai a,b , Varanuj Chatsudthipong a,b , Chatchai Muanprasat a,b,∗ a

Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand Research Center of Transport Protein for Medical Innovation, Faculty of Science, Mahidol University, Bangkok, Thailand c Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand b

a r t i c l e

i n f o

Article history: Received 2 December 2011 Received in revised form 8 March 2012 Accepted 19 March 2012 Keywords: Inflammatory bowel disease Intestinal epithelial cell Chitosan oligosaccharide NF-␬B Apoptosis

a b s t r a c t Inflammatory bowel disease (IBD) results from intestinal epithelial barrier defect and dysregulated mucosal immune response. This study aimed to evaluate the therapeutic potential of chitosan oligosaccharide (COS), a biodegradation product of dietary fiber chitosan, in the treatment of IBD and to elucidate its possible mechanisms of action. Oral administration of COS protected against mortality and intestinal inflammation in a mouse model of acute colitis induced by 5% dextran sulfate sodium (DSS). The most effective dose range of COS was 10–20 mg/kg/day. In addition, nuclear factor kappa B (NF-␬B) activation, and levels of tumor necrosis factor-␣ (TNF-␣) and interleukin-6 (IL-6) in colonic tissues were suppressed in mice receiving COS. Similar protective effect of COS against mortality and intestinal inflammation was observed in another mouse model of acute colitis induced by rectal instillation of 4% acetic acid. Importantly, COS administration after colitis induction was effective in ameliorating intestinal inflammation in both acute colitis models induced by 5% DSS and chronic colitis models induced by cycles of 2.5% DSS. In human colonic epithelial cells (T84 cells), COS treatment prevented NF-␬B activation, production of TNF-␣ and IL-6, and loss of epithelial barrier integrity under both lipopolysaccharide (LPS) and TNF-␣-stimulated conditions. Furthermore, binding of LPS to T84 cells, and TNF-␣ and oxidative stressinduced apoptosis of T84 cells were prevented by treatment with COS. These results suggest that COS may be effective in the treatment of IBD through inhibition of NF-␬B signaling and apoptosis of intestinal epithelial cells. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Inflammatory bowel disease (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC) is characterized by mucosal damage of the intestine due to chronic intestinal inflammation [1]. Several lines of evidence have indicated that chronic inflammation in IBD is underlined by a defect in barrier function of the intestinal epithelium as well as dysregulation of intestinal mucosal immunity [2,3]. Intestinal epithelial cells (IEC), being the principal component of the intestinal epithelial barrier, serve as the first sensor that

Abbreviations: COS, chitosan oligosaccharide; IEC, intestinal epithelial cell; TNF␣, tumor necrosis factor-␣; IL-6, interleukin-6; NF-␬B, nuclear factor kappa B; LPS, lipopolysaccharide; DAPI, 4 ,6 -diamidino-2 -phenylindoledihydrochloride; TLR4, Toll-like receptor 4; DSS, dextran sulfate sodium. ∗ Corresponding author at: Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Road, Rajathevi, Bangkok 10400, Thailand. Tel.: +66 2201 5615; fax: +66 2354 7154. E-mail address: [email protected] (C. Muanprasat). 1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2012.03.013

elicits mucosal immune defense against invading pathogens. Bacterial products such as lipopolysaccharides (LPS) are recognized by IEC via Toll-like receptor 4 (TLR4), leading to stimulation of NF-␬B-mediated production of proinflammatory cytokines and subsequent recruitment of immune cells. Indeed, NF-␬B activation in IEC and increased levels of IEC-derived proinflammatory cytokines, particularly TNF-␣ and IL-6, have been detected in intestinal biopsies of IBD patients [1]. The secreted proinflammatory cytokines (e.g. TNF-␣) and reactive oxygen species produced by recruited immune cells (e.g. neutrophils) subsequently cause apoptosis of IEC, which results in loss of intestinal epithelial barrier integrity and exaggerated mucosal inflammatory response in IBD [4]. It has been reported that small molecules or antibodies targeting NF-␬B and proinflammatory cytokines such as TNF-␣, IL-6 and their signaling pathways were highly effective in attenuating the inflammation-associated intestinal damages in IBD patients and a number of animal models of colitis [5,6]. Chitosan oligosaccharide (COS), an oligomer of d-glucosamine, is derived from decomposition of chitosan or deacetylation and degradation of chitin, which is the structural element in the

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exoskeleton of crustaceans. Due to its high solubility, non-toxicity and biocompatibility, COS has been extensively studied for its biological activities. In particular, COS exhibits anti-inflammatory activities both in vitro [7,8] and in vivo [9,10]. Interestingly, antiinflammatory effect of COS in vitro is positively correlated with its molecular weight and degree of deacetylation [11]. Nonetheless, protective effect of COS against intestinal inflammation in IBD has never been investigated. Herein, we evaluated the therapeutic efficacy of high molecular weight COS (MW 5000–10,000 Da, >90% degree of deacetylation) in two different mouse models of experimental colitis, dextran sulfate sodium (DSS) and acetic acidinduced colitis. Furthermore, possible mechanisms by which COS exerted its action were studied in human colonic epithelial cell line (T84 cells).

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was also evaluated in DSS acute and chronic colitis by administering COS for 7 and 5 days after colitis induction, respectively. Vehicle (100 ␮l water) was given to the colitis control group. 2.4. Assessment of severity of colitis

2. Materials and methods

Disease severity was evaluated daily using the disease activity index (DAI) as described previously [14]. Colonic damage was evaluated from both length and histological analysis of the colon. For colon length, mice were sacrificed at days 8 and 3 after colitis induction with DSS and acetic acid respectively, colon was excised, and the distance from the ileocecal junction to the anal verge was measured. For histological evaluation, H&E-stained sections from distal colon were microscopically analyzed in double-blinded fashion by three independent pathologists using the scoring system described previously [14].

2.1. Animal and cell line

2.5. Assay of myeloperoxidase (MPO) activity

ICR male mice (weight 30–35 g) were obtained from the National Laboratory Animal Center, Nakornpathom, Thailand. All animal protocols were approved by the Laboratory Animal Ethical Committee of the Faculty of Science, Mahidol University, Thailand. T84 cells (American Type Culture Collection, Virginia, USA) were grown in a mixture of 1:1 DMEM and Ham’s F-12 medium (Invitrogen Co., USA), supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin and maintained at 37 ◦ C in a humidified CO2 incubator. To form polarized monolayers, T84 cells were seeded in the Transwell insert (Corning Glass Works, Corning, NY, USA) at a density of 5 × 105 cells/insert and cultured for 14 days (transepithelial electrical resistance (TEER) > 1000 .cm2 ) with culture medium being replaced daily.

Neutrophil infiltration into inflamed colonic mucosa was quantified by measuring MPO activity as previously described [15]. The change in absorbance was measured at 460 nm using a fluorescence plate reader (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany).

2.2. Preparation of COS COS, with the molecular weight of 5000–10,000 Da at >90% degree of deacetylation, was prepared by enzymatic hydrolysis of shrimp shell chitosan by chitinase enzymes as described previously with some modifications [12]. One hundred grams of shrimp shell was deproteinized by soaking in 1 l of 1 N NaOH solution for 24 h, and then demineralized by soaking in 1 N HCl for 24 h (NaOH and HCl solutions were changed every 8 h). Pigments and other lipid soluble substances were removed by extraction with 95% ethanol at 75 ◦ C. The chitin product was deacetylated in 50% (w/w) NaOH solution until 85–90% degree of deacetylation was achieved. The chitosan product was then solubilized in 1% acetic acid solution and subjected to enzymatic hydrolysis. The COS was purified by precipitation with NaOH and washed extensively with distilled water until pH was neutral. Then, the COS was lyophilized and kept at room temperature. Size of COS was determined by gel permeation chromatography. Percent degree of deacetylation was determined by UV spectroscopic method. 2.3. Induction of colitis and treatment with COS In the DSS acute colitis model, mice were given 5% (w/v) DSS (mol wt. 36–50 kDa, MP Biomedicals, OH, USA) in drinking water for 8 days (for analysis of disease severity) or 14 days (for survival study). Acetic acid colitis model was established as described previously [13]. Chronic colitis in mice was induced by giving 2 cycles of 7-day 2.5% DSS in drinking water interrupted by 5 days of normal water. To evaluate the prophylactic effect of COS, the DSS acute colitis and acetic acid colitis mice were intragastrically administered with COS at various doses for 3 days before induction of colitis and thereafter until the end of the study. Therapeutic potential of COS

2.6. Measurement of cytokine levels Colonic samples were homogenized in 50 mM Tris–HCl containing 10 ␮g/ml protease inhibitor cocktail (Sigma–Aldrich Co., USA) on ice. Homogenates were centrifuged at 30,000 × g (4 ◦ C) for 20 min. For measurements of cytokine release in T84 cells, T84 cell monolayers were stimulated apically with serum-free medium containing LPS (10 ␮g/ml; List Biological Laboratories, CA, USA), and basolaterally with TNF-␣ (10 ng/ml; EMD Biosciences, CA, USA) with vehicle or COS (20, 100, and 500 ␮g/ml) added to the apical side. At 24 and 72 h after treatment, media in the apical side were harvested and centrifuged at 7500 × g (4 ◦ C) for 5 min. Supernatants after centrifugation of both types of samples were then assayed for TNF-␣ and IL-6 levels using a specific sandwich ELISA kit (ELISA Ready-SET-GO, eBioscience, CA, USA). Cytokine levels were normalized to the amount of protein in the colonic samples as measured by a modified Lowry method [16]. Transepithelial electrical resistance was measured in T84 monolayers with Epithelial Voltammeter (EVOM2 : World Precision Instruments, FL, USA). 2.7. NF-B (p65) DNA binding activity NF-␬B (p65) binding activity in nuclear extracts was measured using the NF-␬B (p65) transcription factor assay kit (Cayman Chemical Company, MI, USA). For in vitro experiments in T84 cells, cells (1 × 106 cells/ml) were seeded in petri dishes and grown overnight before 24-h treatment with TNF-␣ (10 ng/ml) and LPS (10 ␮g/ml) with or without COS (100 ␮g/ml). Colon samples and harvested T84 cells were homogenized, and nuclear extracts were prepared from the pellets by a nuclear extraction kit (Cayman Chemical, MI, USA). Assay for NF-␬B (p65) transcriptional activity was performed according to the manufacturer’s instructions. Results were normalized to sample total protein contents estimated by Lowry method. 2.8. Immunofluorescence staining of NF-B T84 cells (3 × 105 cells/ml) were seeded on coverslips. Cells were treated with vehicle or COS (100 ␮g/ml) in the presence or absence of LPS (10 ␮g/ml; 12 h) or TNF-␣ (10 ng/ml; 6 h), then fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X100.

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Body weight change (%)

105

Normal DSS DSS + COS (1 mg/kg) DSS + COS (5 mg/kg) DSS + COS (10 mg/kg) DSS + COS (20 mg/kg) DSS + COS (50 mg/kg) DSS + COS (100 mg/kg)

(B)

DSS COS

4.0

*** ***

100

**

95

* 90

Normal DSS DSS + COS (1 mg/kg) DSS + COS (5 mg/kg) DSS + COS (10 mg/kg) DSS + COS (20 mg/kg) DSS + COS (50 mg/kg) DSS + COS (100 mg/kg)

85

80 0

1

2

3

4

ns

3.5

Disease Activity Index

(A)

3.0 2.5

*

2.0

**

1.5 1.0

*** ***

0.5 0.0

5

6

7

8

9

10

1

11

2

Days

3

4

5

6

7

8

Days after induction of DSS colitis

(D)

(C) 100

100

80

80

% Survival

% Protection

ns

60

40

20

60

40 DSS DSS + COS (20 mg/kg) DSS + COS (100 mg/kg)

20 Log 10 scale

0 0.1

0

0.3

1

3.2

10

31.6

100

316.2

1000

Dose of COS (mg/kg)

0

2

4

6

8

10

12

14

Days after induction of DSS colitis

Fig. 1. Treatment with COS improved clinical symptoms of DSS-induced colitis. Mice were orally administered with COS at 1, 5, 10, 20, 50 mg/kg/day and 100 mg/kg/day for 3 days before and during the 8-day period of 5% dextran sulfate sodium (DSS) administration in drinking water. (A) Percent change in body weight of mice over time. (B) Disease activity index (DAI) of mice with indicated treatments after induction of DSS-induced colitis. Data were expressed as mean ± SE (n = 6). ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001 compared with DSS alone. (C) Dose–response curve of the effects of selected doses of COS on DAI at day 8 after induction of DSS-induced colitis. Results were expressed as mean ± SE (n = 6). (D) Effect of treatment with COS on survival rate of mice at 14 days after induction of DSS-induced colitis (n = 6).

For immunofluorescence staining of NF-␬B, cells were incubated overnight with rabbit-anti-NF-␬B p65 (Cell Signaling Technology, MA, USA) at 4 ◦ C. Samples were then washed with PBS, incubated for 1 h with a secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, CA, USA), followed by nuclear staining for 15 min with 4 ,6 -diamidino-2 -phenylindoledihydrochloride (DAPI; 1 ␮M) (Invitrogen, CA, USA). Slides then were mounted in 50% glycerol-PBS, covered and analyzed by fluorescence microscope Nikon TE 2000-S (Nikon, Tokyo, Japan). For each sample, 5 images were taken and analyzed with Image J 1.42 software. 2.9. Cell viability assay T84 cells were seeded in 96-well plates (Corning Glass Works, Corning, NY, USA) at a density of 6 × 104 cells/well and grown overnight at 37 ◦ C under an atmosphere of 5% CO2 and 95% O2 . Cells were then treated for 24 h with serum-free medium containing COS (20, 100, 500 ␮g/ml). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays as described previously [17]. 2.10. Analysis of cell apoptosis by flow cytometry T84 cells were seeded on 6-well plates and incubated for 2 h with H2 O2 (500 ␮M) in the presence or absence of COS (100 ␮g/ml). Cells were then washed with PBS and trypsinized. For detection of apoptosis and necrosis, cells (105 cells) were incubated in the dark for 15 min at room temperature with fluorescein isothiocyanate

(FITC)-annexin V (Sigma–Aldrich, MO, USA) and propidium iodide (PI: EMD Biosciences, CA, USA). Early apoptotic cells were annexin V positive and PI negative, whereas necrotic cells and late apoptotic cells were both annexin V and PI positive [18]. Cells were analyzed by a BD FACScantoTM flow cytometer (BD Bioscience, CA, USA). Data acquisition and analysis were performed using BD FACSDIva.6.1.1 software. 2.11. Measurement of cell apoptosis by DAPI staining T84 cells grown on coverslips were treated for 6 h with vehicle and TNF-␣ (10 ng/ml) in the presence or absence of COS (50 ␮g/ml). After incubation, cells were washed with phosphatebuffered saline (PBS) and fixed with 2% (wt/vol) paraformaldehyde in PBS (pH 7.4). The cells were then washed twice with PBS and stained with 0.2 ␮g/ml of DAPI for 15 min at room temperature in the dark. Then, cells were washed in PBS and mounted on a glass slide using 50% glycerol-PBS, covered and analyzed by fluorescence microscope Nikon TE 2000-S (Nikon, Tokyo, Japan). The number of apoptotic cells, which was characterized by condensed chromatin and fragmented nuclei, per 100 counted cells was used to estimate apoptosis. 2.12. Detection of LPS binding to T84 cell After seeding and growing T84 cells (1 × 106 cells/ml) in petri dishes for 48 h, cells were harvested and re-suspended in DMEM serum-free medium. For LPS binding assay, cells were incubated

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Fig. 2. Effect of treatment with COS on histopathological alterations and inflammatory infiltrates in DSS-induced colitis. (A) Effect of COS treatment on the colon length in colitis mice. (Left) Representative photographs and (Right) summary of changes in colon length at day 8. (B) Histological analysis of colon. Representative H&E-stained colon sections from mice treated as indicated at day 8 were shown (20×). Arrow indicates inflammatory infiltrates and epithelial degeneration. Scale bars = 50 ␮m. (C) Histological score analysis of colonic damage in colonic sections of mice treated as indicated. Score was rated by 3 independent pathologists in a blinded fashion. (D) Myeloperoxidase (MPO) activity of colonic tissues measured at day 8. Data were expressed as mean ± SE (n = 4–6). ns, Non-significant; ***P < 0.001 compared with non-colitis (normal) mice. ### P < 0.001 compared with DSS colitis control.

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Fig. 3. Oral administration of COS improved survival and attenuated intestinal inflammation in mice with acetic acid-induced colitis. Mice were pretreated with COS at 20 and 100 mg/kg/day for 3 days before colitis induction by rectal instillation of 4% acetic acid and thereafter for 7 more days. (A) Effect of COS treatment on the survival rate of mice with acetic acid-induced colitis. Data were expressed as % survival of mice (n = 6). (B) Histological analysis of colonic tissues. Representative H&E-stained colon sections from all groups at day 3 after induction of colitis were shown (20×). Arrow indicates inflammatory infiltrates and epithelial degeneration. Scale bars = 50 ␮m. (C) Histological score analysis of colonic tissues rated by 3 independent pathologists (n = 6). (D) MPO activity assays in colonic samples. MPO activity was measured at day 3 in colonic samples of mice with indicated treatments. Data were expressed as mean ± SE (n = 6). ns, Non-significant; **P < 0.01; ***P < 0.001 compared with normal non-colitis mice. # P < 0.05; ### P < 0.001 compared with vehicle-treated colitis mice.

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2.13. Detection of intracellular reactive oxygen species (ROS) T84 cells were plated in 96 well plates and treated with vehicle or COS (100 ␮g/ml) for 2 h. Then, cells were pulsed with 10 ␮M/2 ,7 -dichlorofluorescin diacetate (DCF-DA; Sigma–Aldrich, MO, USA), challenged with H2 O2 (500 ␮M), and analyzed as described previously [19].

DSS control

3.1. Amelioration of mucosal damage in DSS-induced acute colitis by oral administration of COS Prophylactic effect of COS in the treatment of IBD was first evaluated in the DSS-induced acute colitis model, in which mice were given 5% DSS in drinking water. COS was given 3 days prior to colitis induction and thereafter for 8 days. As depicted in Fig. 1A and B, at the end of the experiment, oral administration of all doses of COS except 100 mg/kg/day significantly reduced the clinical signs of colitis compared with control, based on the body weight loss and DAI. Grading of the magnitude of protection by COS was 10 mg/kg/day = 20 mg/kg/day > 5 mg/kg/day > 50 mg/kg/day > 1 mg/kg/day > 100 mg/kg/day. Of note, the protective effect of COS against DSS colitis exhibited a bell shaped dose–response relationship with maximal effective doses being ∼10–20 mg/kg/day and ED50 of ∼3 mg/kg/day (Fig. 1C). According to the above dose–response relationship, subsequent survival studies and intestinal inflammation analyses were performed using two doses of COS, low dose (20 mg/kg/day) and high dose (100 mg/kg/day). The former dose was selected to delineate the maximal efficacy of COS against DSS colitis, while the latter dose was chosen to confirm that high dose of COS was less effective. To evaluate the effect of COS on survival rate of mice with DSS colitis, the duration of exposure to DSS was extended to 14 days with the same COS pretreatment protocol as in the previous experiment. We found that the survival rate of the colitis control mice was 0% at day 14 of colitis induction (Fig. 1D), whereas pretreatment with COS at 20 and 100 mg/kg/day increased the survival rate to 65% and 18%, respectively, at day 14. Next, the effect of COS on DSS-induced acute intestinal inflammation was examined based on colon length, histological score analysis, and measurement of MPO activity. The colon length, a macroscopic indicator of mucosal injury, exhibited a remarkable shortening in DSS control mice compared with normal mice (Fig. 2A). Notably, COS treatment at 20 mg/kg/day significantly preserved the colon length in colitis mice compared with DSS control mice. However, colon length of colitis mice treated with 100 mg/kg/day COS was not statistically different from that of DSS

3.5 3.0 2.5 2.0 1.5

***

1.0 0.5 0.0 0

1

2

3

4

5

6

Tissue extract (μg/well)

(B)

Normal

DSS + COS (20 mg/kg)

DSS

DSS + COS (100 mg/kg)

800

Concentration (pg/mg protein)

3. Results

DSS + COS (20 mg/kg)

4.0

2.14. Statistical analysis All presented data are expressed as means ± standard error (S.E.). Statistical analysis for multiple comparisons in each study was determined by the analysis of variance (one- or two-way ANOVA) followed by the Bonferroni analysis using Prism 5.0 (GraphPad Software). For survival studies, Log-rank (Mantel-Cox) test was used as appropriate at each time point. For cytokines levels, data are expressed as mean, median, and upper and lower quartile values using Mann–Whitney U test. A P-value of <0.05 was considered statistically significant.

Normal

(A)

Absorbance (450 nm)

for 15 min at 37 ◦ C with FITC-conjugated LPS (LPS-FITC; 10 ␮g/ml; Escherichia coli 055:B5, Sigma–Aldrich, MO, USA) plus vehicle, COS (20 and 100 ␮g/ml) or purified LPS (10 ␮g/ml). Then, cells were centrifuged, washed twice in ice-cold PBS, and analyzed by a BD FACScantoTM flow cytometer (BD Bioscience, CA, USA).

71

#

ns

***

***

##

###

600

**

*

400 ns

ns

200

0 TNF-α

IL-6

Fig. 4. COS suppressed the mucosal inflammatory responses in DSS-induced colitis. (A) COS repressed NF-␬B activation in colitis mice. Mice were orally administered with COS at 20 mg/kg/day for 3 days before and during the 8-day period of 5% DSS administration in drinking water. Nuclear NF-␬B (p65) binding activity in mouse colonic tissues was determined by Cayman NF-␬B (p65) transcription factor assays. Results (absorbance at 450 nm) were normalized to sample protein. Data were expressed as mean ± SE (n = 5). ***P < 0.001 compared with DSS-induced colitis mice. (B) Effect of COS on inflammatory cytokine production in colonic tissues. Levels of TNF-␣ and IL-6 were measured by ELISA kits at day 8 of DSS treatment. Data were presented as mean (+), median (–), and upper and lower quartile values (n = 6). Data were analyzed by Mann–Whitney U test. ns, Non-significant; *P < 0.05; **P < 0.01; ***, P < 0.001 compared with non-colitis (normal) mice. # P < 0.05; ## P < 0.01; ### P < 0.001 compared with DSS colitis control.

control mice. Histological examination of the distal colon of DSS control mice showed transmural inflammation, mucosal ulceration (affecting >75% of the epithelial surface) and crypt hyperplasia accompanied by extensive goblet cell depletion. Evident edema between the mucosa and the muscular layer, and marked infiltration of inflammatory cells were also observed (Fig. 2B; arrows). In contrast, COS treatment at low dose (20 mg/kg/day) significantly ameliorated the histological alterations induced by DSS as shown in the representative images (Fig. 2B) and histological score analysis (Fig. 2C). Interestingly, COS at low dose produced more protective effect against mucosal injury than the high dose. DSS-induced intestinal inflammation was accompanied by mucosal infiltration of inflammatory cells, especially neutrophils [20]. We therefore measured the activity of myeloperoxidase, an enzyme abundantly present in neutrophil granulocytes, to quantify the neutrophil infiltrates in the colonic tissues of the mice. As presented in

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Fig. 5. COS promoted recovery after DSS-induced acute and chronic colitis. (A) Effect of COS on body weight changes in DSS-induced acute colitis. Mice were exposed to 5% DSS in drinking water for 7 days, after which DSS was removed and mice were orally administered with COS (20 mg/kg) for 7 days. Body weight of mice was recorded daily during the whole study period. Data were expressed as mean % body weight ± SE (n = 4–6). *P < 0.05 compared with colitis mice. Effect of COS on body weight changes (B), colon length (C), colon histology (D) and histological score (E) in mice with DSS-induced chronic colitis. Chronic colitis in mice was induced by 2 cycles of 2.5% DSS in drinking waster interrupted with 5-day normal drinking water (with no DSS). After the end of the second cycles, mice were orally administered with COS (20 mg/kg/day) for 5 days. Colon length measurement and histological analysis were done at the end of study (after 5 days of COS treatment). Representative H&E stained colon sections were shown (20×). Arrow indicates fibrosis. Histological score was rated by 3 independent pathologists in a blinded fashion. Data were expressed as mean ± SE (n = 4–6). ns, Non-significant; *P < 0.05, **P < 0.01 and ***P < 0.001 compared with non-colitis (normal) mice; # P < 0.05 and ## P < 0.01 compared with DSS colitis control.

Fig. 2D, colonic tissues obtained from DSS control mice exhibited a 4-fold increase in MPO activity compared with normal mice. Interestingly, only treatment with COS at 20 mg/kg/day significantly diminished MPO activity in colitis mice compared with DSS control. Together, these findings provided compelling evidence of the anti-inflammatory effects of COS. 3.2. Abolishment of acetic acid-induced colitis in mice by oral administration of COS To confirm that the anti-inflammatory efficacy of COS was not model specific, effects of oral administration of COS on mice survival rate and intestinal inflammation were evaluated in mouse colitis model induced by intrarectal instillation of 4% acetic acid. As shown in Fig. 3A, at day 7, after a challenge with acetic acid, 100% of mice treated with vehicle and 100 mg/kg/day COS died, whereas pretreatment with COS at 20 mg/kg/day rescued 50% of the mice. Evidence of reduced injury was provided by microscopic analysis of colonic sections at day 3 after colitis induction, which showed that COS at 20 mg/kg/day markedly preserved the epithelial architecture in colitis mice (Fig. 3B and C). Likewise, MPO activity in colonic tissues of colitis mice treated with both doses of COS was significantly reduced compared with colitis control mice (Fig. 3D). Collectively, our results indicated that low dose COS (20 mg/kg/day) provides better therapeutic outcome than the high dose COS (100 mg/kg/day), a conclusion consistent with the results from the DSS-induced acute colitis model. 3.3. COS attenuated the mucosal inflammatory response in mice with DSS-induced colitis To determine whether suppression of NF-␬B activation pathways contributed to the protective effect of COS against colitis, NF-␬B activation in mouse colon was determined by measurements

of nuclear NF-␬B (p65) DNA binding activity. As seen in Fig. 4A, nuclear NF-␬B (p65) DNA binding activity in colonic tissues from DSS colitis mice receiving COS at 20 mg/kg/day was significantly lower than that from DSS control mice, indicating suppression of NF-␬B activation by COS treatment. Furthermore, the markedly elevated levels of NF-␬B-regulated proinflammatory cytokines, TNF-␣ and IL-6, in colonic tissues from DSS mice were almost completely restored to the normal level by low dose COS treatment (Fig. 4B). Of note, treatment with COS at high dose significantly suppressed the level of IL-6 but not TNF-␣. These findings indicated that COS exerted preventive action against the inflammatory responses in intestinal mucosa via inhibition of NF-␬B activation. 3.4. Therapeutic potential of COS in the treatment of IBD In addition to protective action of COS, potential application of COS in the treatment of IBD was evaluated using post-treatment protocol, in which COS was administered after induction of either acute or chronic intestinal inflammation. As shown in Fig. 5A, body weight of mice receiving COS at 20 mg/kg/day for 7 days after induction of acute colitis by 5% DSS was significantly higher than those of DSS control, indicating that COS post-treatment enhanced recovery of body weight in acute colitis models. In order to gain better insight into potential therapeutic utility of COS in the treatment of IBD, effect of COS post-treatment on chronic intestinal inflammation induced by 2 cycles of 2.5% DSS was investigated. As depicted in Fig. 5B, body weight of mice receiving COS at 20 mg/kg/day was significantly increased compared with that of DSS control mice. Colon shortening, an index of intestinal mucosal damage, in DSS colitis mice treated with COS at 20 mg/kg/day was reduced compared with DSS colitis control (Fig. 5C). Histological examination of colon showed reduced crypt depth, submucosal edema, inflammatory cell infiltrates and fibrosis (arrow) in DSS control mice, all of

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Fig. 6. COS inhibited LPS-stimulated inflammatory responses in T84 cells. (A) Effect of COS on LPS-induced NF-␬B nuclear translocation in T84 cells. T84 cells treated for 12 h with vehicle, purified LPS (10 ␮g/ml), and LPS plus COS (100 ␮g/ml) before co-localization study of NF-␬B activation. Representative pictures were presented. Images were adjusted and merged using Imag J software (n = 3–5). Scale bars = 100 ␮m. (B) Effect of COS on LPS-induced production of inflammatory cytokines in T84 cells. Levels of TNF-␣ and IL-6 measured by ELISA in T84 cell monolayers after 1–3 days of stimulation with LPS with or without concomitant treatment with COS at indicated doses. Data were presented as mean (+), median (−), and upper and lower quartile values (n = 4). Mann–Whitney U test was used for statistical analysis of the results. ns, Nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001 compared with LPS-treated control. (C) Effect of COS on T84 epithelial integrity. Transepithelial electrical resistance (TEER) of T84 cell monolayers was measured by epithelial tissue voltammeter (EVOM) at day 0 to day 3 of treatment with vehicle, LPS, and LPS plus COS at indicated concentrations. *P < 0.05; ***P < 0.001 compared with LPS-treated control. (D) Effect of COS on LPS-induced NF-␬B activation in T84 cells. Cells were seeded on petri dishes and incubated with vehicle or LPS (10 ␮g/ml) in presence or absence of COS (100 ␮g/ml). DNA binding activity of nuclear NF-␬B (p65) was detected using nuclear extraction and NF-␬B (p65) transcription factor assay kits. Results were normalized to sample protein. Data were expressed as mean ± SE (n = 4). ***P < 0.001 compared with LPS-treated control.

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Fig. 7. COS suppressed TNF-␣-induced inflammatory responses and preserved the epithelial barrier integrity in T84 cells. (A) Inhibition of NF-␬B nuclear translocation by COS in T84 cells. T84 cells were treated for 6 h with vehicle, TNF-␣ (10 ng/ml), and TNF-␣ (10 ng/ml) plus COS (100 ␮g/ml) before co-localization analysis of NF-␬B activation. Representative pictures were shown. Photos were adjusted and merged using ImagJ software (n = 5). Scale bars = 100 ␮m. (B) Effect of COS on TNF-␣-stimulated production of inflammatory cytokines in T84 cells. Levels of TNF-␣ and IL-6 were measured by ELISA kits in T84 cell monolayers after 1–3 days of stimulation with TNF-␣ with simultaneous treatment with vehicle or COS at indicated concentrations. Data were presented as mean (+), median (−), and upper and lower quartile values (n = 4). Data were analyzed by Mann-Whitney U test. ns, Non-significant; *P < 0.05; **P < 0.01; ***P < 0.001 compared with TNF-␣-treated control. (C) Effect of COS on TNF-␣-induced loss of intestinal barrier integrity. Transepithelial electrical resistance (TEER) of T84 cell monolayers was measured by epithelial tissue voltammeter (EVOM) at day 0 to day 3 of treatment with vehicle, TNF-␣, and TNF-␣ plus COS at indicated concentrations. ns, Non-significant; **P < 0.01; ***P < 0.001 compared with TNF-␣-treated control. (D) Effect of COS on TNF-␣-induced NF-␬B activation in T84 cells. Cells were seeded on petri dishes and incubated with vehicle or TNF-␣ (10 ng/ml) in the presence or absence of COS (100 ␮g/ml). DNA binding activity of nuclear NF-␬B (p65) was detected using nuclear extraction and NF-␬B (p65) transcription factor assay kits. Results were normalized to sample protein. Results were presented as mean ± SE (n = 4). *P < 0.05 compared with TNF-␣-treated control.

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Fig. 8. COS inhibited binding of LPS to T84 cells. T84 cells were incubated with LPS-FITC plus COS (20 and 100 ␮g/ml) or purified LPS (5 ␮g/ml) for 15 min at 37 ◦ C. Binding of LPS-FITC to T84 cells was detected by flow cytometry (n = 3). Data were analyzed by BD FACSDiva 6.1.1 software.

which was alleviated by post-treatment with COS (20 mg/kg/day) for 5 days (Fig. 5D). Agreeably, histological score analysis showed that COS was effective in alleviating intestinal inflammation in this model (Fig. 5E). 3.5. COS protected against LPS- and TNF-˛-induced inflammatory responses and mucosal barrier disruption in IEC In IBD, activation of NF-␬B signaling in IEC has been regarded as an early pathogenic event leading to mucosal immune activation and destructive inflammation in the intestine [21]. We therefore hypothesized that COS exerted anti-inflammatory action in vivo by acting at IEC. The direct anti-inflammatory effect of COS was next investigated in human colonic epithelial cell line (T84 cells). Bacterial lipopolysaccharide (LPS) and the inflammatory cytokine TNF-␣ are critically implicated in the pathogenesis and perpetuation of IBD [22]. Both stressors involve activation of NF-␬B, through TLR4 and TNF-␣ receptors respectively, resulting in stimulation of the inflammatory responses by IEC, mucosal immune activation, and subsequent epithelial barrier breakdown [23,24]. Therefore, the effect of COS on LPS- and TNF-␣induced NF-␬B activation and its consequences, increased cytokine production and epithelial barrier disruption, were investigated. Immunofluorescence staining of active NF-␬B p65 revealed that LPS and TNF-␣ induced nuclear translocation of NF-␬B p65 in T84 cells (Figs. 6A and 7A; arrows). Simultaneous treatment with COS (100 ␮g/ml) suppressed both LPS- and TNF-␣-induced NF-␬B nuclear translocation (Figs. 6A and 7A). In addition, LPS- and TNF␣-stimulated induction of TNF-␣ and IL-6 in T84 cell monolayers were sharply reduced by the same COS concentration at day 1 and day 3 (Figs. 6B and 7B). Surprisingly, the bell shaped dose–response relationship was also observed with this effect of COS. We further determined, electrophysiologically, the effect of COS on LPS- and TNF-␣-induced epithelial barrier disruption in T84 cell

monolayers by measuring the transepithelial electrical resistance (TEER) as an index of epithelial barrier integrity. We observed that, concomitant incubation with COS at all concentrations (20, 100 and 500 ␮g/ml) significantly restored the LPS- and TNF-␣-induced reduction in TEER to normal level at day 3 of incubation, with the greatest effect being observed at 100 ␮g/ml of COS (Figs. 6C and 7C). In addition, effect of COS on LPS- and TNF-␣-induced NF-␬B activation in T84 cells was measured using assays of DNA binding activity of nuclear NF-␬B (p65). As shown in Figs. 6D and 7D, it was found that COS treatment (100 ␮g/ml) significantly reduced both LPSand TNF-␣-induced DNA binding activity of nuclear NF-␬B (p65), indicating that COS acted at NF-␬B activation pathways in IEC. 3.6. COS inhibited LPS binding to IEC Due to the large molecular size and polycationicity of COS, we speculated that COS may interact with receptors on IEC surface to inhibit NF-␬B-mediated signaling pathways, leading to amelioration of intestinal inflammation in vivo. To test this hypothesis, we performed flow cytometric analysis of T84 cell staining with FITCtagged LPS (LPS-FITC). As shown in Fig. 8, co-incubation of T84 cells with COS at 20 and 100 ␮g/ml prevented LPS-FITC binding to T84 cells in a concentration-dependent manner. As a control experiment, purified LPS was co-administered with LPS-FITC, and it was found that LPS-FITC staining of T84 cells was markedly diminished in the presence of purified LPS. These findings suggested that COS may interact with LPS receptors on the surface of IEC and prevent LPS-induced NF-␬B activation. 3.7. COS suppressed TNF-˛- and H2 O2 -induced apoptosis in T84 cells TNF-␣- and oxidative stress-induced apoptosis of IEC have been reported to contribute to the barrier dysfunction of colonic

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Fig. 9. Treatment with COS protected against TNF-␣-induced apoptosis in T84 cells. (A) Effect of COS on T84 viability determined by MTT assays. T84 cell were incubated for 24 h with vehicle or COS at indicated concentrations. Data were expressed as mean ± SE (n = 3). ns, Non-significant compared with vehicle-treated control. (B) Inhibition by COS of TNF-␣-induced apoptosis. T84 cells were treated for 6 h with vehicle, TNF-␣ (50 ng/ml) plus vehicle or COS (100 ␮g/ml), and incubated for 15 min with DAPI. Representative images were shown (n = 5). Scale bars = 25 ␮m. Arrow indicated apoptotic cells (C) Effect of COS on apoptosis index. Percent apoptotic T84 cells were obtained by counting at least 400 cells/sample. Data were expressed as mean ± SE (n = 3–5). ns, Non-significant; *P < 0.05; ***P < 0.001 compared with vehicle-treated control. ### P < 0.001 compared with TNF-␣-treated control.

epithelium in IBD [24–26]. Therefore, we investigated the effect of COS on TNF-␣- and oxidative stress-induced apoptosis in T84. Prior to the experiment, we examined the toxicity of COS on T84 cells by MTT assay. We found that, COS at concentrations ranging from 20 to 500 ␮g/ml had no effect on T84 cell viability (Fig. 9A). Then, the effect of COS on TNF-␣-induced apoptosis in T84 cells was examined by the nuclear staining with DAPI. The major criteria for identifying apoptotic cells by DAPI staining are signs of chromatin condensation and DNA fragmentation, indicators of early steps in apoptosis, presented as reduced size of nuclei and increased fluorescent intensity compared with control. As shown in Fig. 9B, COS (100 ␮g/ml) markedly diminished the TNF-␣-induced T84 cell apoptosis from 52.67 ± 3.2% (control) to 21.3 ± 2.1%.

The effect of COS on oxidative stress-induced T84 cell apoptosis was studied using flow cytometry analysis of FITC-Annexin V and propidium iodide (PI) staining. Cells stained only with FITC-Annexin V were considered early apoptotic stage and cells stained with both Annexin V and PI were considered necrotic or late apoptotic stage. Cells treated with H2 O2 (500 ␮M) showed a dramatic increase in the proportion of both Annexin V-positive and Annexin V/PI positive cells after 2-h incubation as compared with vehicle treated cells (26% vs 16.3%; P < 0.01 and 2.7% vs 0.9%; P < 0.001 respectively: Fig. 10). Interestingly, co-incubation with COS (100 ␮g/ml) caused a marked decline in the number of Annexin V-positive (15.8% vs 26%; P < 0.01) and Annexin V/PI positive cells compared to H2 O2 treated cells (1.2% vs 2.7%; P < 0.01: respectively; Fig. 10). In addition, the effect of COS on

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Fig. 10. COS inhibited oxidative stress-induced apoptosis in T84 cells. Non-confluent monolayers of T84 cells were incubated for 2 h with vehicle, H2 O2 (500 ␮M), and H2 O2 plus COS (100 ␮g/ml). Cells were stained with FITC-annexin V alone (abscissa), to detect early apoptosis, or in combination with PI (ordinate) to detect late apoptosis and necrosis. Representative density blots and histograms of flow cytometric analysis of apoptosis in T84 cells were shown. Number in the lower right quadrant was percentage of apoptotic cells stained with FITC-annexin V, whereas, percent of late apoptotic and necrotic cells was represented by numbers in the upper right quadrant (n = 3). Data were analyzed by BD FACSDiva 6.1.1 software.

H2 O2 -induced production of ROS in T84 cells was investigated using dichlorofluorescein (DCF) assays. As illustrated in Fig. 11, COS (100 ␮g/ml) abolished the increased ROS production by H2 O2 at all study time points, suggesting an anti-oxidation activity of COS in IEC.

4. Discussion In the present study, we demonstrated for the first time that oral administration of COS prevented inflammation-associated intestinal damage and mortality in two experimental models of acute

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DCF fluorescence intensity

140

Vehicle H2 O2 H2 O2 + COS

***

120 100

** ##

80

#

60 40 20 0

30 min

90 min

3h

Fig. 11. COS attenuated H2 O2 -induced production of ROS in T84 cells. Cells were treated with vehicle or COS (100 ␮g/ml) and incubated for 2 h in a CO2 incubator at 37 ◦ C. Subsequently, cells were incubated for 1 h with DCF-DA dye (10 ␮M) before being challenged with H2 O2 (500 ␮M). DCF fluorescent intensity (in arbitrary unit) was detected at indicated time points after H2 O2 challenges by a microplate reader. Data were expressed as mean ± SE (n = 3). **P < 0.01; ***P < 0.001 compared with vehicle-treated control. # P < 0.05; ## P < 0.01 compared with H2 O2 -treated groups.

colitis. In addition, COS administered after colitis induction was effective in reducing intestinal inflammation in models of both acute and chronic intestinal inflammation. Importantly, we found that COS suppressed NF-␬B activation, attenuated proinflammatory cytokine production, inhibited LPS binding, and inhibited TNF-␣ and oxidative stress-induced apoptosis in IEC. These effects may account for the therapeutic efficacy of COS observed in vivo. In our study, colitis was induced by challenging the mice with DSS in drinking water or rectal instillation of acetic acid. These wellestablished models are close in their pathogenic mechanisms to IBD in human, i.e. epithelial barrier disruption inducing exaggerated mucosal inflammatory response. By disrupting intestinal epithelial barrier (which allows exposure of immune cells to luminal bacteria), DSS ingestion can provoke intestinal inflammation as well as clinical and pathological manifestations of colitis [27,28]. Rectal instillation of acetic acid induces extensive erosion of colonic epithelium, which brings about acute intestinal injury and subsequent inflammation [29]. Of particular relevance to the application in the treatment of IBD, COS given after induction of colitis was effective in alleviating intestinal inflammation and body weight loss in mouse models of acute and chronic intestinal inflammation. This finding together with the preventive effect of COS in the two experimental models of acute colitis supports future prospects for therapeutic utility of COS in treatment of IBD. Since COS is an oligomer of d-glucosamine, which is resistant to degradation by digestive enzymes, we therefore speculated that ingested COS could reach colon and provides protection against colitis by acting directly on the IEC. It has been shown that IEC expresses Toll-like receptors (e.g. TLR4) and many other cytokine and chemokine receptors [30]. LPS and TNF-␣, the major stimulators of aberrant inflammation in IBD, interact with their respective receptors (TLR4 and TNFR 1 and 2) and elicit NF-␬Bmediated production of proinflammatory cytokines in IEC [31,32]. In IBD, increased expression of both TLR4 and TNFR in IEC has been linked to exaggerated IEC-mediated mucosal inflammatory responses and, consequently, intestinal epithelial barrier dysfunction [31,33,34]. We found that COS treatment markedly attenuated both LPS and TNF-␣-stimulated NF-␬B activation, as well as resultant TNF-␣ and IL-6 production in human colonic epithelial cell-like T84 cells. As IEC-derived TNF-␣ has recently been shown to suffice to cause Crohn-like pathology in mice [33], it is therefore suggested that the inhibitory effect of COS on NF-␬B activation and

proinflammatory cytokine production in IEC could account for the anti-inflammatory effect of COS observed in mouse colitis models. Importantly, our results indicated that COS may suppress NF-␬B activation in IEC at the very early step by inferring with binding of bacterial products (i.e. LPS) to its membrane receptors (e.g. TLR4), leading to alleviation of mucosal immune activation in mouse models of colitis. In addition, we demonstrated that treatment with COS prevented the LPS- and TNF-␣-induced decrease in transepithelial electrical resistance and inhibited TNF-␣ and H2 O2 -induced apoptosis in T84 cells. Furthermore, the anti-oxidative activity of COS against H2 O2 -induced ROS production was also demonstrated by DCF assays. These findings suggest that oral administration of COS could preserve the intestinal epithelial barrier integrity through anti-oxidative and anti-apoptotic mechanisms, thus conferring protection against initiation and progression of colitis in vivo. Fructo-oligosaccharide, an oligomer of fructose, (at dose of 10 g/day) has been demonstrated to alleviate DSS-induced colitis in mice via prebiotic mechanism (i.e. stimulating growth/activity of health-promoting intestinal bacteria) [35]. COS has also been found to exhibit prebiotic properties, although being much less effective than fructo-oligosaccharide [36]. The dose of COS reported to exhibit prebiotic properties is 1 g/kg/day. In the present study, COS prevented colitis at a much lower dose range (1–100 mg/kg/day), thus was not expected to produce a significant prebiotic effect in this study. Another point of interest was the bell-shaped pattern of doseresponse relationship of COS seen both in vivo and in vitro. This biphasic effect of COS could be attributed to the capacity of COS to stimulate two different signaling pathways resulting in opposing effects, so called functional antagonism. The sum of the two doseresponse curves therefore determined the net effect of COS and yielded the bell-shaped dose-response curve. This functional antagonism model has been used to describe biphasic effects elicited by some receptor–ligand interactions including effect of morphine on heart rate [37] and effect of prostaglandin on adenylate cyclase activity [38]. 5. Conclusions COS is effective in preventing experimental colitis in vivo. Our results indicate that COS protect against colitis by inhibiting NF␬B activation and preventing TNF-␣ and oxidative stress-induced apoptosis of IEC. Further development of this natural carbohydrate oligomer may provide a new effective therapeutic modality for IBD. Acknowledgements We thank Professor Nateetip Krishnamra for critical reading of the manuscript. This work was supported by the Thailand Research Fund (TRF), the Office of the Higher Education Commission, Mahidol University (through grant MRG5380125 to CM), Mahidol University research grant (to CM), Faculty of Science Mahidol University and the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative (to CM). References [1] Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu Rev Immunol 2010;28:573–621. [2] Sartor RB. Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol 2006;3:390–407. [3] Strober W, Fuss I, Mannon P. The fundamental basis of inflammatory bowel disease. J Clin Invest 2007;117:514–21. [4] Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 2011;474:298–306. [5] Atreya R, Mudter J, Finotto S, Mullberg J, Jostock T, Wirtz S, et al. Blockade of interleukin 6 trans signaling suppresses t-cell resistance against apoptosis in

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