The mechanisms responsible for human inflammatory bowel disease remain poorly understood. The pathogenic factors for dextran sulfate sodium (DSS)-induced colitis, one of the experimental animal colitis models, also remain unknown. Furthermore, detailed studies on DSS metabolism in the gut lumen have not been reported. Therefore, we investigated DSS metabolism in the mouse gut lumen and report the mechanisms which induce colitis. DSS was labeled with 2-aminopyridine (pyridylamino-DSS, PA-DSS). PA-DSS was administered orally to male BALB/cA Jcl mice. The metabolites and histological findings were observed using HPLC and light or fluorescence microscopy. PA-DSS with Mr 5000 was depolymerized rapidly in the gastric lumen, and the depolymerized PA-DSS was absorbed in the small intestine. Therefore, the majority of the PA-DSS in the cecal contents returned to Mr 5000 PA-DSS, escaping absorption in the small intestine. Mr 5000 DSS induced severe colitis, and immunostaining using an anti-mouse Ki-67 antibody and the TUNEL assay showed that DSS arrested the cell cycle at the G0 phase and induced apoptosis of the colonic epithelium. Mr 2500 PA-DSS, however, induced these same effects weakly. During these processes, we observed that the epithelial cells can depolymerize DSS themselves. An
In human inflammatory bowel disease (IBD), the subtypes of ulcerative colitis (UC) and Crohn’s disease are chronic, relapsing, and remitting conditions that characterized by diarrhea, bloody stools, abdominal pain and weight loss. Currently, UC is believed to be caused by multiple factors, including genetic and environmental factors. Histologically, UC is characterized by crypt abscesses, crypt distortion and loss, ulceration, and by the infiltration of large numbers of neutrophils, monocytes and lymphocytes. IBD affects at least 1 in 1,000 people in Western countries (
Experimental animal models of colitis have been developed in order to investigate the underlying physiologic mechanisms and to improve medical therapies for IBD. In the most commonly-used models, colitis is induced by administering sulfated polysaccharides such as dextran sulfate sodium (DSS) or carrageenan (
Mr 5000 and 500k DSS and 2-aminopyridine were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Mr 8000, 10000 DSS and D-glucose 3-sulfate were obtained from Sigma Chemical Co. (St. Louis, MO). Mr 2500 DSS was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
The pyridylamination of the reducing termini of sugar chains has been useful for the structural analysis and metabolic studies on
Since PA-DSS is strongly negatively charged in aqueous solution, a strong interaction between PA-DSS and the stationary phase is present. Therefore, 0.2 M phosphate buffer at pH 3.0 was used as the mobile phase according to our previous report (
Specific pathogen-free male BALB/cA Jcl mice, 6-week old, were purchased from Nippon Clea Inc. (Tokyo, Japan). They were housed in a room with controlled temperature (20–22°C), humidity (50–60%) and a preset light-dark cycle (12 h:12 h). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of the Shiga University of Medical Science (Permit no: 2006-7-6). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
At the beginning of the experiment, the mice were fed the standard diet (MF, Oriental Yeast Co,. Ltd., Tokyo, Japan) containing 5% (w/w of diet) Mr 5000 or 2500 PA-DSS for 8 days (n=5). During the experimental period, body weight was measured every other day. On the final day, blood samples were collected by cardiac puncture. The contents of the gut lumen were removed, diluted adequately by PBS, centrifuged to remove any insoluble material (14,000 rpm, for 20 min), and then the supernatants were used for the HPLC analysis. Each organ was resected, irrigated with chilled PBS, placed in 0.5 ml of a hexadecyltrimethylammonium bromide solution (0.5%, w/w), homogenized, sonicated and subjected to three rapid cycles of freezing and thawing. The samples were then centrifuged and the supernatants were used for HPLC analysis. The blood samples were centrifuged, and the supernatants were used for HPLC analysis. On the other hand, a specimen at 2 cm distance from the anal margin (middle colon) was removed, frozen and then cut into 5 μm sections. The sections were observed under a fluorescence microscope, or stained with hematoxylin and eosin (H&E) to observe under light microscopy. The mucosal damage was determined according to a previously described method (
Caco-2 (a human colon cancer cell line) cells were purchased from the American Type Culture Collection (Rockville, USA). The cells were cultured in Dulbecco’s modified Eagle’s minimum essential medium (DMEM, pH 7.4) supplemented with 25 mM glucose, 10% inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin and 1% non-essential amino acid solution at 37°C in a humidified 5% CO2 atmosphere.
We reported that the Mr 5000 DSS was depolymerized to Mr 1800, and ~70% of the sulfate groups were depleted from each DSS molecule following autoclave treatment (at 115°C for 15 min, 1.7 atm) (
When confluent and allowed to mature on permeable inserts, the Caco-2 monolayer formed tight junctions and attained many of the morphological and functional characteristics of the intestinal epithelium with normal barrier function (
We observed each sample using immunohistochemical staining. Anti-mouse or anti-human Ki-67 antibodies (Dako Cytomation, Denmark) and the TdT-FragEL™ DNA Fragmentation Detection kit (Calbiochem, USA) were used to analyze the cell cycle and apoptosis, respectively according to the manuals, provided by the manufacturers.
The results are presented as means ± SEM. The variance was analyzed by the F test. Subsequently, the Student’s t-test for unpaired values was performed to compare the means of the normally distributed data. The Mann-Whitney U test was also performed to compare the means of non-parametric or abnormally distributed data. Differences were regarded as statistically significant if the P-values were <0.05.
First, we investigated the metabolism of DSS in the gut lumen using 2-aminopyridine labeling and HPLC systems. No PA-DSS was recognized in the esophageal mucosa. Surprisingly, Mr 5000 PA-DSS was depolymerized in the gastric contents (
Next, we observed DSS-induced colitis both macroscopically and microscopically. After the Mr 5000 PA-DSS administration, diarrhea occurred on Days 3–4. Macroscopic examination of the colon revealed hyperemia, erosions and occasional tiny blood clots in the distal colon on Day 5. Using H&E staining, there was obvious evidence of inflammatory cell infiltration into the mucosa and submucosa. Crypt shortening, entire crypt loss, and earthenware mortar-like deformity of the crypts, surface epithelial loss and mucosal edema were also evident (
We next investigated whether this difference in the molecular mass distribution of PA-DSS in the lumen explain the fact that Mr 5000 PA-DSS mainly induces inflammation in the colon, but not in the stomach or small intestine. Specifically, we examined whether Mr 2500 PA-DSS could induce colitis or not. After the Mr 2500 PA-DSS administration, the appearance of diarrhea was delayed until Days 6–7. The body weight loss was also less than that in mice fed Mr 5000 PA-DSS (
Subsequently, we examined how DSS contributes to the induction of colitis. We previously found that DSS strongly and rapidly inhibited the intracellular energy metabolism in Caco-2 cells (
In the control mice, there was sporadic mitosis present in the crypts using H&E staining (
On the other hand, we observed the cell cycle status of the colonic epithelium.
It has become clear that DSS arrested the cell cycle of the colonic epithelium. Therefore, we investigated whether DSS induces apoptosis of the colonic epithelium or not as the dynamics of the epithelial cells.
There were a few apoptotic bodies present, especially at the top of the villi, in the control mice (
The molecular mass in the feces was predominantly Mr 5000, whereas that in the mucosa of the colon was below Mr 2000. Does this result mean that some of the DSS was depolymerized when it passed through the epithelial cells? In addition, can DSS also induce cell cycle arrest and apoptosis in culture cells? We investigated the metabolism of PA-DSS using Caco-2 cells in order to clarify these possibilities. In general, a mixture of DSS and toluidine blue exhibits a color change from blue to violet called metachromasia. This color change is derived from the shift of the UV absorbance from 620 to 560 nm and a minimum molecular mass of 2,500 Da was required for a metachromatic reaction according to our previous study (
The Mr 5000 PA-DSS was also depolymerized while it passed through the Caco-2 monolayer.
It has become clear that Caco-2 cells can also depolymerize PA-DSS. We thus investigated if DSS can also induce the cell cycle arrest and apoptosis in Caco-2 cells.
When investigating the pathogenesis of DSS-induced colitis, the metabolism of DSS is a problem that cannot be avoided. However, there has been no report that shows in detail the metabolites of DSS found in the biological materials, including the contents of the gut lumen. First, Mr 5000 PA-DSS was administer orally, and was rapidly depolymerized in the stomach. We previously reported that acidic but not alkaline conditions can depolymerize PA-DSS (
In addition, the intestinal epithelial cells are able to uptake and depolymerize PA-DSS. On the other hand, even Mr 500k DSS could enter Caco-2 cells, and the nucleus exhibited metachromasia (data not shown). DSS could also enter other types of cells, such as IEC-6 (a rat intestinal epithelial cell line) and Hep G2 (a human hepatoblastoma cell line) cells, and became depolymerized (data not shown). Since dextran (non-sulfated polysaccharide) cannot enter these cells nor the epithelium, these characteristics of DSS are quite remarkable. This DSS pathway, however, has not been clarified yet. In addition, it is still unclear how the DSS was depolymerized or what enzymes were involved.
There have been a few previous reports showing in part the metabolism of DSS in the biological materials. In those reports, DSS was detected mainly using a metachromatic reaction (
In the present study, the precise mechanisms responsible for the induction of cell cycle arrest and apoptosis remain unclear. Some previous reports, however, have suggested an association between DSS and the cell cycle arrest. For example, the cell cycle of B lymphocytes was arrested by DSS (
With respect to apoptosis, a few previous reports showed the involvement of apoptosis in DSS-induced colitis (
It is also very interesting that T and B cells are not required for acute DSS-induced colitis, since DSS also produces colitis in severe combined immunodeficient (SCID) mice (
In addition, carrageenan is produced from seaweed and is used as a gelling agent in food. Carrageenan is another type of sulfated polysaccharide consisting of anhydro-D-galactose (α-1,3-galactosidic link) and sulfate (over Mr 100k to 800k), and also induces colitis (
Finally, several types of bacteria, including
In conclusion, we proposed one plausible mechanism responsible for an early stage event in DSS-induced colitis. DSS is depolymerized rapidly in the mouse stomach. This depolymerized DSS cannot induce severe inflammation in the stomach or small intestine. However, the majority of the DSS in the colonic lumen returned to the Mr 5000 form. This Mr 5000 DSS induced even more severe colitis through cell cycle arrest and apoptosis than the depolymerized DSS.
Metabolism of Mr 5000 PA-DSS. (A) Structure of DSS. DSS is a sulfated polysaccharide consisting of anhydro-D-glucose (α-1,6-glucosidic link) and sulfate. (B) Chromatogram of PA-DSS in the gastric contents. The molecular masses were approximately Mr 2000 (peak a) and 1200 (peak c), respectively. (C) Chromatogram of PA-DSS in the contents from the small intestine. The molecular masses were approximately Mr 2000 (peak a), 1500 (peak b), and 1200 (peak c), respectively. (D) Chromatogram of PA-DSS in the cecal contents. A main peak was recognized at approximately Mr 5000. Smaller peaks of Mr 1200 (peak c) and 750 PA-DSS (peak d) were also recognized. (E and F) Quantification of PA-DSS in each organ and content, respectively. These data show the amount of Mr 5000 PA-DSS regardless of the degree of depolymerization. All values are expressed as means ± SEM.
Mr 5000 PA-DSS-induced colitis. (A-C) Microscopic findings from the H&E staining or (D and E) fluorescence microscopy of the colon in a Mr 5000 PA-DSS-induced colitis mouse. (F) Fluorescence microscopy of the colon in a control mouse. (A-C) Original magnification, ×100; (D-F), ×40.
Mr 2500 PA-DSS-induced colitis. (A) Body weight losses in the control, Mr 2500 and 5000 DSS-induced colitis mice. (B-D) Microscopic findings of the colon in a control, Mr 2500 and 5000 DSS-induced colitis mice. Original magnification, ×100 using H&E staining. (E) The mucosal damage was quantified by a scoring system (
Mitosis and cell cycle in PA-DSS-induced colitis. (A) Microscopic findings of the colon in a control mouse. There was sporadic mitosis in the crypts (arrow). (B) Microscopic findings of the colon in a Mr 5000 DSS-induced colitis mouse. There were far fewer mitoses. (A and B) Original magnification, ×200 using H&E staining. (C) To quantify this mitotic activity, the ratio of the crypt counts with mitosis to the total cell count in the crypt was calculated. All values are expressed as means ± SEM. *P<0.05. (D and E) Immunohistochemical staining of the colon in a control mouse. There were many anti-Ki-67 immunopositive cells in the lower part of the crypt. (F and G) Immunohistochemical staining in the colon of a Mr 5000 DSS-induced colitis mouse. There were far fewer anti-Ki-67 immunopositive cells in the lower part of the crypt. (D and F) Original magnification, ×100 using anti-Ki-67 immunostaining. (E and G) Original magnification, ×200 using anti-Ki-67 immunostaining. (H) To quantify these anti-Ki-67 immunopositive cells, the ratio of the crypt counts with the anti-Ki-67 immunopositive cell count to the total cell count in the crypt was calculated. All values are expressed as means ± SEM. *P<0.05.
Apoptosis in the crypts in Mr 5000 or 2500 PA-DSS-induced colitis mice. (A) Microscopic findings of the colon in a control mouse. There were a few apoptotic bodies, especially at the top of the villi (arrow). (B and C) Microscopic findings of the colon in a Mr 5000 DSS-induced colitis mouse. There were far more apoptotic bodies recognized (arrow). (D) Immunohistochemical staining of the colon in a control mouse. There were a few TdT positive cells in the epithelium (arrow). (E) Immunohistochemical staining in the colon of a Mr 5000 DSS-induced colitis mouse. There were far more TdT positive cells recognized (arrow). (A and B) Original magnification, ×100, and (C) ×400 using H&E staining. (D and E) Original magnification, ×100 using TdT staining. (F) To quantify these TdT positive cells, the ratio of the TdT positive cell count to the total cell count in the crypt was calculated. All values are expressed as means ± SEM. *P<0.05.
Metabolism of Mr 5000 PA-DSS in Caco-2 cells. (A) Toluidine blue staining of CaCo-2 cells incubated with 3% Mr 5000 DSS for 24 h. (B) Toluidine blue staining of Caco-2 cells for 3 h after replacing with medium alone. (C) Toluidine blue staining of Caco-2 cells for 24 h after replacing with medium alone. (D) Fluorescence microscopic image of control CaCo-2 cells. (E-G) Fluorescence microscopic images of CaCo-2 cells incubated with 3% Mr 5000 PA-DSS for 24 h. (A-C) Original magnification, ×100; (D, F and G), ×400; (E), ×100. (H) Chromatogram of PA-DSS permeated outside a porous filter after 5 h. The molecular masses were approximately Mr 1500 (peak b), 1200 (peak c) and 750 (peak d), respectively. The data show the amount of Mr 5000 PA-DSS regardless of the degree of depolymerization.
Cell cycle arrest and apoptosis in Caco-2 cells. Caco-2 cells were co-incubated with medium alone, 3% Mr 5000 or 2500 DSS for 5 days. (A) Immunohistochemical staining of the Caco-2 cells using an anti-mouse Ki-67 antibody. (B) Immunohistochemical staining of Caco-2 cells using the TUNEL assay (TdT). (C and D) To quantify the anti-Ki-67 immunopositive cells or TdT positive cells, the ratio of the anti-Ki-67 immunopositive cell count or TdT positive cell count to the total cell count was calculated. All values are expressed as means ± SEM. *P<0.05 vs. control and **P<0.05 vs. Mr 2500 or control.