Biochemical and histological changes in the small intestine of mice with dextran sulfate sodium colitis

The dextran sulfate sodium (DSS) model of colitis has been commonly utilized in mice to assess novel treatments for ulcerative colitis. Recent studies have indicated that morphological and biochemical changes extend to the small intestine (SI). This study aimed to characterize histological and biochemical changes in the SI during DSS colitis in wild‐type (WT) and DPIV knock‐out (DPIV^?/?) mice treated with saline or the DPIV inhibitors, Ile‐Pyrr‐(2‐CN)*TFA or Ile‐Thia. Groups (n = 10) of DPIV^?/? and WT mice were orally gavaged twice daily with saline, Ile‐Pyrr‐(2‐CN)*TFA or Ile‐Thia. Mice consumed 2% DSS in drinking water for 6 days to induce colitis. Small intestinal tissue was assessed for histological changes, sucrase, and DPIV activity and neutrophil infiltration. Jejunal villus length was increased in all groups after 6 days DSS consumption (P < 0.05). Jejunal DPIV activity was significantly lower by 35% in WT mice receiving Ile‐Pyrr‐(2‐CN)*TFA compared to saline controls. Jejunal MPO activity was significantly increased in the WT + saline and DPIV^?/? + saline groups following DSS consumption, compared to WT and DPIV^?/? controls at day 0. Increased sucrase activity was apparent at day 0 in DPIV^?/? compared to WT mice (P < 0.05). We conclude that DSS‐induced damage is not restricted to the colon, but also extends to the small intestine. Furthermore, reduced or absent DPIV activity resulted in functional adaptations to brush border enzyme activity. DPIV inhibitors are now a recognized therapy for type‐II diabetes. The work presented here highlights the need to delineate any long‐term effects of DPIV inhibitors on SI function, to further validate their safety and tolerability. J. Cell. Physiol. 226: 3219–3224, 2011. © 2011 Wiley Periodicals, Inc.     

Milk fat globule-EGF factor 8 is a critical protein for healing of dextran sodium sulfate-induced acute colitis in mice

Milk fat globule-EGF factor 8 (MFG-E8) has been shown to play an important role in maintaining the integrity of the intestinal mucosa and to accelerate healing of the mucosa in septic mice. Herein, we (a) analyzed the expression of MFG-E8 in the gut of wild-type (WT) C57BL/6 (MFG-E8(+/+)) mice with and without dextran sulfate sodium (DSS)-induced colitis, (b) characterized the pathological changes in intestinal mucosa of MFG-E8(+/+) and MFG-E8(-/-) mice with DSS-induced colitis and (c) examined the therapeutic role of MFG-E8 in inflammatory bowel disease by using DSS-induced colitis model. Our data documented that there was an increase in colonic and rectal MFG-E8 expression in MFG-E8(+/+) mice during the development of DSS colitis. MFG-E8 levels in both tissues decreased to below baseline during the recovery phase in mice with colitis. Changes in MFG-E8 gene expression correlated to the levels of inflammatory response and crypt-epithelial injury in both colonic and rectal mucosa in MFG-E8(+/+) mice. MFG-E8(-/-)mice developed more severe crypt-epithelial injury than MFG-E8(+/+) mice during exposure to DSS with delayed healing of intestinal epithelium during the recovery phase of DSS colitis. Administration of MFG-E8 during the recovery phase ameliorated colitis and promoted mucosal repair in both MFG-E8(-/-) and MFG-E8(+/+) mice, indicating that lack of MFG-E8 causes increased susceptibility to colitis and delayed mucosal healing. These data suggest that MGF-E8 is an essential protective factor for gut epithelial homeostasis, and exogenous administration of MFG-E8 may represent a novel therapeutic target in inflammatory bowel disease.     

Enteric neural pathways mediate the anti-inflammatory actions of glucagon-like peptide 2

Glucagon-like peptide-2 (GLP-2) is an important regulator of nutritional absorptive capacity with anti-inflammatory actions. We hypothesized that GLP-2 reduces intestinal mucosal inflammation by activation of vasoactive intestinal polypeptide (VIP) neurons of the submucosal plexus. Ileitis or colitis was induced in rats by injection of trinitrobenzene sulfonic acid (TNBS), or colitis was induced by administration of dextran sodium sulfate (DSS) in drinking water. Subsets of animals received (1-33)-GLP-2 (50 mug/kg sc bid) either immediately or 2 days after the establishment of inflammation and were followed for 3-5 days. The involvement of VIP neurons was assessed by concomitant administration of GLP-2 and the VIP antagonist [Lys(1)-Pro(2,5)-Arg(3,4)-Tyr(6)]VIP and by immunohistochemical labeling of GLP-2-activated neurons. In all models, GLP-2 treatment, whether given immediately or delayed until inflammation was established, resulted in significant improvements in animal weights, mucosal inflammation indices (myeloperoxidase levels, histological mucosal scores), and reduced levels of inflammatory cytokines (IFN-gamma, TNF-alpha, IL-1beta) and inducible nitric oxide synthase, with increased levels of IL-10 in TNBS ileitis and DSS colitis. Reduced rates of crypt cell proliferation and of apoptosis within crypts in inflamed tissues were also noted with GLP-2 treatment. These effects were abolished with coadministration of GLP-2 and the VIP antagonist. GLP-2 was shown to activate neurons and to increase the number of cells expressing VIP in the submucosal plexus of the ileum. These findings suggest that GLP-2 acts as an anti-inflammatory agent through activation of enteric VIP neurons, independent of proliferative effects. They support further studies to examine the role of neural signaling in the regulation of intestinal inflammation.     

细胞粘附分子CHL1缺失对炎症性肠病的影响

目的：探讨细胞粘附分子CHL1缺失对炎症性肠病（IBD）的影响。方法：采用葡聚糖硫酸钠（DSS）建立CHL1（-/-）炎症性肠病小鼠模型,将10只C57/BL6为遗传背景的CHL1（+/+）型小鼠分为对照组、DSS模型组（n=5）;将10只C57/BL6为背景的CHL （-/-）型小鼠分为CHL1缺失组、CHL1缺失DSS模型组（n=5）。DSS模型组和CHL1缺失DSS模型组自主饮用7 d含1.5% DSS蒸馏水,随后改用蒸馏水饲喂2 d;对照组和CHL1缺失组连续饮用蒸馏水9 d,观察小鼠体重、血便、生存率及结肠组织长度的变化。结果：CHL1缺失DSS模型组小鼠在炎症性肠病的发生中便血明显,从应激的第7天开始,体重明显减轻,在第9天,CHL1缺失DSS模型组型小鼠死亡率明显增加;结肠长度明显缩短,损伤加重。结论：细胞粘附分子CHL1缺失加重炎症性肠病的发生。     

DA-6034, a derivative of flavonoid, prevents and ameliorates dextran sulfate sodium-induced colitis and inhibits colon carcinogenesis

Previously, we have shown that DA-6034, a synthetic derivative of flavonoid eupatilin, inhibited NF-kappaB activation in colon epithelial cells and prevented trinitrobenzene sulfonic acid-induced rat colitis. The aim of this study was to investigate the preventive and therapeutic effect of DA-6034 on dextran sulfate sodium (DSS)-induced colitis and on inflammation-related cancer. C57BL/6 mice were given 4% DSS for 5 days with and without DA-6034 in the acute preventive model. In the acute therapeutic model, mice were given 4% DSS for 5 days followed by rectal administration of DA-6034. Colitis was quantified by body weight, disease activity index (DAI), colon length, and histology. In the inflammation-related cancer model, mice were given a single intraperitoneal injection of azoxymethane, then three cycles of 2% DSS for 5 days, then 2 weeks of free water consumption. Apoptosis was determined by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, and the expression of Ki-67, phospho-kappaB kinase alpha (IKKalpha), and COX-2 were evaluated by immunohistochemistry. In both the acute preventive and acute therapeutic models, DA-6034 significantly attenuated DSS-induced weight loss, an increase in DAI, and a shortening of colon length. DA-6034-treated mice maintained crypt architecture and revealed a scanty infiltration of inflammatory cells in both the preventive and therapeutic models. In the inflammation-related cancer model, DA-6034 reduced the number of colon tumors and ameliorated weight loss and shortening of colon length. DA-6034 strongly enhanced apoptosis and inhibited the expression of COX-2 and phospho-IKKalpha in inflammation-related colon cancer models. Our results suggest that DA-6034 prevents acute murine colitis and inhibits inflammation-related colon carcinogenesis. DA-6034 could be a potential therapeutic agent for inflammatory bowel disease.     

TGF-beta signaling-dependent alleviation of dextran sulfate sodium-induced colitis by mesenchymal stem cell transplantation

Alleviation of dextran sulfate sodium (DSS)-induced colitis was shown after by transplantation of bone marrow derived cells in mice. Nevertheless, the underlying mechanism remains elusive. In the present study, we transplanted primary mouse mesenchymal stem cells (MSC) into isogeneic mice with DSS-induced colitis. We found that MSC transplantation significantly alleviated the DSS-induced colitis. Inhibition of transforming growth factor beta (TGF-beta) signaling abrogated the therapeutic effect of MSC transplantation on DSS-colitis, suggesting a TGF-beta signaling-dependent manner. Moreover, MSC transplantation seemed to induce M2 macrophage polarization, which appeared to be the major source of TGF-beta in this model. Our data thus demonstrate that MSC transplantation may activate TGF-beta signaling pathways to promote the recovery of DSS-colitis.     

Alteration of colonic stem cell gene signatures during the regenerative response to injury

Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n-3 polyunsaturated fatty acids (n-3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creER(T2) mice were fed diets enriched with n-3 PUFA vs n-6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5(+) stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5(+) stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n-3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5(+) stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.     

鼠衣原体改善DSS诱导的小鼠溃疡性结肠炎的作用研究

【目的】探讨鼠衣原体(Chlamydia muridarum)对小鼠溃疡性结肠炎的作用。【方法】取15只雌性C57BL/6J小鼠随机分为3组,每组5只动物,分别为空白对照组(Control)、肠炎模型组(DSS)、实验组(CM+DSS)。选取CM+DSS组小鼠予以2×10~5 IFU的鼠衣原体灌胃处理,并在其感染后第29天开始,给予DSS组和CM+DSS组的小鼠2%DSS饮水,持续5d,每天监测小鼠体重和肠炎疾病评分,实验结束后检测小鼠结肠长度和结肠组织炎性改变。【结果】肠炎模型组的小鼠均表现出典型的肠炎症状(包括体重减轻、肠炎疾病评分、结肠长度和组织炎性改变);而经鼠衣原体预处理的小鼠(CM+DSS组)肠炎症状显著减轻,表现在肠炎疾病评分降低,体重和结肠长度有所恢复,肠组织炎性损伤减轻。【结论】鼠衣原体对DSS诱导的小鼠溃疡性结肠炎具有改善作用。     

胰高血糖素样肽-2对小鼠小肠缺血/再灌注损伤的保护作用

目的:观察胰高血糖素样肽-2(GLP-2)对缺血/再灌注损伤小鼠小肠的保护效应.方法:采用肠缺血/再灌注(I/R)模型,将32只小鼠随机分为4组(n=8)假手术(Sham)组、I/R组、I/R GLP-2保护组和I/R 谷氨酰胺(GLN)阳性对照组.光镜观察小肠黏膜形态学改变.检测小肠绒毛高度和隐窝深度;小肠组织二胺氧化酶(DAO)活性;肠系膜淋巴结(MLN)细菌易位率.结果:与假手术组相比,I/R组部分小肠绒毛坏死脱落,绒毛高度下降,隐窝变浅(P＜0 01);小肠组织DAO活性降低(P＜0.01);MLN细菌易位率增加(P＜0.05).与I/R组比,GLP-2组肠绒毛损害明显减轻,DAO活性回升(P＜0.01),细菌易位率回降(P＜0.05).结论:GLP-2对缺血/再灌注损伤小鼠小肠的形态结构及肠屏障功能具有保护作用.     

Interleukin-10-independent anti-inflammatory actions of glucagon-like peptide 2

Glucagon-like peptide 2 (GLP-2) is an important intestinal growth factor with anti-inflammatory activity. We hypothesized that GLP-2 decreases mucosal inflammation and the associated increased epithelial proliferation by downregulation of Th1 cytokines attributable to reprogramming of lamina propria immune regulatory cells via an interleukin-10 (IL-10)-independent pathway. The effects of GLP-2 treatment were studied using the IL-10-deficient (IL-10(-/-)) mouse model of colitis. Wild-type and IL-10(-/-) mice received saline or GLP-2 (50 microg/kg sc) treatment for 5 days. GLP-2 treatment resulted in significant amelioration of animal weight loss and reduced intestinal inflammation as assessed by histopathology and myeloperoxidase levels compared with saline-treated animals. In colitis animals, GLP-2 treatment also reduced crypt cell proliferation and crypt cell apoptosis. Proinflammatory (IL-1beta, TNF-alpha, IFN-gamma,) cytokine protein levels were significantly reduced after GLP-2 treatment, whereas IL-4 was significantly increased and IL-6 production was unchanged. Fluorescence-activated cell sorting analysis of lamina propria cells demonstrated a decrease in the CD4(+) T cell population following GLP-2 treatment in colitic mice and an increase in CD11b(+)/F4/80(+) macrophages but no change in CD25(+)FoxP3 T cells or CD11c(+) dendritic cells. In colitis animals, intracellular cytokine analysis demonstrated that GLP-2 decreased lamina propria macrophage TNF-alpha production but increased IGF-1 production, whereas transforming growth factor-beta was unchanged. GLP-2-mediated reduction of crypt cell proliferation was associated with an increase in intestinal epithelial cell suppressor of cytokine signaling (SOCS)-3 expression and reduced STAT-3 signaling. This study shows that the anti-inflammatory effects of GLP-2 are IL-10 independent and that GLP-2 alters the mucosal response of inflamed intestinal epithelial cells and macrophages. In addition, the suggested mechanism of the reduction in inflammation-induced proliferation is attributable to GLP-2 activation of the SOCS-3 pathway, which antagonizes the IL-6-mediated increase in STAT-3 signaling.     

Hyaluronic acid regulates normal intestinal and colonic growth in mice

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.     

Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice.

This study presents a histological examination of dextran sodium sulfate (DSS)-induced colitis in germ-free (GF) mice. A comparison of the pathology between GF and conventionalized mice (CVz) was made to determine the role that intestinal microflora play in DSS-induced colitis. To induce colitis, GF and CVz IQI/Jic mice were given either 5% or 1% DSS orally. Administration of 5% DSS, a common concentration used to induce colitis in mice, caused gross rectal bleeding and a marked decrease in hematocrit as early as day one in GF mice. These mice died on day three due to massive bleeding into the intestinal lumen. In contrast, CVz mice did not die during the seven-day experimental period. Histopathological examination three days after administration of 5% DSS did not reveal any colitis lesions in GF mice, but CVz mice had developed moderate colitis in the large intestine. Administration of a low concentration of DSS (1%), which only induces mild basal crypt loss in CVz mice, caused severe colitis in the distal colon in GF mice, and they died on day 14. These data suggest that intestinal microflora are not necessary for the induction of colitis. Furthermore, DSS may be highly toxic to GF mice, and when given at a concentration of 5% it causes massive bleeding into the intestinal lumen resulting in death prior to development of colitis.     

Efficacy of an inhibitor of adhesion molecule expression (GI270384X) in the treatment of experimental colitis

Modulation of adhesion molecule expression or function is regarded as a promising therapy for inflammatory conditions. This study evaluates the effects of an inhibitor of adhesion molecule expression (GI270384X) in two experimental models of colitis. Colitis of different severity was induced in C57BL/6J mice by administering 1, 2, or 3% dextran sulfate sodium (DSS). GI270384X (3, 10, or 25 mg.kg(-1).day(-1)) was administered as pretreatment or started 3 days after colitis induction. In IL-10-deficient mice, the highest dose was given for 2 wk. The clinical course of colitis, pathological changes, serum inflammatory biomarkers, expression of adhesion molecules, and leukocyte-endothelial cell interactions in colonic venules were measured in mice treated with vehicle or with active drug. In the most severe forms of colitis (2% and 3% DSS and IL-10-deficient mice), the magnitude of colonic inflammation was not modified by treatment with GI270384X. In a less severe form of colitis (1% DSS), GI270384X treatment dose dependently ameliorated the clinical signs of colitis, colonic pathological changes, and serum levels of biomarkers (IL-6 and serum amyloid A). Administration of 25 mg.kg(-1).day(-1) GI270384X abrogated upregulation of ICAM-1 in the inflamed colon but had no effect on VCAM-1 or E-selectin expression. This was associated with a significant reduction in number of rolling and firmly adherent leukocytes in colonic venules. These results indicate that GI270384X is effective in the treatment of experimental colitis of moderate severity. Reduced adhesion molecule expression and leukocyte recruitment to the inflamed intestine contribute to this beneficial effect.     

PACAP provides colonic protection against dextran sodium sulfate induced colitis

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a crucial role in immunity and inflammation. Our aim was to obtain insight in the role of PACAP in experimental colitis in mice and thus its possible role in inflammatory bowel disease. PACAP-deficient (PACAP-/-) mice and wild-type control mice were challenged by colitis-inducing agent, dextran sulfate sodium (DSS). We monitored clinical symptoms, intestinal morphology, and difference of cytokine production in the proximal and distal colon. After DSS administration, mortality was more severe in PACAP-/- mice versus wild-type control mice. The histological score and the disease activity index of PACAP-/- mice were significantly higher than those of wild-type control mice. In proximal colon, production of IL-1beta and IL-6 in PACAP-/- mice were significantly upregulated on day 8 after DSS administration, compared to wild-type control mice. In distal colon, furthermore, production of IFNgamma, IL-1beta, IL-6, IL-12, and KC were significantly higher in PACAP-/- mice than in wild-type control mice on day 4. Our findings indicate that PACAP regulates the production of pro-inflammatory cytokine in the experimental colitis.     

Targeted delivery of vitamin D to the colon using β-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether β-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-β-glucuronide [β-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of β-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested β-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or β-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of β-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-β-glucuronide [β-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with β-gluc-1,25(OH)(2)D alone or in combination with β-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. β-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.     

Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier

Emerging evidence supports a pathological link between vitamin D deficiency and the risk of inflammatory bowel disease (IBD). To explore the mechanism we used the dextran sulfate sodium (DSS)-induced colitis model to investigate the role of the vitamin D receptor (VDR) in mucosal barrier homeostasis. While VDR(+/+) mice were mostly resistant to 2.5% DSS, VDR(-/-) mice developed severe diarrhea, rectal bleeding, and marked body weight loss, leading to death in 2 wk. Histological examination revealed extensive ulceration and impaired wound healing in the colonic epithelium of DSS-treated VDR(-/-) mice. Severe ulceration in VDR(-/-) mice was preceded by a greater loss of intestinal transepithelial electric resistance (TER) compared with VDR(+/+) mice. Confocal and electron microscopy (EM) revealed severe disruption in epithelial junctions in VDR(-/-) mice after 3-day DSS treatment. Therefore, VDR(-/-) mice were much more susceptible to DSS-induced mucosal injury than VDR(+/+) mice. In cell cultures, 1,25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] markedly enhanced tight junctions formed by Caco-2 monolayers by increasing junction protein expression and TER and preserved the structural integrity of tight junctions in the presence of DSS. VDR knockdown with small interfering (si)RNA reduced the junction proteins and TER in Caco-2 monolayers. 1,25(OH)(2)D(3) can also stimulate epithelial cell migration in vitro. These observations suggest that VDR plays a critical role in mucosal barrier homeostasis by preserving the integrity of junction complexes and the healing capacity of the colonic epithelium. Therefore, vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and increased risk of IBD.