The effect of essential fatty acid deficiency on eicosanoid production in the inflamed rat colonic mucosa

Eicosanoids are potent mediators of inflammation and are synthesized in increased quantity in active ulcerative colitis. To elucidate the role of prostaglandin E2, thromboxane A2, prostaglandin I2, and leukotriene B2 in acute chemical colitis induced by 4% acetic acid, we utilized an animal model which has a deficiency of arachidonic acid, the precursor of eicosanoids due to an essential fatty acid deficient diet. Forty-eight hours after colitis was induced, mucosal synthesis of the cyclooxygenase products, prostaglandin E2, thromboxane A2, and prostaglandin I2, was significantly decreased in essential fatty acid deficient rats compared to normal controls. However, the 5-lipoxygenase product, leukotriene B4, was not different between groups. The decrease in cyclooxygenase products did not correlate with any change in the severity of colonic inflammation as assessed by gross morphology, histology, or myleoperoxidase activity. Thus inhibition of formation of the cyclooxygenase products of arachidonate metabolism does not appear to improve the degree of inflammation under the experimental conditions employed in this study.     

Leukotriene synthesis by human gastrointestinal tissues

The prostaglandin and leukotriene synthesizing capacity of human gastrointestinal tissues obtained at surgery was investigated using radioimmunoassay for prostaglandin E2, leukotriene B4 and sulfidopeptide leukotrienes. The leukotriene immunoassay data were validated by high-pressure liquid chromatography (HPLC). During incubation at 37 degrees C, fragments of human gastric, jejuno-ileal and colonic mucosa released considerably larger amounts of prostaglandin E2 than of leukotriene B4 and sulfidopeptide leukotrienes. Gastrointestinal smooth muscle tissues released even larger amounts of prostaglandin E2, but smaller amounts of leukotrienes than the corresponding mucosal tissues. Adenocarcinoma tissue released larger amounts of leukotriene B4, sulfidopeptide leukotrienes and prostaglandin E2 than normal colonic mucosa. Ionophore A23187 (5 micrograms/ml) did not stimulate release of prostaglandin E2 from any of the tissues investigated, but enhanced release of leukotriene B4 and sulfidopeptide leukotrienes. HPLC analysis demonstrated that immunoreactive leukotriene B4 co-chromatographed almost exclusively with standard leukotriene B4, while immunoreactive sulfidopeptide leukotrienes consisted of a mixture of leukotrienes C4, D4 and E4. Leukotriene synthesis by human gastrointestinal tissues was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the dual enzyme inhibitor BW755C (3-amino-1-(trifluoromethylphenyl)-2-pyrazoline hydrochloride). Synthesis of prostaglandin E2 was inhibited by the cyclooxygenase inhibitor indomethacin as well as by BW755C. Incubation of gastrointestinal tissues in the presence of glutathione decreased the amounts of leukotrienes D4 and E4, while release of leukotriene C4 was simultaneously increased. On the other hand, incubation of tritiated leukotriene C4 with incubation media from human gastric or colonic mucosa resulted in conversion of the substrate to [3H]leukotriene D4 and [3H]leukotriene E4. The results indicate the capacity of human gastrointestinal tissues to synthesize the 5-lipoxygenase-derived products of arachidonate metabolism, leukotriene B4 and sulfidopeptide leukotrienes, in addition to larger amounts of prostaglandin E2. Furthermore, considerable activities of the sulfidopeptide leukotriene-metabolizing enzymes gamma-glutamyl transpeptidase and dipeptidase were detected in human gastrointestinal tissues. These enzymes might play an important role in biological inactivation and/or change of biological profile of sulfidopeptide leukotrienes generated in the human gastrointestinal tract.     

Preventive effects of Goji berry on dextran-sulfate-sodium-induced colitis in mice

Goji berry (Lycium barbarum) exerts immune modulation and suppresses inflammation in vitro and in vivo. We hypothesized that Goji berry had beneficial effects on dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice through suppressing inflammation. Six-week-old male C57BL/6 mice were supplemented with a standard AIN-93G diet with or without 1% (w/w) Goji berry for 4 weeks. Then, colitis was induced by supplementing 3% DSS in drinking water for 7 days, followed by 7 days of remission period to mimic ulcerative colitis symptoms. Goji berry supplementation ameliorated DSS-induced body weight loss, diminished diarrhea and gross bleeding, and resulted in a significantly decreased disease activity index, as well as DSS-associated colon shortening. Moreover, 30% mortality rate caused by DSS-induced colitis was avoided because of Goji berry supplementation. Histologically, Goji berry ameliorated colonic edema, mucosal damage and neutrophil infiltration into colonic intestinal tissue in response to DSS challenge, which was associated with decreased expression of chemokine (C-X-C motif) ligand 1 and monocyte chemoattractant protein-1, as well as inflammatory mediators interleukin-6 and cyclooxygenase-2. In conclusion, Goji supplementation confers protective effects against DSS­induced colitis, which is associated with decreased neutrophil infiltration and suppressed inflammation. Thus, dietary Goji is likely beneficial to inflammatory bowel disease patients as a complementary therapeutic strategy.     

氯化两面针碱对小鼠溃疡性结肠炎的干预作用及其机制*

目的:探讨氯化两面针碱(NC)通过靶向miR-31对葡聚糖硫酸钠(DSS)诱发小鼠结肠炎的保护作用及其机制。方法:用1%DSS诱发小鼠溃疡性结肠炎(UC)。30只雄性C57BL/6小鼠随机分为正常对照组(Control)(n=7),DSS组(n=8),DSS+NC组(7.27 mg/kg)(n=8)和NC组(n=7),饮水给予DSS,灌胃给予氯化两面针碱。造模周期为3周,分别为Control组和NC组每天饮用无菌水,DSS组和DSS+NC组第一周饮用1% DSS水,第2周正常饮水,第3周1% DSS水。造模最后一周给予Control组和DSS组小鼠0.5% 羧甲基纤维素钠(CMC-Na)灌胃,DSS+NC组和NC组给予NC灌胃。造模完成后,观察小鼠结肠炎相关的疾病活动指数(DAI),HE染色进行结肠组织病理评分,qPCR检测小鼠结肠组织miR-31的表达水平,Western blot检测小鼠结肠组织炎症蛋白NF-κB和COX-2的表达情况。结果:①与DSS组相比,DSS+NC组的 DAI 显著降低(P＜0.01),结肠病理损伤明显改善;②与Control组相比,DSS组小鼠结肠组织miR-31表达显著升高(P＜0.01),DSS+NC组miR-31的表达水平显著低于DSS组(P＜0.05);③与DSS组相比,DSS+NC组中的炎症蛋白NF-κB和COX-2表达水平显著下降(P＜0.05)。结论:氯化两面针碱对DSS诱导的小鼠溃疡性结肠炎有明显的治疗作用,其抗炎机制与下调miR-31的表达有关。     

Sequential release of cytokines, lipid mediators and nitric oxide in experimental colitis

The object of this study was to establish whether different pro- and anti-inflammatory mediators were formed in colonic tissue from experimental colitis depending on the course of the disease. Concentrations of mediators of inflammation were examined in colonic tissue in dextran induced colitis in mice. Initial inflammation was produced by 5 days treatment of 10% dextran sodium sulfate (DSS) in drinking water, followed by a further 9 day period of 2% DSS in an attempt to produce a milder chronic inflammation. The degree of inflammation was scored by a standardized macroscopic and histological examination. Initially, a 60% maximum inflammation score was observed at day 4. At this time inflammation was associated with the release of interleukin-lbeta (IL-1beta) and tumour necrosis factor-alpha (TNFalpha), whereas both prostaglandins 6kPGF(1alpha) and PGE(2) and nitric oxide (NO) markedly decreased. Then a 25% inflammation score was reached which coincided with an increased production of platelet-activating factor (PAF). No significant changes were observed in leukotriene B(4) and C(4) formation. In conclusion, pro-inflammatory cytokines IL-1beta and TNFalpha are considered to be primary mediators, whereas PAF, eicosanoids and NO may reflect secondary mediators in experimental colitis.     

Involvement of nitric oxide with activation of Toll-like receptor 4 signaling in mice with dextran sodium sulfate-induced colitis

Ulcerative colitis is an inflammatory bowel disease characterized by acute inflammation, ulceration, and bleeding of the colonic mucosa. Its cause remains unknown. Increases in adhesion molecules in vascular endothelium, and activated neutrophils releasing injurious molecules such as reactive oxygen species, are reportedly associated with the pathogenesis of dextran sodium sulfate (DSS)-induced colitis. Nitric oxide (NO) production derived from inducible NO synthase (iNOS) via activation of nuclear factor κB (NF-κB) has been reported. It is also reported that stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide can activate NF-κB. In this study, we investigated the involvement of NO production in activation of the TLR4/NF-κB signaling pathway in mice with DSS-induced colitis. The addition of 5% DSS to the drinking water of male ICR mice resulted in increases in TLR4 protein in colon tissue and NF-κB p65 subunit in the nuclear fraction on day 3, increases in colonic tumor necrosis factor-α on day 4, and increases in P-selectin, intercellular adhesion molecule-1, NO₂⁻/NO₃⁻, and nitrotyrosine in colonic mucosa on day 5. These activated inflammatory mediators and pathology of colitis were completely suppressed by treatment with a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, as well as an iNOS inhibitor, aminoguanidine. Conversely, a NO-releasing compound, NOC-18, increased TLR4 levels and nuclear translocation of NF-κB p65 and exacerbated mucosal damage induced by DSS challenge. These data suggest that increases in TLR4 expression induced by drinking DSS-treated water might be directly or indirectly associated with NO overproduction.     

Effects of 16,16-dimethyl prostaglandin E2 and indomethacin on leukotriene B4 and inflammation in rabbit colitis

The role of increased prostaglandin production and the effects of exogenous prostaglandins on inflammation of colitis are not established. We administered intramuscular 16,16-dimethyl prostaglandin E2 (DiM-PGE2) and indomethacin to rabbits with formalin immune-complex colitis and measured leukotriene B4 (LTB4), prostaglandin E2 (PGE2) and severity of inflammation. DiM-PGE2 (100 micrograms/kg/BID) reduced LTB4 production (from 401 +/- 108 to 216 +/- 58 pg/ml) and infiltration of neutrophils, mucosal necrosis, inflammatory exudate and edema (all P less than 0.05). Other studies determined that parenteral DiM-PGE2 did not reduce the initial chemical damage induced by formalin, suggesting that cytoprotection of chemical insult was not the mechanism of suppressed inflammation in the immune colitis model. Indomethacin (10 mg/kg/d) reduced endogenous PGE2 by 80%, but did not reduce leukotriene production or inflammation. Exogenous prostaglandins cause a dose-dependent suppression of inflammation in experimental colitis, by a mechanism other than cytoprotection of chemical-induced mucosal injury.     

Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis

The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D(2) has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D(2) derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D(2) synthesis was elevated >4-fold above controls within 1-3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D(2) synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D(2) or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D(2) synthesis via cyclooxygenase-2. Prostaglandin D(2) downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.     

Bovine milk-derived α-lactalbumin inhibits colon inflammation and carcinogenesis in azoxymethane and dextran sodium sulfate-treated mice

Cyclooxygenase-2 is expressed early in colon carcinogenesis and plays crucial role in the progress of the disease. Recently, we found that α-lactalbumin had anti-inflammatory activity by inhibiting cyclooxygenase-2. In experiment 1, we investigated the effects of α-lactalbumin on the colon carcinogenesis initiated with azoxymethane (AOM) followed by promotion with dextran sodium sulfate (DSS) in mice. Dietary treatment with α-lactalbumin decreased fecal occult blood score at 3?days after DSS intake. α-Lactalbumin also decreased the colon tumor at week 9. In experiment 2, AOM-treated mice were sacrificed at 7?days after DSS intake. The plasma and colon prostaglandin E2 (PGE2) levels in AOM/DSS-treated mice were higher than those in the DSS-treated mice without initiation by AOM. α-Lactalbumin decreased PGE2 in both plasma and colon. These results suggest that α-lactalbumin effectively inhibited colon carcinogenesis, and the inhibition may be due to the decreased PGE2 by inhibiting cyclooxygenase-2 at cancer promotion stages.     

Effects of 16,16-dimethyl prostaglandin E2 and indomethacin on leukotriene B4 and inflammation in rabbit colitis

The role of increased prostaglandin production and the effects of exogenous prostaglandins on inflammation of colitis are not established. We administered intramuscular 16,16-dimethyl prostaglandin E2 (DiM-PGE2) and indomethacin to rabbits with formalin immune-complex colitis and measured leukotriene B₄ (LTB4), prostaglandin E2 (PGE2) and severity of inflammation. DiM-PGE2 (100 ug/kg/BID) reduced LTB4 production (from 401±108 to 216±58 pg/ml) and infiltration of neutrophils, mucosal necrosis, inflammatory exudate and edema (all P<0.05). Other studies determined that parenteral DiM-PGE2 did not reduce the initial chemical damage induced by formalin, suggesting that cytoprotection of chemical insult was not the mechanism of suppressed inflamation in the immune colitis model. Indomethacin (10 mg/kg/d) reduced endogenous PGE2 by 80%, but did not reduce leukotriene production or inflammation. Exogenous prostaglandins cause a dose-dependent suppression of inflammation in experimental colitis, by a mechanism other than cytoprotection of chemical-induced mucosal injury.     

EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

Prostaglandin E₂ plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis.     

Anti-inflammatory effects and changes in prostaglandin patterns induced by 7beta-hydroxy-epiandrosterone in rats with colitis

High dose levels of dehydroepiandrosterone and its 7-hydroxylated derivatives have been shown to reduce oxidative stress and inflammatory responses in dextran sodium sulfate (DSS)-induced colitis in rats. Another endogenous steroid, 7beta-hydroxy-epiandrosterone (7beta-hydroxy-EpiA) has been shown to exert neuroprotective effects at much smaller doses. Our aims were to evaluate whether 7beta-hydroxy-EpiA pre-treatment prevents DSS-induced colitis and to determine whether the effects involve changes in anti-inflammatory prostaglandin (PG) D(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) levels. Rats were administered 0.01, 0.1 and 1mg/kg 7beta-hydroxy-EpiA i.p. once a day for 7 days. Thereafter, colitis was induced by administration of 5% DSS in drinking water for 7 days. Levels of the PGs and the expression of cyclooxygenase (COX-2) and PG synthases were assessed during the course of the experiment. Administration of 7beta-hydroxy-EpiA caused a transient increase in COX-2 and PGE synthase expression within 6-15h and augmented colonic tissue levels of 15d-PGJ(2) levels starting at day 2. Treatment with DSS resulted in shortened colon length, depleted mucus in goblet cells and induced oxidative stress. COX-2 and mPGES-1 synthase expression were enhanced and accompanied by increased PGE(2), D(2) and 15d-PGJ(2) production. Although all dose levels of 7beta-hydroxy-EpiA reduced PGE(2) production, only the lowest dose (0.01mg/kg) of the steroid completely prevented colitis damage and tissue inflammation. 7beta-Hydroxy-EpiA pre-treatment prevents the occurrence of DSS-induced colitis through a shift from PGE(2) to PGD(2) production, associated with an early but transient increase in COX-2 expression and a sustained increase in the production of the anti-inflammatory prostaglandin 15d-PGJ(2).     

Indomethacin worsens and a leukotriene biosynthesis inhibitor accelerates mucosal healing in rat colitis.

The implication of leukotrienes as mediators of inflammation and recent evidence that prostaglandin analogues provide a beneficial effect during experimental colitis led to the speculation that (i) leukotrienes may be injurious and (ii) prostaglandins may be protective to colonic mucosa. Using a 2% acetic acid induced rat colitis model, we administered specific cyclooxygenase (indomethacin) and leukotriene biosynthesis inhibitors (MK-886) to examine the effect of endogenous prostaglandins and leukotrienes on colonic macroscopic injury, mucosal inflammation as measured by myeloperoxidase activity, net in vivo intestinal fluid absorption, and colonic PGE2 and LTB4 levels as measured by in vivo rectal dialysis. Indomethacin treatment prior to induction of colitis reduced endogenous mucosal PGE2 levels and exacerbated macroscopic ulceration and net fluid absorption. Addition of the exogenous PGE1 analogue misoprostol to the indomethacin-exacerbated colitis completely healed colonic macroscopic ulceration and inflammation but only partially improved fluid absorptive injury. The specific leukotriene biosynthesis inhibitor MK-886 administered prior to induction of colitis healed macroscopic ulceration and inflammation but not fluid absorptive injury. This mucosal reparative effect of MK-886 occurred at a dose that reduced colonic LTB4 synthesis while concomitantly enhancing PGE2 levels. Combining MK-886 with misoprostol treatment improved not only macroscopic ulceration and inflammation but also provided a synergistic effect that maintained net colonic fluid absorption at noncolitic control levels. These studies suggest that, during the induction of experimental colitis, endogenous prostaglandins play a pivotal role in providing a mucosal healing effect, and that leukotriene biosynthesis inhibitor may manifest part of its beneficial effect by shifting arachidonic acid metabolism towards production of prostaglandins.     

Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils

Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.     

Prolonged lipopolysaccharide inhibits leukotriene synthesis in peritoneal macrophages: mediation by nitric oxide and prostaglandins

Resident rat peritoneal macrophages synthesize a variety of prostanoids and leukotrienes from arachidonic acid. Overnight treatment with lipopolysaccharide (LPS) induces the synthesis of cyclooxygenase-2 (COX-2) and an altered prostanoid profile that emphasizes the preferential conversion of arachidonic acid to prostacyclin and prostaglandin E2. In these studies, we report that exposure to LPS also caused a strong suppression of 5-lipoxygenase but not 12-lipoxygenase activity, indicated by the inhibition of synthesis of both leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE), but not of 12-HETE. Inhibition of 5-lipoxygenase activity by LPS was both time- and dose-dependent. Treatment of macrophages with prostaglandin E2 partially inhibited leukotriene synthesis, and cyclooxygenase inhibitors partially blocked the inhibition of leukotriene generation in LPS-treated cells. In addition to COX-2, nitric oxide synthase (NOS) was also induced by LPS. Treatment of macrophages with an NO donor mimicked the ability of LPS to significantly reduce leukotriene B4 synthesis. Inhibition of NOS activity in LPS-treated cells blunted the suppression of leukotriene synthesis. Inhibition of both inducible NOS and COX completely eliminated leukotriene suppression. Finally, macrophages exposed to prolonged LPS demonstrated impaired killing of Klebsiella pneumoniae and the combination of NOS and COX inhibitors restored killing to the control level. These results indicate that prolonged exposure to LPS severely inhibits leukotriene production via the combined action of COX and NOS products. The shift in mediator profile, to one that minimizes leukotrienes and emphasizes prostacyclin, prostaglandin E2 and NO, provides a signal that reduces leukocyte function, as indicated by impaired killing of Gram-negative bacteria.     

Chemically induced mouse models of intestinal inflammation

Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and ulcerative colitis, the two major forms of inflammatory bowel disease in humans. Here, we provide protocols for establishing murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-, oxazolone- and both acute and chronic dextran sodium sulfate (DSS) colitis, the most widely used chemically induced models of intestinal inflammation. In the former two models, colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens. In the DSS model, mice are subjected several days to drinking water supplemented with DSS, which seems to be directly toxic to colonic epithelial cells of the basal crypts. The procedures for the hapten models of colitis and acute DSS colitis can be accomplished in about 2 weeks but the protocol for chronic DSS colitis takes about 2 months.     

CXCR4 antagonist AMD3100 modulates claudin expression and intestinal barrier function in experimental colitis

Ulcerative colitis is a gastrointestinal disorder characterized by local inflammation and impaired epithelial barrier. Previous studies demonstrated that CXC chemokine receptor 4 (CXCR4) antagonists could reduce colonic inflammation and mucosal damage in dextran sulfate sodium (DSS)-induced colitis. Whether CXCR4 antagonist has action on intestinal barrier and the possible mechanism, is largely undefined. In the present study, the experimental colitis was induced by administration of 5% DSS for 7 days, and CXCR4 antagonist AMD3100 was administered intraperitoneally once daily during the study period. For in vitro study, HT-29/B6 colonic cells were treated with cytokines or AMD3100 for 24 h until assay. DSS-induced colitis was characterized by morphologic changes in mice. In AMD3100-treated mice, epithelial destruction, inflammatory infiltration, and submucosal edema were markedly reduced, and the disease activity index was also significantly decreased. Increased intestinal permeability in DSS-induced colitis was also significantly reduced by AMD3100. The expressions of colonic claudin-1, claudin-3, claudin-5, claudin-7 and claudin-8 were markedly decreased after DSS administration, whereas colonic claudin-2 expression was significantly decreased. Treatment with AMD3100 prevented all these changes. However, AMD3100 had no influence on claudin-3, claudin-5, claudin-7 and claudin-8 expression in HT-29/B6 cells. Cytokines as TNF-α, IL-6, and IFN-γ increased apoptosis and monolayer permeability, inhibited the wound-healing and the claudin-3, claudin-7 and claudin-8 expression in HT-29/B6 cells. We suggest that AMD3100 acted on colonic claudin expression and intestinal barrier function, at least partly, in a cytokine-dependent pathway.     

Interleukin-1beta targets interleukin-6 in progressing dextran sulfate sodium-induced experimental colitis

Inflammatory bowel disease (IBD) is an immunologically mediated disorder that is characterized by chronic, relapsing, and inflammatory responses. Dextran sulfate sodium (DSS)-induced experimental colitis in mice has been recognized as a useful model for human IBD and interleukin (IL)-1beta is a key cytokine in the onset of IBD. The purpose of the present study was to clarify which pro-inflammatory mediators are targeted by IL-1beta in mice with DSS-induced colitis. First, we found that DSS markedly induced IL-1beta production in both dose- and time-dependent manners (P < 0.05 and P < 0.01, respectively) in murine peritoneal macrophages (pMphi), while that of tumor necrosis factor-alpha was insignificant. Further, the expressions of mRNA and protein for IL-1beta were increased in colonic mucosa and pMphi from mice that received drinking water containing 5% DSS for 7 days (P < 0.01, each). In addition, the expressions of IL-6, granulocyte macrophage-colony stimulating factor, inducible nitric oxide synthase, and cyclooxygenase-2 mRNA were also time dependently increased (P < 0.01, each). Furthermore, administration of rIL-1beta (10 microg/kg, i.p.) significantly induced the expressions of IL-1beta and IL-6 mRNA in colonic mucosa from non-treated mice (P < 0.01). Anti-mIL-1beta antibody treatments (50 microg/kg, i.p.) attenuated DSS-induced body weight reduction and shortening of the colorectum (P < 0.05, each), and abrogated the expressions of IL-1beta and IL-6 mRNA in colonic mucosa (P < 0.01, each). Our results evidently support the previous findings that IL-1beta is involved in the development of DSS-induced experimental colitis in mice, and strongly suggest that IL-1beta targets itself and IL-6 for progressing colonic inflammation.     

Importance and regulation of the colonic mucus barrier in a mouse model of colitis

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.