Effects of anthocyanin-rich tea "Sunrouge" on dextran sodium sulfate-induced colitis in mice

Sunrouge, an anthocyanin-rich tea, has similar levels of catechins as "Yabukita," the most popular green tea cultivar consumed in Japan. Green tea polyphenols (GTPs) have attracted interest due to their potent antioxidative activities combined with a lack of side effects in humans at normal consumption levels. However, we previously reported that high doses (0.5 and 1%) of dietary GTPs can result in deterioration of colitis and failed to prevent colon carcinogenesis in inflamed colons. In the present study, we determined the inhibitory effects of Sunrouge on colitis in dextran sodium sulfate (DSS)-treated and untreated control mice. Five-week-old female ICR mice were administered a single dose of Yabukita or Sunrouge (extracts in 1 mL distilled water) via a stomach tube for 3 weeks. After 1 week of treatment, the mice were divided into four groups (two Yabukita and two Sunrouge groups) and given drinking water with or without 3% DSS for 2 weeks, then they were euthanized. Those treated with DSS developed watery diarrhea and bloody stools, and showed body weight loss, spleen hypertrophy, and shortening of the colon, as well as deteriorations in survival rate, liver function, colon mucosal interleukin-1β level and expression of phase II detoxification enzyme mRNA. Sunrouge improved these DSS-induced symptoms, at least in part, whereas Yabukita showed either no effect or adverse effects in regard to some those parameters. It is suggested that the differences between Yabukita and Sunrouge on DSS-induced colitis might be due to the high levels of anthocyanins found in Sunrouge tea.     

Dual role of endogenous nitric oxide in development of dextran sodium sulfate-induced colitis in rats.

The role of nitric oxide (NO) in the etiology of ulcerative colitis is controversial with reports of the improvement and aggravation of colonic lesions by inducible NO synthase (iNOS) inhibitors. In the present study, we compared the effect of the selective iNOS inhibitor aminoguanidine and the nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on a dextran sulfate sodium (DSS)-induced model of colitis in rats. Experimental colitis was induced by a 3% DSS-solution added to drinking water for 7 days. Aminoguanidine (5 approximately 20 mg/kg) and L-NAME (10 mg/kg) were administered p.o. twice daily for the first 3 days, the last 3 days or all 6 days of DSS treatment. Body weight and severity of colitis (diarrhea, bloody feces) were observed over a period of 7 days. DSS treatment resulted in severe colonic lesions, accompanied by diarrhea, bloody feces, decrease of body weight and colon shortening. All of the parameters investigated improved significantly with aminoguanidine treatment at 20 mg/kg for 6 days or the last 3 days of DSS-treatment, but L-NAME did not significantly affect the colitis during these periods. When L-NAME or aminoguanidine was given in the first 3 days of DSS treatment, the colonic lesions were slightly aggravated by L-NAME but not affected by aminoguanidine. The expression of iNOS mRNA was observed from the 3(rd) day of DSS treatment. These results suggested that endogenous NO exerts a biphasic influence on DSS-induced colitis, depending on the NOS isoenzyme; a beneficial effect of NO derived from constitutive NOS and a detrimental effect of NO produced by iNOS in the development of colitis.     

Dietary tryptophan alleviates dextran sodium sulfate-induced colitis through aryl hydrocarbon receptor in mice

Ulcerative colitis is the typical progression of chronic inflammatory bowel disease. Amino acids, particularly tryptophan, have been reported to exert a protective effect against colitis induced by dextran sodium sulfate (DSS), but the precise underlying mechanisms remain incompletely clarified. Tryptophan metabolites are recognized to function as endogenous ligands for aryl hydrocarbon receptor (Ahr), which is a critical regulator of inflammation and immunity. Thus, we conducted this study to investigate whether dietary tryptophan supplementation protects against DSS-induced colitis by acting through Ahr. Female wild-type (WT) and Ahr-deficient (knockout; KO) mice (10–12 weeks old) were divided into four groups and fed either a control or 0.5% tryptophan diet. The tryptophan diet ameliorated DSS-induced colitis symptoms and severity in WT mice but not in KO mice, and the diet reduced the mRNA expression of Il-6, Tnfα, Il-1β and the chemokines Ccl2, Cxcl1 and Cxcl2 in the WT groups. Furthermore, Il-22 and Stat3 mRNA expression in the colon was elevated in WT mice fed with the tryptophan diet, which mainly protected epithelial layer integrity, and Ahr also modulated immune homeostasis by regulating Foxp3 and Il-17 mRNA expression. These data suggest that tryptophan-containing diet might ameliorate DSS-induced acute colitis and regulate epithelial homeostasis through Ahr. Thus, tryptophan could serve as a promising preventive agent in the treatment of ulcerative colitis.     

Protection against dextran sodium sulfate-induced colitis by dehydroepiandrosterone and 7alpha-hydroxy-dehydroepiandrosterone in the rat

In this study the anti-oxidant effect of DHEA and 7alpha-hydroxy-DHEA against oxidative stress induced by colitis was investigated in vivo in rats. The two steroids were intraperitoneally injected once daily (50 mg/kg body weight) for 7 days before the induction of colitis that was effected by a daily treatment of 5% (w/v) dextran sodium sulfate (DSS) in drinking water for 7 days. This was quantified by the evidence of weight loss, rectal bleeding, increased wall thickness, and colon length. The inflammatory response was assessed by neutrophil infiltration after a histological examination and myeloperoxidase (MPO) activity measurement. Two markers of oxidative damage were measured in colon homogenates after the onset of DSS treatment: protein carbonyls and thiobarbituric acid-reacting substances. The colonic metabolism of corticosterone by 11beta-hydroxysteroid dehydrogenases types 1 and 2 (11beta-HSD) was investigated in control and treated animals. Results indicated that colitis caused a decrease in body weight and colon length. Severe lesions were observed in the colon with a reduced number of goblet cells which contained less mucins. The lesions were associated with increased MPO activity and oxidative damage. Colonic inflammation down and up regulated the 11beta-HSD2 and 11beta-HSD1, respectively. Treatments by DHEA and 7alpha-hydroxy-DHEA attenuated the inflammatory response when MPO activity decreased; but this did not increase the colonic oxidation of corticosterone into 11-dehydrocorticosterone. Both DHEA and 7alpha-hydroxy-DHEA exerted a significant anti-oxidant effect against oxidative stress induced by colitis through reducing the oxidative damage to proteins and lipids. This resulted in a moderate increase in the amount of colonic mucus. Both DHEA and 7alpha-hydroxy-DHEA may prove useful in the prevention or treatment of colitis.     

NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats

Although substance P (SP) has been implicated as a mediator of neurogenic inflammation in the small intestine, little information is available regarding the role of SP in the pathogenesis of chronic ulcerative colitis. In this study, our aim was to investigate whether the intraperitoneal administration of a nonpeptide neurokinin-1 (NK-1) antagonist, CP-96345, which antagonizes the binding of SP to its NK-1 receptor, results in the attenuation of colonic inflammation induced in rats by 5% dextran sodium sulfate (DSS) in drinking water for 10 days compared with an inactive enantiomer, CP-96344. Disease activity was assessed daily for 10 days, after which colonic tissue damage was scored and myeloperoxidase activity and colon and urinary 8-isoprostanes were measured. Animals receiving DSS exhibited marked physical signs of colitis by day 5 compared with controls. Chronic administration of the NK-1 antagonist significantly reduced the disease activity index, mucosal myeloperoxidase activity, colonic tissue damage score, and mucosal and urinary levels of 8-isoprostanes compared with inactive enantiomer- or vehicle-injected (saline) animals receiving DSS alone. These data indicate that the administration of an NK-1 antagonist can attenuate colonic inflammation and oxidative stress and suggest a novel therapeutic approach in the treatment of chronic ulcerative colitis.     

Effect of cinnamon essential oil on gut microbiota in the mouse model of dextran sodium sulfate-induced colitis

Increasing evidence has confirmed that the antimicrobial and anti-inflammatory effects of cinnamon essential oil (CEO) contribute to protection against inflammatory bowel disease (IBD). The dextran sodium sulfate (DSS)-induced colitis mouse model was established to investigate the correlation between the protective effects of CEO and the regulation of intestinal microflora. The symptoms of IBD were assessed by measuring the hemoglobin content, myeloperoxidase activity, histopathological observation, cytokines, and toll-like receptor (TLR4) expression. The alteration of the fecal microbiome composition was analyzed by 16S rRNA gene sequencing. The results indicated that the oral administration of CEO enriched with cinnamaldehyde effectively alleviated the development of DSS-induced colitis. In contrast to the inability of antibiotics to regulate flora imbalance, the mice fed with CEO had an improved diversity and richness of intestinal microbiota, and a modified community composition with a decrease in Helicobacter and Bacteroides and an increase in Bacteroidales_S24-7 family and short-chain fatty acids (SCFA)-producing bacteria (Alloprevotella and Lachnospiraceae_NK4A136_group). Moreover, the correlation analysis showed that TLR4 and tumor necrosis factor-α was positively correlated with Helicobacter, but inversely correlated with SCFA-producing bacteria. These findings indicated from a new perspective that the inhibitory effect of CEO on IBD was closely related to improving the intestinal flora imbalance.     

Changes in colonic mucosal permeability in mouse colitis induced with dextran sulfate sodium.

In this study we examined changes in colonic mucosal permeability induced by dextran sulfate sodium (DSS) during the acute phase of mouse colitis. To induce colitis, the mice were given drinking water containing 5% (w/v) DSS (MW = 40,000) ad libitum. Colonic mucosal permeability was evaluated by the permeation of Evans blue (EB) from the lumen into the wall of the colon on 1, 2, 3 and 7 days postadministration of DSS. Mucosal changes were also histologically examined daily for 7 days postadministration. The permeation of EB increased significantly by days 3 and 7 postadministration. Histological analysis showed that crypt loss was the initial change, with no inflammatory process and the surface mucosal epithelial cells remained morphologically intact. These histological changes developed on 2 to 3 days postadministration. Erosion was first recognized at 5 days postadministration. These findings indicated that the increase in colonic mucosal permeability may have occurred in 3 days postadministration, and the increase in mucosal permeability occurred before the appearance of the inflammatory process. This suggests that an increase in colonic mucosal permeability, leading to the destruction of mucosal barrier function, may play an important role in the induction of DSS-induced murine colitis.     

Inhibition of myeloid differentiation 1 specifically in colon with antisense oligonucleotide exacerbates dextran sodium sulfate-induced colitis

Myeloid differentiation 1 (MD-1), also known as lymphocyte antigen 86 (Ly86), is a soluble protein homologous to MD-2 and forms a complex with radioprotective 105 (RP105). RP105/MD-1 complex negatively regulates toll-like receptor 4 (TLR4) signaling and is involved in several immune disorders. However, the precise role of MD-1 in inflammatory bowel diseases (IBD) remains poorly understood. To further investigate the involvement of MD-1 in IBD, we inhibited MD-1 in colon with antisense oligonucleotide (AS-ODN) and assessed the effect of MD-1 inhibition on dextran sodium sulfate (DSS)-induced colitis. We discovered that MD-1 protein expression was remarkably decreased in both patients with ulcerative colitis and mice with DSS-induced colitis. For the first time, we showed that oral administration of MD-1 AS-ODN to mice significantly suppressed the MD-1 protein levels in colon rather than systemic tissues. Subsequently, we found that MD-1 AS-ODN treated mice were more susceptible to DSS-induced colitis based on loss of body weight, colon length, histological scores, and disease activity index. MD-1 inhibition also significantly enhanced inflammatory cytokines production such as IL-6 and IL-1β in colons. Finally, mice treated with MD-1 AS-ODN exhibited increased messenger RNA levels of TLR4 and MyD88 after DSS exposure and showed enhanced nuclear factor (NF)-κB activation compared with the control. Taken together, specifically suppression of MD-1 in colon tissues with AS-ODN exacerbates DSS-induced experimental colitis in mice, which is possibly related to activation of TLR4/NF-κB signaling.     

mTOR inhibitor INK128 attenuates dextran sodium sulfate-induced colitis by promotion of MDSCs on Treg cell expansion

Accumulating evidence has shown that mammalian target of rapamycin (mTOR) pathway and myeloid-derived suppressor cells (MDSCs) are involved in pathogenesis of inflammatory bowel diseases (IBDs). INK128 is a novel mTOR kinase inhibitor in clinical development. However, the exact roles of MDSCs and INK128 in IBD are unclear. Here, we showed that the INK128 treatment enhanced the resistance of mice to dextran sodium sulfate (DSS)–induced colitis and inhibited the differentiation of MDSCs into macrophages. Moreover, interferon (IFN)-α level was elevated in INK128-treated colitis mice. When stimulated with IFN-α in vitro, MDSCs showed a superior immunosuppression activity. Of note, the regulatory T cells (Tregs) increased but Th1 cells decreased in INK128-treated colitis mice. These results indicate that mTOR inhibitor INK128 attenuates DSS-induced colitis via Treg expansion promoted by MDSCs. Our work provides a new evidence that INK128 is potential to be a therapeutic drug on DSS-induced colitis via regulating MDSCs as well as maintaining Treg expansion.     

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.     

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.     

The deficit in folate and vitamin B12 triggers liver macrovesicular steatosis and inflammation in rats with dextran sodium sulfate-induced colitis

The risks of nonalcoholic steatohepatitis (NASH) and deficiency in vitamin B12 and folate (methyl donor deficiency, MDD) are increased in inflammatory bowel disease (IBD). We investigated the influence of MDD on NASH in rats with DSS-induced colitis. Two-month-old male Wistar rats were subjected to MDD diet and/or ingestion of DSS and compared to control animals. We studied steatosis, inflammation, fibrosis, plasma levels of metabolic markers, cytokines and lipopolysaccharide, and inflammatory pathways in liver. MDD triggered a severe macrovesicular steatosis with inflammation in DSS animals that was not observed in animals subjected to DSS or MDD only. The macrovesicular steatosis was closely correlated to folate, vitamin B12, homocysteine plasma level and liver S-adenosyl methionine/S-adenosyl homocysteine (SAM/SAH) ratio. Liver inflammation was evidenced by activation of nuclear factor kappa B (NFκB) pathway and nuclear translocation of NFκB phospho-p65. MDD worsened the increase of interleukin 1-beta (IL-1β) and abolished the increase of IL10 produced by DSS colitis. It increased monocyte chemoattractant protein 1 (MCP-1). MDD triggers liver macrovesicular steatosis and inflammation through imbalanced expression of IL-1β vs. IL10 and increase of MCP-1 in DSS colitis. Our results suggest evaluating whether IBD patients with MDD and increase of MCP-1 are at higher risk of NASH.     

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.     

The protective and healing effects of a natural antioxidant formulation based on ubiquinol and Aloe vera against dextran sulfate-induced ulcerative colitis in rats

Oxygen/nitrogen reactive species (ROS/RNS) are currently implicated in the pathogenesis of ulcerative colitis, drawing attention on the potential prophylactic and healing properties of antioxidants, scavengers, chelators. We evaluated the possible protective/curative effects of a natural antioxidant preparation based on Aloe vera and ubiquinol, against intestinal inflammation, lesions, and pathological alterations of the intestinal electrophysiological activity and motility, in a rat model of DSS-induced colitis. 5% dextrane sulfate (DDS) (3 days), followed by 1% DSS (4 days) was administered in drinking water. The antioxidant formulation (25 mg/kg) was delivered with a pre-treatment protocol, or simultaneously or post-colitis induction. Spontaneous and acetylcholine-stimulated electrical activity were impaired in the small intestine and in distal colon, upon exposure to DSS only. Severe inflammation occurred, with increased myeloperoxidase activity, and significant alterations of the oxidant/antioxidant status in colonic tissue and peritoneal cells. Lipoperoxidation, superoxide production, glutathione peroxidase and glutathione-S-transferase activities, and reduced glutathione content increased, whilst superoxide dismutase and catalase activities were sharply suppressed in colon tissue. ROS/RNS formation in peritoneal cells was strongly inhibited. Inflammation, electrical/mechanical impairment in the gut, and a great majority of oxidative stress parameters were improved substantially by pre-treatment with the antioxidant preparation, but not by simultaneous administration or post-treatment.     

The detrimental effect of nitric oxide on tissue is associated with inflammatory events in the vascular endothelium and neutrophils in mice with dextran sodium sulfate-induced colitis

Abstract

Nitric oxide (NO) is thought to be a key molecule in the progression of ulcerative colitis and experimental colitis induced by dextran sodium sulfate (DSS). However, the detrimental effect of DSS-induced NO production on the colonic mucosa is incompletely understood. Increases in the expression of adhesion molecules in the vascular endothelium and activated neutrophils (thereby releasing injurious molecules such as reactive oxygen species) are reportedly associated with the pathogenesis of DSS-induced colitis. We investigated if the detrimental effect of NO production on the colonic mucosa was attributable to the activation of neutrophil infiltration by NO in mice with DSS-induced colitis. NO₂^?/NO₃^? content in the middle and distal colon was increased on days 5 and 7, but alterations in the proximal colon were not observed. Myeloperoxidase (MPO) activity and expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) were significantly increased in the entire colon, whereas TNF-α levels were significantly increased only in the middle and distal colon on day 7. The pathology of colitis and increases in colonic MPO activity, P-selectin, ICAM-1, and TNF-α levels were suppressed by the inducible NO synthase (iNOS)-specific inhibitor aminoguanidine and NO scavenger c-PTIO, whereas all but TNF-α levels were increased by the non-specific NOS inhibitor L-NAME. These findings suggest that iNOS-derived NO increases TNF-α levels in the middle and distal colon and increased TNF-α levels induce expression of P-selectin and ICAM-1, thereby promoting the infiltration of activated neutrophils, which leads to damage to colonic tissue.