Dextran Sulfate Sodium Treatment Research Articles

Growth hormone-releasing hormone (GHRH) deficiency promotes inflammation-associated carcinogenesis

The somatotropic axis, in addition to its well-known metabolic and endocrine effects, plays a pivotal role in modulation of inflammation. Moreover, growth hormone (GH)-releasing hormone (GHRH) has been involved in the development of various human tumors. In this work we aimed to investigate the consequences of GHRH deficiency on the development of inflammation-associated colon carcinogenesis in a mouse model of isolated GH deficiency due to generalized ablation of the GHRH gene [GHRH knock out (GHRHKO)]. Homozygous GHRHKO (-/-) male mice and wild type (C57/BL6, +/+) male mice as control group, were used.After azoxymetane (AOM)/dextran sodium sulfate (DSS) treatment -/- mice displayed higher Disease Activity Index (DAI) score, and more marked weight loss compared to +/+ animals. Additionally, -/- mice showed a significant increase in total tumors, in particular of large size predominantly localized in distal colon. In colonic tissue of AOM/DSS-treated -/- mice we found the presence of invasive adenocarcinomas, dysplasia and colitis with mucosal ulceration. Conversely, AOM/DSS-treated +/+ mice showed only presence of adenomas, without invasion of sub-mucosa.Treatment with AOM/DSS significantly increased prostaglandin (PG)E2 and 8-iso-PGF2α levels along with cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) gene expression, in colon specimens. The degree of increase of all these parameters was more markedly in -/- than +/+ mice.In conclusion, generalized GHRH ablation increases colon carcinogenesis responsiveness in male mice. Whether this results from lack of GH or GHRH remains to be established.

Preventive Effects of Pyungwi-san against Dextran Sulfate Sodium- and Clostridium difficile-Induced Inflammatory Bowel Disease in Mice.

Several lines of evidence indicate that inflammatory bowel disease (IBD) is associated with Clostridium difficile (CD) infection as a consequence of gut dysbiosis. Currently available treatments of IBD are either not very effective or have adverse effects. Pyungwi-san (PWS), a traditional Chinese herbal formulation, has long been used to treat gastrointestinal disorders. The present study was conducted to investigate the efficacy of PWS against dextran sulfate sodium (DSS) + CD-induced IBD in mice. The animals received DSS in drinking water for seven days to produce DSS-induced acute colitis. In the DSS + CD group, the DSS-fed animals were orally administered with CD spores twice during the DSS treatment period. We observed that exposure of DSS + CD-treated animals to PWS significantly decreased the disease activity index; prevented the shortening of colonic length and increases in spleen size and weight; restored colonic histological parameters by significantly increasing mucus thickness, crypt depth, and goblet cell numbers; protected the tight junction proteins; improved the profiles of pro-inflammatory and anti-inflammatory cytokines; and normalized the abundance ratio of the Firmicutes/Bacteroidetes in the gut. Thus, PWS exerted a number of protective effects on DSS + CD-induced colitis, which might be mediated via restoration of a balance in gut microbial communities.

135 FH and FGSH prepared from fish skin gelatin using ginger protease alleviates DSS-induced Colitis via Nrf2 pathway

Abstract Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disease affecting the colon, and its incidence is rising worldwide. Hence, it is important to apply new strategies to cope with the IBD. We previously found that hydrolysate fractions (HF) and fish skin gelatin hydrolysate (FSGH), isolated from fish skin gelatin hydrolysate using ginger protease, could exert antioxidant effects in IPEC-J2 cells. Here, we aimed to evaluate the effects of FH and FGSH in a mouse model of dextran sodium sulfate (DSS)-induced colitis. Mouse were treated with 3% DSS in their drinking water for 7 days to induce acute colitis. FH (200 mg/kg) and FGSH (200 mg/kg) was administered for 7 days before and during DSS treatment via oral gavage once per day. Mouse were sacrificed at day 7 after colitis induction. The results showed that FH and FGSH significantly ameliorated the clinical symptoms of DSS-induced mice colitis, such as weight loss, disease activity index (DAI), colon shortening, spleen hypertrophy and histological scores. For MPO activity, an important indicator of neutrophil infiltration and acute inflammation in DSS-induced mouse colitis, FH and FGSH significantly reduced the level of DSS-induced hyperactivated MPO. Treatment with FH and FGSH also significantly inhibited the secretion proinflammatory cytokines TNF-α, IL-8 and IL-12 in DSS-induced mouse colitis. And FH and FGSH administration significantly increased expression of antioxidant enzymes GCLM, GCLC and GPX4 and maintained tight junction in colon tissues. Furthermore, we found that FH and FGSH significantly increased the level of Nrf2 in colon tissues, it indicated that FH and FGSH may alleviated colitis by Nrf2 pathway. Overall, these results suggested that FH and FGSH could be a potential promising candidate for the treatment of IBD.

Azathioprine Has a Deleterious Effect on the Bone Health of Mice with DSS-Induced Inflammatory Bowel Disease.

Patients with inflammatory bowel disease (IBD) often present poor bone health and are 40% more at risk of bone fracture. Studies have implicated autophagy in IBD pathology and drugs used to treat IBD stimulate autophagy in varying degrees, however, their effect on the skeleton is currently unknown. Here, we have utilised the dextran sulphate sodium (DSS) model of colitis in mice to examine the effects of the thiopurine drug azathioprine on the skeleton. Ten-week-old male mice (n = 6/group) received 3.0% DSS in their drinking water for four days, followed by a 14-day recovery period. Mice were treated with 10 mg/kg/day azathioprine or vehicle control. Histopathological analysis of the colon from DSS mice revealed significant increases in scores for inflammation severity, extent, and crypt damage (p < 0.05). Azathioprine provided partial protection to the colon, as reflected by a lack of significant difference in crypt damage and tissue regeneration with DSS treatment. MicroCT of vehicle-treated DSS mice revealed azathioprine treatment had a significant detrimental effect on the trabecular bone microarchitecture, independent of DSS treatment. Specifically, significant decreases were observed in bone volume/tissue volume (p < 0.01), and trabecular number (p < 0.05), with a concurrent significant increase in trabecular pattern factor (p < 0.01). Immunohistochemical labelling for LC3 revealed azathioprine to induce autophagy in the bone marrow. Together these data suggest that azathioprine treatment may have a deleterious effect on IBD patients who may already be at increased risk of osteoporotic bone fractures and thus will inform on future treatment strategies for patient stratification.

Retinoid acid induced 16 deficiency aggravates colitis and colitis-associated tumorigenesis in mice

Inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC) is a serious health issue, but etiopathological factors remain unclear. Although some studies reported the roles of Retinoid acid induced 16 (RAI16) in the tumorigenesis of hepatocellular carcinoma and PKA signaling, the roles of RAI16 in IBD and CRC are undressed. RAI16−/− mice were generated and the roles of RAI16 were addressed in dextran sodium sulfate (DSS) or azoxymethane (AOM)-DSS induced IBD or CAC mouse models, respectively. At first, RAI16−/− mice were viable, fertile with no apparent defects. Then, it was found that RAI16−/− mice were more susceptibility to colitis induced by DSS than wild type (WT) littermates, which was evaluated by disease activity index and histological score. Furthermore, the expressions of tissues repair associated molecules Cox2, Ereg and MMP-10 were significantly decreased in RAI16−/− colon under DSS treatment. Gut barrier related genes including antimicrobial peptides Reg3b and Reg3g and intestinal mucus genes Muc4, Muc6 and Muc20 were reduced in RAI16−/− colon. These findings indicated that RAI16 may function to affect genes involved in intestinal barrier function and immunoprotective inflammation. Accordingly, RAI16−/− mice displayed significantly increased tumor burden compared with WT mice assessed in CAC model induced by AOM/DSS. Much more Ki67 + nuclei were observed in RAI16−/− tumors suggesting RAI16 to be critical in colonic cell proliferation during tumorigenesis. Conclusively, we demonstrate the roles of RAI16 in colonic inflammation and inflammation-associated tumorigenesis by using a novel RAI16−/− mouse model for the first time.

Inflammation-induced colon cancer in uPA-deficient mice is associated with a deregulated expression of Notch signaling pathway components.

Notch is an evolutionarily conserved signaling pathway with an important role in development and cell fate determination. Deregulation of Notch signaling has been associated with several pathological conditions, including cancer. Acting as an oncogene in some types of cancers and as a tumor suppressor in other, Notch effects seem to be highly context-dependent in solid tumors. In the present study, we aimed to investigate gene expression levels of Notch pathway constituents, including ligands, receptors, and target genes, during the early stages of inflammation-associated intestinal carcinogenesis. To achieve so, we used our recently developed mouse model, in which colon cancer arises in the absence of urokinase-type plasminogen activator (uPA) due to colitis induced by dextran sodium sulfate (DSS) treatment. Among the cell surface components, ligands Jag1/Jag2 and receptors Notch1/Notch2 were found to be significantly upregulated in the uPA-deficient protumorigenic inflammatory microenvironment. Moreover, several intracellular Notch modulators, i.e. Hes1, Hey1, and Klf4, were also shown to be deregulated with inflammation, yet irrespective of uPA status. Sox9 transcription factor, however, was significantly downregulated in the uPA-deficient/DSS-treated mice that developed colon adenomas as compared to the wild-type/DSS-treated group with no neoplasia identified. The latter finding supports a tumor suppressive role of Sox9 in intestinal carcinogenesis. Our results point towards an early activation of Notch signaling pathway at the receptor-ligand level in inflammation-associated colon neoplasmatogenesis developed in the absence of uPA. Interestingly, such activation may not be accompanied by deregulation of downstream Notch-target genes, possibly due to the effects of other inter-related signaling pathways.

An orally administered butyrate-releasing xylan derivative reduces inflammation in dextran sulphate sodium-induced murine colitis

Butyrate has been shown to be effective in ulcerative colitis (UC). However, its oral administration is rare due to its rancid odour and unpleasant taste. In this study, the effect of a butyrate-releasing polysaccharide derivative, xylan butyrate ester (XylB), was evaluated in a dextran sodium sulphate (DSS)-induced UC model in C57BL/6 mice. Linear xylan was extracted from corn cobs. The C-2 and C-3 positions of the linear xylan were esterified with butyrate, forming XylB. The protective and therapeutic effects of XylB against UC were determined in a DSS-induced mouse model. The results showed that XylB treatments reversed the imbalance between pro- and anti-inflammatory cytokines. Moreover, XylB rebalanced the gut microbiota that interfered with DSS treatment and significantly decreased the relative abundance of the genera Oscillibacter, Ruminococcaceae UCG-009, Erysipelatoclostridium, and Defluviitaleaceae UCG-01. XylB increased butyrate content in the colon, upregulated G-protein coupled receptor 109A protein expression, inhibited histone deacetylase (HDAC) activity, and exerted anti-inflammatory activity through autophagy pathway activation and nuclear factor-κB (NF-κB) inhibition. XylB reduces inflammatory intestinal damage in mice, suggesting that it would be a potential drug for the treatment of UC and could be used to overcome the limitations of the oral administration of sodium butyrate.

Chitosan Ameliorates DSS-Induced Ulcerative Colitis Mice by Enhancing Intestinal Barrier Function and Improving Microflora.

Ulcerative colitis (UC) has been identified as one of the inflammatory diseases. Intestinal mucosal barrier function and microflora play major roles in UC. Modified-chitosan products have been consumed as effective and safe drugs to treat UC. The present work aimed to investigate the effect of chitosan (CS) on intestinal microflora and intestinal barrier function in dextran sulfate sodium (DSS)-induced UC mice and to explore the underlying mechanisms. KM (Kunming) mice received water/CS (250, 150 mg/kg) for 5 days, and then received 3% DSS for 5 days to induce UC. Subsequently, CS (250, 150 mg/kg) was administered daily for 5 days. Clinical signs, body weight, colon length, and histological changes were recorded. Alterations of intestinal microflora were analyzed by PCR-DGGE, expressions of TNF-α and tight junction proteins were detected by Western blotting. CS showed a significant effect against UC by the increased body weight and colon length, decreased DAI (disease activity index) and histological injury scores, and alleviated histopathological changes. CS reduced the expression of TNF-α, promoted the expressions of tight junction proteins such as claudin-1, occludin, and ZO-1 to maintain the intestinal mucosal barrier function for attenuating UC in mice. Furthermore, Parabacteroides, Blautia, Lactobacillus, and Prevotella were dominant organisms in the intestinal tract. Blautia and Lactobacillus decreased with DSS treatment, but increased obviously with CS treatment. This is the first time that the effect of original CS against UC in mice has been reported and it is through promoting dominant intestinal microflora such as Blautia, mitigating intestinal microflora dysbiosis, and regulating the expressions of TNF-α, claudin-1, occludin, and ZO-1. CS can be developed as an effective food and health care product for the prevention and treatment of UC.

Hypothalamic paraventricular nucleus neurons activated by estrogen GPER1 receptors promote anti-inflammation effects in the early stage of colitis.

The hypothalamus-pituitary-adrenal (HPA) axis is known to mediate gut-brain interaction, and the pathological inflammatory process in the intestine can induce HPA axis involved 'fight or flight' response to suppress or facilitate intestinal inflammation. Hypothalamic paraventricular nucleus (PVN) neurons are responsible for controlling the HPA axis activity, but their exact role in modulating intestinal inflammation remains unclear. In this study, we used the dextran sulfate sodium (DSS)-induced mice colitis model, gene editing, and RNA interference to determine the effects of PVN neurons on intestinal inflammation. We found that at the early stage (third day) after DSS treatment, there was a mild inflammation in the colorectal area and an increased neuron activation in the PVN but not in the adjacent area. At the same time, ~80% of activated PVN neurons also expressed novel estrogen GPER1 receptor. The colitis noticeably worsened in GPER1-knockout mice and local PVN GPER1-knockdown mice. These results indicated that PVN GPER1 positive neurons potentially have a protective function during the early stages of DSS-induced colitis, and this may be a mechanism by which the central nervous system attempts to suppress intestinal inflammation to achieve self-protection.

Wheel running behaviour in group-housed female mice indicates disturbed wellbeing due to DSS colitis.

Voluntary wheel running (VWR) behaviour is a sensitive indicator of disturbed wellbeing and used for the assessment of individual experimental severity levels in laboratory mice. However, monitoring individual VWR performance usually requires single housing, which itself might have a negative effect on wellbeing. In consideration of the 3Rs principle, VWR behaviour was evaluated under group-housing conditions. To test the applicability for severity assessment, this readout was evaluated in a dextran sodium sulphate (DSS) induced colitis model. For continuous monitoring, an automated system with integrated radio-frequency identification technology was used, enabling detection of individual VWR. After a 14-day adaptation period mice demonstrated a stable running performance. Analysis during DSS treatment in combination with repeated facial vein phlebotomy and faecal sampling procedure resulted in significantly reduced VWR behaviour during the course of colitis and increased VWR during disease recovery. Mice submitted to phlebotomy and faecal sampling but no DSS treatment showed less reduced VWR but a longer-lasting recovery. Application of a cluster model discriminating individual severity levels based on VWR and body weight data revealed the highest severity level in most of the DSS-treated mice on day 7, but a considerable number of control mice also showed elevated severity levels due to sampling procedures alone. In summary, VWR sensitively indicated the course of DSS colitis severity and the impact of sample collection. Therefore, monitoring of VWR is a suitable method for the detection of disturbed wellbeing due to DSS colitis and sampling procedure in group-housed female laboratory mice.

Effects of kampo medicine keishikashakuyakuto on large intestine disease in mice

ABSTRACTAimThe aim of this study was to examine the effects of keishikashakuyakuto (KST) on large intestine diarrhea and inflammation in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) mouse models.MethodsThis study examined the effects of KST on diarrhea and inflammatory bowel syndrome using 5‐hydroxytryptamine (5‐HT)‐induced diarrhea and 3% dextran sulfate sodium (DSS)‐treated colitis models.ResultsKeishikashakuyakuto (200 and/or 500 mg/kg) and ramosetron (5 μg/kg) prolonged the onset‐time of 5‐HT‐induced diarrhea in mice. KST and ramosetron prevented diarrhea and the stool score was decreased 60 min after 5‐HT treatment. The disease activity index in the 3% DSS‐treated mice was lower after oral KST than in mice given 3% DSS only. Furthermore, KST inhibited the decreases in red cells and platelets, and in hemoglobin and hematocrit that were observed in DSS‐treated mice. On colon histology using hematoxylin–eosin and direct fast scarlet staining, KST prevented the mucosa membrane ulceration and eosinophil infiltration of the mucosa membrane caused by DSS treatment. The increase in colonic monocyte chemoattractant protein 1, interleukin‐1β and tumor necrosis factor‐α in DSS‐treated mice was reduced by oral KST.ConclusionKeishikashakuyakuto may be effective for IBS and IBD.

Effects of Exposure to Mild Hyperbaric Oxygen on DSS-Induced Colonic Inflammation and Diarrhea in Rats.

PurposeIn rodents, dextran sulfate sodium (DSS)-induced diarrhea and colonic inflammation have similar symptoms to those of ulcerative colitis in humans. We examined the effects of exposure to mild hyperbaric oxygen (MHO) at an atmospheric pressure of 1317 hPa with 40% oxygen on DSS-induced diarrhea and colonic inflammation in rats.MethodsFive-week-old male Kyoto Apc Delta (KAD) rats (n = 12) were administered 2% DSS through drinking water for 1 week. Subsequently, DSS-treated male rats were not subjected to any further treatment (n = 6) or exposed to MHO (n = 6) for 2 weeks. Age-matched KAD rats not subjected to DSS treatment or exposed to MHO were used as the control group (n = 6).ResultsControl rats did not exhibit diarrhea and colonic inflammation. However, DSS-treated rats exhibited diarrhea and colonic inflammation, regardless of exposure to MHO. Exposure to MHO for 2 weeks led to decreased incidence of diarrhea in DSS-treated rats (p < 0.05). Exposure to MHO had no effect on colonic inflammation in DSS-treated rats (p = 0.12).ConclusionExposure to MHO for 2 weeks can improve diarrhea but cannot attenuate colonic inflammation, possibly due to the short exposure duration (2 weeks) used in this study.

Intestinal proteomic analysis of a novel non-human primate model of experimental colitis reveals signatures of mitochondrial and metabolic dysfunction.

Animal models recapitulating features of chronic colitis, such as ulcerative colitis, Crohn's disease, or HIV infection, are critical to study disease pathogenesis and test novel therapeutics. In this study, we used a proteomics approach to explore the molecular intestinal response in two rhesus macaque (RM) animal models of experimentally induced colitis using dextran sulfate sodium (DSS) and simian immunodeficiency virus (SIV) infection. Proteomic analysis detected more than 2500 proteins in colonic tissue collected from 30 RMs. Differential protein expression analysis revealed a protein expression pattern in DSS-treated RMs resembling the proteome of human ulcerative colitis. In a group of 12 DSS-treated RMs compared to 6 with no treatment, decrease in expression of proteins related to mitochondrial energy metabolism, including fatty acid metabolism was noted, while innate immune activation pathways, including complement and coagulation proteins were upregulated. SIV infection of RMs resulted in increased innate immune responses related to viral defense. Proteomic signatures of barrier damage were apparent in both DSS treatment or SIV infection. These results demonstrate that DSS treatment in a non-human primate model resembles features of human ulcerative colitis, making this a promising tool to study important immunological mechanisms in inflammatory bowel disease.

Jasonia glutinosa (L.) DC., a traditional herbal medicine, reduces inflammation, oxidative stress and protects the intestinal barrier in a murine model of colitis.

Jasonia glutinosa (L.) DC., known as rock tea (RT), is traditionally used in Spain as a digestive due to its beneficial properties in bowel disorders. The pharmacological nature of these properties has not been established yet. The aim of this work was to evaluate the therapeutic utility of RT in experimental colitis and to identify chemical constituents with anti-inflammatory and/or anti-oxidative properties. RT extract was prepared with ethanol in a Soxhlet apparatus and analysed by HPLC-DAD. Superoxide radical scavenging properties, xanthine oxidase and lipoxygenase (5-LOX) inhibitory activity, and capability to lower nitric oxide (NO) and tumor necrosis factor α (TNF-α) levels were measured in cell-free and cell-based assays. In the 2.5%-dextran-sodium sulphate (DSS) injury-repair model of ulcerative colitis (UC), mice were daily treated with sulfasalazine (SSZ, as reference drug, 100mg/kg bw), RT (5, 25 and 50mg/kg bw, p.o.), or vehicle over 20 days. Colitis was scored daily. Colon samples were examined macroscopically and histopathologically. Protein levels of myeloperoxidase (MPO), interleukins 6, and 10 (IL-6, IL-10), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied as markers of oxidative stress and inflammatory activity. The integrity of the apical epithelial layer was assessed by immunofluorescence staining of zonula ocludens-1 (ZO-1). Finally, intestinal contractility was also evaluated by isometric myography. Fifteen phenolic compounds and three pigments were identified and quantified, of which caffeoylquinic acids, and the flavonoid, quercetin-3-O-galactoside, were the most abundant. RT extract significantly scavenged superoxide radicals, inhibited 5-LOX activity, and lowered NO and TNF-α levels. DSS-treated mice receiving RT scored clinically lower than controls during the first 3 days of DSS treatment and during the recovery period. SSZ was less effective than RT. Anatomical and histological examination of colon samples revealed that RT significantly prevented colon shortening, increased colon thickness, and lowered the macroscopic damage score. RT also significantly prevented the increase of MPO activity, IL-6 levels, iNOS and COX-2 expression, the loss of ZO-1 apical expression, and normalized contractility disturbances. In conclusion, daily administration of RT showed therapeutic properties in the DSS-model of UC. The benefits of RT can likely be attributed to its anti-inflammatory and antioxidant phenolic and flavonoid constituents.

Increased pain and inflammatory sensitivity in growth hormone-releasing hormone (GHRH) knockout mice

Growth hormone (GH) and GH-releasing hormone (GHRH), in addition to metabolic and endocrine effects, play a role in the modulation of pain and inflammation. We aimed to elucidate the consequences of GHRH deficiency on acute nociceptive stimulation and on both acute and chronic inflammatory stimuli in a mouse model of GH deficiency. Mice with generalized ablation of the GHRH gene (GHRH knock out, GHRHKO, -/-) were compared to wild type (GHRH +/+) mice.Responsiveness to acute nociceptive stimulation and to acute inflammatory stimulation was evaluated by conventional hot plate apparatus and formalin test, respectively. We also evaluated responsiveness to colonic inflammation induced both in vivo, after dextran sodium sulfate (DSS) treatment, or ex vivo, by incubating colon segments with bacterial lipopolysaccaride (LPS). Macroscopical and histological examinations were performed, prostaglandin (PG) E2 and 8-iso-PGF2α levels and cyclooxigenase (COX)-2 and tumor necrosis factor (TNF)-α gene expression were measured.Compared to controls, -/- mice showed decreased response latency during the hot plate test, and increased licking/biting time in formalin test, particularly in the second phase of inflammation. DSS treated -/- mice showed a significant increase of colonic inflammation compared to controls. Moreover DSS treatment increased PGE2 and 8-iso-PGF2α levels, along with COX-2 and TNF-α gene expression more markedly in colon specimens of -/- mice compared to controls. LPS-induced PGE2 and 8-iso-PGF2α production from colonic segments incubated ex vivo was also increased in -/- mice.Generalized GHRH gene ablation increases sensitivity to thermal pain and both acute and persistent inflammatory stimuli in male mice.

The Ussing chamber system for measuring intestinal permeability in health and disease

BackgroundThe relationship between intestinal epithelial integrity and the development of intestinal disease is of increasing interest. A reduction in mucosal integrity has been associated with ulcerative colitis, Crohn’s disease and potentially could have links with colorectal cancer development. The Ussing chamber system can be utilised as a valuable tool for measuring gut integrity. Here we describe step-by-step methodology required to measure intestinal permeability of both mouse and human colonic tissue samples ex vivo, using the latest equipment and software. This system can be modified to accommodate other tissues.MethodsAn Ussing chamber was constructed and adapted to support both mouse and human tissue to measure intestinal permeability, using paracellular flux and electrical measurements. Two mouse models of intestinal inflammation (dextran sodium sulphate treatment and T regulatory cell depletion using C57BL/6-FoxP3DTR mice) were used to validate the system along with human colonic biopsy samples.ResultsDistinct regional differences in permeability were consistently identified within mouse and healthy human colon. In particular, mice showed increased permeability in the mid colonic region. In humans the left colon is more permeable than the right. Furthermore, inflammatory conditions induced chemically or due to autoimmunity reduced intestinal integrity, validating the use of the system.ConclusionsThe Ussing chamber has been used for many years to measure barrier function. However, a clear and informative methods paper describing the setup of modern equipment and step-by-step procedure to measure mouse and human intestinal permeability isn’t available. The Ussing chamber system methodology we describe provides such detail to guide investigation of gut integrity.

Mitigation of Colonic Inflammation in Dextran Sulfate Sodium-treated Mice by Dietary Supplementation with Soybean Fiber (P06-079-19)

ObjectivesSoybean fiber (SBF) is an insoluble, but highly fermentable dietary fiber. Previous in vitro fermentation studies with human feces have shown that SBF produces 1.5–8 times more acetate, propionate, and butyrate than oat bran, corn bran, or wheat bran fiber. Short chain fatty acids (SCFA) have been reported to play a key role in maintaining colon health and reducing inflammation. The impact of dietary SBF on colonic inflammation has not previously been examined. Our objective was to determine the anti-inflammatory efficacy of dietary supplementation with SBF in a mouse model of acute colonic inflammation. MethodsMale C57BL/6 J mice (5 weeks old) were randomized to AIN93G diet (CTL) or diet where 40% of the fiber was replaced with SBF (SBF-Hi). After 2 weeks of pretreatment, mice were given 2% dextran sulfate sodium (DSS) as the sole source of drinking fluid for 1 week to induce colonic inflammation. During DSS treatment, mice were maintained on their diet treatments. After DSS treatment, mice were euthanized and colonic inflammation was assessed. ResultsDSS-treated mice had significantly larger spleens and shorter colons than mice treated with water. SBF-Hi mitigated DSS-induced increases in spleen weight (20% lower) and colon shortening (15% longer). Quantitative, reverse transcriptase PCR analysis showed that DSS-treatment increased colonic mRNA expression of interleukin-6 (Il6) and tumor necrosis factor-a (Tnfa) by 3-fold compared to water-treated mice. Dietary supplementation with SBF blunted these increases in Il6 and Tnfa by 87% and 71%, respectively. ConclusionsOur results suggest that dietary supplementation with SBF may be a useful approach to mitigate colonic inflammation. On-going studies are focused on determining fecal levels of SCFA and measuring protein markers of inflammation and gut barrier function. Future studies are needed to evaluate whether the protective effects observed in this study are maintained in situations of more chronic colonic inflammation. Funding SourcesThis work was funded by a grant from the Pennsylvania Soybean Board and by USDA National Institute of Food and Agriculture Appropriations under Project PEN04565.

Ameliorative Effect of High-Dose Vitamin C Administration on Dextran Sulfate Sodium-Induced Colitis Mouse Model.

Vitamin C is a natural nutrient with antioxidant properties and is used as a health supplement. In this study, we examined the effects of intraperitoneal administration of high-dose vitamin C (4 g/kg) on dextran sodium sulfate (DSS)-induced ulcerative colitis. We prepared a mouse ulcerative colitis model by administering DSS for 7 d along with high-dose vitamin C each day during DSS treatment. Ulcerative colitis induced by DSS was ameliorated by high-dose vitamin C administration. Blood levels of interleukin-6, tumor necrosis factor-α, hydrogen peroxide (H2O2), and iron were elevated in DSS-treated mice but lowered by high-dose vitamin C administration. Contrarily, the levels of H2O2 and iron and the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells in the colon were further increased by high-dose vitamin C administration. The expression levels of fibroblasts, collagen type I, and collagen type III decreased in the DSS-treated mice but increased in mice administered high-dose vitamin C. These results suggest that high-dose vitamin C administration can improve ulcerative colitis.

A Grape Powder Attenuates Colonic Inflammation, Inhibits Colitis-associated Colorectal Cancer Development and Causes Favorable Changes of Gut Microbiota in Mice (OR04-07-19)

ObjectivesRecent discoveries suggest that gut microbiota is involved in the progression of colitis-associated colorectal cancer (CAC) and natural products like polyphenols can modulate gut microbiota. Polyphenol components of grape like resveratrol have been shown to have anti-colorectal cancer effects in animal models, but the underlying mechanism is not completely understood. The objective of this study is to examine the chemo-preventive effect of a whole grape powder (GP) on tumorigenesis in a mouse CAC model and evaluated the impact of GP on gut microbiota as a potential anti-CAC mechanism. To dissect the role of polyphenols in the GP, we compared GP at 3 and 10% diet to calorie, fiber, sugar and organic acid-matched placebo. MethodsWe used male Balb/c mice and divided them into diseased groups treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) and healthy groups, both of which had placebo control diet, GP at 3% or 10% diet. To induce tumorigenesis, we injected AOM at 9.5 mg/kg bw at 6 weeks of age, followed by 2-cycle DSS (1.5% in drinking water). During the study, we monitored animals’ body weight and food consumption weekly, and evaluated their colitis symptoms during DSS treatments. All animals were sacrificed at 16 weeks of age and 24-hr accumulative fecal samples were collected prior to sacrifice for gut microbial analysis. ResultsCompared to the control diet, 10% GP diet alleviated colitis symptoms including rectal bleeding and diarrhea, and reduced total tumor multiplicity by 29% (P < 0.05). GP diet increased microbial alpha-diversity and significantly shifted the gut microbial composition in both healthy and diseased groups. Under both conditions, 10% GP diet increased the abundance of various taxa belonging to Lachnospiraceae family. Canonical correspondence analysis (CCA) of gut microbiota indicated that increased GP supplementation was associated with healthier animal status. In particular, we observed that the predicted functional profile of gut microbiota from diseased mice with 10% GP diet was similar to those from healthy mice with the control diet. Conclusions10% GP diet showed CAC chemo-preventive effects and modulated gut microbiota under both healthy and diseased conditions, and appeared to prevent CAC-associated gut microbiota changes. Funding SourcesCalifornia Table Grape Commission.

Probiotic Mixture VSL#3 Alleviates Dextran Sulfate Sodium-induced Colitis in Mice by Downregulating T Follicular Helper Cells.

Clinical trials have shown beneficial effects of probiotics on inflammatory bowel diseases (IBD), although the exact mechanism remains unknown. VSL#3, a mixture of 8 probiotic bacteria, has been confirmed to have adjunctive therapeutic effects on colitis. T follicular helper (Tfh) cells, a new separate subset of CD4+ T helper cells, have been proved to play a vital role in autoimmunity. The present study aimed to identify the beneficial effect of the probiotic mixture VSL#3 on the mouse model of colitis by regulating Tfh cells. Dextran sulfate sodium (DSS) was used to induce chronic colitis in C57BL/6 mice. VSL#3 (3×109 live bacteria) was given to C57BL/6 mice every other day for 60 days by gavage. The disease activity index (DAI), histological activity index (HAI), colon length and myeloperoxidase (MPO) activity were detected. Immunofluorescence was used to visualize the location of Tfh cells. Immunoglobulins, Tfh cells and plasma cells were quantified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, real-time PCR or Western blotting. The results showed that after DSS treatment, the humoral immunity was disordered in C57BL/6 mice, with increased IgM, IgG and IgA levels in colonic mucus and increased Tfh cells in mesenteric lymph nodes (MLN). VSL#3 treatment showed anti-inflammatory effects as evidenced by reduced DAI score, HAI score and MPO activity. IgM, IgG and IgA levels were significantly reduced in colon mucus, and the number of Tfh cells was markedly decreased in MLN after VSL#3 treatment. It was concluded that VSL#3 alleviates DSS-induced colitis by downregulating Tfh cells, and Tfh cells may become a potential therapeutic target for IBD.