Administration Of Dinitrobenzene Sulfonic Acid Research Articles

Enterococcus faecalis EF-2001 protects DNBS-induced inflammatory bowel disease in mice model.

Recent studies have demonstrated the immunomodulatory effects of heat-killed lactic acid bacteria. The aim of this study was to evaluate the protective effect of heat-killed Enterococcus faecalis EF-2001 (EF-2001) on a model of inflammatory bowel disease (IBD). A total of 28 female NC/Nga mice were divided into 4 treatment groups. Controls were fed a normal commercial diet. In the experimental groups, colitis was induced by rectal administration of dinitrobenzene sulfonic acid. Two groups were orally administered 2 and 17 mg/kg EF-2001, respectively. EF-2001 treatment decreased the expression of several cytokines, including cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, interleukin (IL)-1β, and IL-6 in inflamed colon compared to the DNBS alone group. In addition, EF-2001 suppressed DNBS-induced colonic tissue destruction. Therefore, this study strongly suggests that EF-2001 could alleviate the inflammation associated with mouse IBD.

A Versatile New Model of Chemically Induced Chronic Colitis Using an Outbred Murine Strain.

Murine colitis models are crucial tools for understanding intestinal homeostasis and inflammation. However, most current models utilize a highly inbred strain of mice, and often only one sex is employed to limit bias. This targeted approach, which in itself is biased, means that murine genetic diversity and sex-related differences are ignored, making it even more difficult to extend findings to humans, who are highly heterogeneous. Furthermore, most models do not examine the chronic form of colitis, an important fact taking into account the chronic nature of the inflammatory bowel diseases (IBD). Here, we attempted to create a more realistic murine colitis model by addressing these three issues. Using chemically induced chronic colon inflammation in an outbred strain of mice (RjOrl:SWISS [CD-1]), we (i) mimicked the relapsing nature of the disease, (ii) better represented normal genetic variability, and (iii) employed both female and male mice. Colitis was induced by intrarectal administration of dinitrobenzene sulfonic acid (DNBS). After a recovery period and 3 days before the mice were euthanized, colitis was reactivated by a second administration of DNBS. Protocol length was 24 days. Colitis severity was assessed using body mass, macroscopic scores, and histological scores. Myeloperoxidase (MPO) activity, cytokine levels, and lymphocyte populations were also characterized. Our results show that the intrarectal administration of DNBS effectively causes colitis in both female and male CD-1 mice in a dose-dependent manner, as reflected by loss of body mass, macroscopic scores and histological scores. Furthermore, colon cytokine levels and mesenteric lymph node characteristics indicate that this model involves immune system activation. Although some variables were sex-specific, most of the results support including both females and males in the model. Our ultimate goal is to make this model available to researchers for testing candidate anti-inflammatory agents, such as classical or next-generation probiotics; we also aim for the results to be more easily transferrable to human trials.

Adelmidrol, a Palmitoylethanolamide Analogue, as a New Pharmacological Treatment for the Management of Inflammatory Bowel Disease.

Leukocyte infiltration, improved levels of intercellular adhesion molecule 1 (ICAM-1), and oxidative stress in the colon are the principal factors in inflammatory bowel disease. The goal of the current study was to explore the effects of adelmidrol, an analog of the anti-inflammatory fatty acid amide signaling molecule palmitoylethanolamide, in mice subjected to experimental colitis. Additionally, to clarify whether the protective action of adelmidrol is dependent on the activation of peroxisome proliferator-activated receptors (PPARs), we investigated the effects of a PPARγ antagonist, GW9662, on adelmidrol action. Adelmidrol (10 mg/kg daily, o.s.) was tested in a murine experimental model of colitis induced by intracolonic administration of dinitrobenzene sulfonic acid. Nuclear factor-κB translocation, cyclooxygenase-2, and phosphoextracellular signal-regulated kinase, as well as tumor necrosis factor-α and interleukin-1β, were significantly increased in colon tissues after dinitrobenzene sulfonic acid administration. Immunohistochemical staining for ICAM-1, P-selectin, nitrotyrosine, and poly(ADP)ribose showed a positive staining in the inflamed colon. Treatment with adelmidrol decreased diarrhea, body weight loss, and myeloperoxidase activity. Adelmidrol treatment, moreover, reduced nuclear factor-κB translocation, cyclooxygenase-2, and phosphoextracellular signal-regulated kinase expression; proinflammatory cytokine release; and the incidence of nitrotyrosine and poly(ADP)ribose in the colon. It also decreased the upregulation of ICAM-1 and P-selectin. Adelmidrol treatment produced a reduction of Bax and an intensification of Bcl-2 expression. This study clearly demonstrates that adelmidrol exerts important anti-inflammatory effects that are partly dependent on PPARγ, suggesting that this molecule may represent a new pharmacologic approach for inflammatory bowel disease treatment.

Strain Specific Persistent Suppression of Visceral Pain Perception After Recovery From a Bout of Colitis

Visceral pain is often experienced by patients with GI inflammation, and some studies suggest that such pain can persist after resolution of inflammation. Colitis was induced in male Wistar and Sprague‐Dawley (SD) rats via intrarectal administration of dinitrobenzene sulfonic acid (DNBS) in 50% ethanol, controls received saline only. At day 15, under general anesthesia, a balloon was inserted into the colon and distended (colo‐rectal distention; CRD), heart rate was recorded continuously. Cardioautonomic responses to CRD were analyzed and expressed as % change from the pre‐CRD heart rate. Colonic injury and inflammation were examined histologically and by measurement of tissue myeloperoxidase activity. By 15 days following DNBS administration, colitis had resolved. CRD caused a significant visceral pain response (bradycardia) in both strains of control rats. However, Wistar rats showed a significant suppression of pain response to CRD, whereas the response in SD rats remained the same after colitis. There was no difference in colonic compliance between control and DNBS groups or the two strains. Our results provide evidence of a strain‐dependent alteration in the pain response to CRD that persists after resolution of colitis, likely due to changes in autonomic sensory function induced by inflammation. Our results are consistent with the variability in pain perception seen in patients with GI inflammation.

P8 Hydrogen sulfide production during colonic inflammation occurs primarily via a pyridoxal-5′-phosphate (P5P)-independent pathway

Hydrogen sulfide (H 2 S) is an important endogenous anti-inflammatory mediator, which has been shown to promote resolution of colitis and healing of ulcers. H 2 S synthesis was believed to occur primarily via two P5P-dependent enzymes (cystathionine- β -synthase (CBS) and cystathionine- γ -lyase (CSE)). In the present study, we examined the possible contribution of a 3 rd pathway for H 2 S synthesis, in the healthy and inflamed colon, which involves the enzymes cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). Methods The capacity of tissue to produce H 2 S was measured from homogenized tissue in the presence of exogenous substrate and/or inhibitors using a zinc-trapping assay. Production of H 2 S via the CAT-3MST pathway was explored using the substrate α -ketoglutarate and the CAT inhibitors l -aspartate and O -carboxymethyl-hydroxylamine hemihydrochloride (CHH). Colitis was induced in rats by intracolonic administration of dinitrobenzene sulfonic acid (DNBS). Rats were euthanized at different time points up to 28 days post-DNBS for measurement of H 2 S synthesis. To assess the contributions of different colonic layers to total colonic H 2 S synthesis, healthy and inflamed colonic sections collected on day 3 after DNBS-instillation were dissected to separate mucosal and smooth muscle layers. Diseased colonic sections were further dissected at the edges of the ulcer yielding distinct ulcerated mucosal and smooth muscle layers. Results Colonic H 2 S synthesis via the CAT-3MST pathway was 5-fold greater than that produced via the previously studied pathways (CBS and CSE) (225.2 ± 58.9 nmol/g/h vs. 41.8 ± 21 nmol/g/h respectively; p 2 S synthesis via CAT-3MST markedly increased (>4-fold) compared to controls (963.6 ± 115.0 nmol/g/h vs. 225.2 ± 58.9 nmol/g/h, respectively; p 2 S synthesis were observed when inflammation was most robust (3 days post DNBS) and were suppressed by both l -aspartate and CHH (1132.3 ± 87.7 nmol/g/h control vs. 424.2 ± 153.4 nmol/g/h; p p 2 S synthesis decreased to control levels. Furthermore, the ability of the ulcerated mucosa to produce H 2 S via the CAT-3MST pathway was markedly increased when compared to smooth muscle layers (both healthy and ulcerated) and the mucosa from healthy colons. The capacity of the ulcerated mucosa to produce H 2 S via the CBS/CSE pathways also increased significantly when compared to smooth muscle sections (both healthy and ulcerated) and healthy mucosal layers. Conclusions These data suggest that in both the healthy and inflamed colon CAT and 3MST represent the primary pathway for H 2 S synthesis. The increased production of H 2 S via the CAT-3MST enzymes appears to be occurring at the site of mucosal ulceration. Interestingly, the CBS/CSE pathways also make important contributions to H 2 S synthesis at the ulcerated mucosa. Further understanding of the pathways for H 2 S production in the colon can present new opportunities for modulating colonic H 2 S production, which may have important implications in the development of novel therapies for inflammatory bowel disease.

Influence of Interleukin-13 in 5-HT Synthesis and Severity of Experimental Colitis

BACKGROUND: Mucosal inflammation in conditions ranging from infective acute enteritis or colitis to inflammatory bowel disease is accompanied by alteration in serotonin (5hydroxytryptamine, 5-HT) content in the gut. In our previous studies, we have shown that 5-HT plays an important role in the pathogenesis of experimental colitis. Dendritic cells (DC) are important innate immune cells and play a key role in the activation of the immune response and generation of gut inflammation. DCs express the 5-HT7 receptor, and we have recently demonstrated that 5-HT plays an important role in DC activation in relation to gut inflammation. In this study, we have evaluated the effect of inhibiting 5-HT signaling by using a 5-HT7 receptor antagonist or 5-HT7 receptor knockout mice in experimental colitis. METHODS: Mice (C57BL/6) were treated with selective 5-HT7 receptor antagonist SB266970 (40mg/kg, ip) or vehicle (saline) starting one day prior to administration of 5% dextran sulfate sodium (DSS) in drinking water and were sacrificed on day 5 post-DSS to assess the extent of colitis, myeloperoxidase (MPO) activity and pro-inflammatory cytokines. Colitis was also induced in 5-HT7 receptor deficient mice on C57BL/6 background (5HT7KO) and wild type mice either by 5% DSS or intrarectal administration of dinitrobenzene sulfonic acid (DNBS, 5mg/mouse) and were sacrificed on day 5 post-DSS and day 3 postDNBS to assess the extent of colitis, MPO activity and pro-inflammatory cytokines. RESULTS: Inhibition of 5-HT signaling by blocking 5-HT7 receptor function with a 5-HT7 antagonist ameliorated DSS-induced colitis. Compared to saline treated mice, we observed significantly lower macroscopic (4.0±0.28 vs. 2.6±0.43) and histological damage scores (4.8±0.59 vs. 2.4±0.54) in antagonist treated mice on day 5 post-DSS. This was associated with downregulation of MPO activity (16.9±0.88 vs. 12.32±1.21, P<0.05) and lower production of IL-1β, TNF-α and IL-6 (P<0.05) post-DSS. There were also significantly lower histological scores, MPO and pro-inflammatory cytokine levels in 5-HT7KOmice as compared to controls post-DSS. DCs isolated from 5-HT7KO mice post-DSS produced lower levels of IL-12 and IL-1β when stimulated with LPS as compared to wild-type mice (P<0.05). We also observed significant reduction in severity of colitis in 5-HT7KO mice following induction of DNBScolitis. CONCLUSION: These results demonstrate that disruption of 5-HT7 receptor function reduces the severity of gut inflammation and identifies a key role of 5-HT7 receptor mediated signaling in the pathogenesis of experimental colitis. These observations provide us with novel information on the role of the 5-HT7 receptor in gut inflammation and highlight the potential benefit of targeting this receptor to alleviate disease severity in intestinal inflammatory conditions including inflammatory bowel disease.

Cannabidiol, a safe and non-psychotropic ingredient of the marijuana plant Cannabis sativa, is protective in a murine model of colitis

Inflammatory bowel disease affects millions of individuals; nevertheless, pharmacological treatment is disappointingly unsatisfactory. Cannabidiol, a safe and non-psychotropic ingredient of marijuana, exerts pharmacological effects (e.g., antioxidant) and mechanisms (e.g., inhibition of endocannabinoids enzymatic degradation) potentially beneficial for the inflamed gut. Thus, we investigated the effect of cannabidiol in a murine model of colitis. Colitis was induced in mice by intracolonic administration of dinitrobenzene sulfonic acid. Inflammation was assessed both macroscopically and histologically. In the inflamed colon, cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) were evaluated by Western blot, interleukin-1beta and interleukin-10 by ELISA, and endocannabinoids by isotope dilution liquid chromatography-mass spectrometry. Human colon adenocarcinoma (Caco-2) cells were used to evaluate the effect of cannabidiol on oxidative stress. Cannabidiol reduced colon injury, inducible iNOS (but not cyclooxygenase-2) expression, and interleukin-1beta, interleukin-10, and endocannabinoid changes associated with 2,4,6-dinitrobenzene sulfonic acid administration. In Caco-2 cells, cannabidiol reduced reactive oxygen species production and lipid peroxidation. In conclusion, cannabidiol, a likely safe compound, prevents experimental colitis in mice.

The Vagus Nerve: A Tonic Inhibitory Influence Associated With Inflammatory Bowel Disease in a Murine Model

The recently proposed Inflammatory Reflex describes an interaction between the vagus nerve and peripheral macrophages, resulting in attenuation of proinflammatory cytokine release in response to systemic exposure to bacterial endotoxin. The purpose of this study was to determine whether a similar vagus/macrophage axis modulates the inflammatory responses in the colon in mice. We assessed the Disease Activity Index (DAI), macroscopic and histologic damage, serum amyloid-P level, and myeloperoxidase activity in colitis induced by administration of dextran sodium sulfate (DSS) in healthy and vagotomized C57BL/6 and in mice deficient in macrophage-colony stimulating factor (M-CSF)-induced and in hapten-induced colitis. A pyloroplasty was performed in vagotomized mice. DAI, macroscopic and histologic scores, myeloperoxidase activity, levels of serum amyloid-P, and colonic tissue levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha were increased significantly in vagotomized mice 5 days post-DSS and 3 days after hapten-induced colitis compared with sham-operated mice that received DSS or the hapten. Pretreatment with nicotine significantly decreased each of these markers in vagotomized mice with DSS colitis, and all markers except DAI and IL-6 in sham-operated DSS-treated mice. Conversely, hexamethonium treatment significantly increased each of these markers in the sham-operated DSS-treated mice. Vagotomy had no effect on the colitis in M-CSF-deficient mice. The vagus nerve plays a counterinflammatory role in acute colitis via a macrophage-dependent mechanism, involving hexamethonium-sensitive nicotinic receptors. The identification of a counterinflammatory neural pathway would open new therapeutic avenues for treating acute exacerbations of inflammatory bowel disease.

Beneficial effects of GW274150 treatment on the development of experimental colitis induced by dinitrobenzene sulfonic acid

Inflammatory bowel disease is associated with inducible nitric oxide synthase (iNOS) expression, oxidative and nitrosative stress, and leukocyte infiltration in the colon. Here, we investigate the effects of the selective iNOS-inhibitor ( S)-2-amino-(1-iminoethylamino)-5-thiopentanoic acid (GW274150) on the development of experimental colitis induced by dinitrobenzene sulfonic acid. When compared to dinitrobenzene sulfonic acid-treated mice, GW274150 (5 mg/kg i.p.)-treated mice subjected to dinitrobenzene sulfonic ACID-induced colitis experienced a significantly lower rate of the extent and severity of the histological signs of colon injury. Dinitrobenzene sulfonic acid-treated mice experienced hemorrhagic diarrhoea and weight loss. At 4 days after the administration of dinitrobenzene sulfonic acid, the mucosa of the colon exhibited large areas of necrosis. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) (PAR) showed an intense staining in the inflamed colon. Treatment of dinitrobenzene sulfonic acid-treated mice with GW274150 significantly reduced the degree of hemorrhagic diarrhoea and weight loss caused by administration of dinitrobenzene sulfonic acid. GW274150 also caused a substantial reduction of (i) the degree of colon injury, (ii) the rise in myeloperoxidase (MPO) activity (mucosa), (iii) the increase in staining (immunohistochemistry) for nitrotyrosine, as well as (iv) PARP activation caused by dinitrobenzene sulfonic acid in the colon. Thus, GW274150 treatment reduced the degree of colitis caused by dinitrobenzene sulfonic acid. We propose that selective inhibition of iNOS activity with GW274150 may be useful in the treatment of inflammatory bowel disease.

Tempol, a membrane-permeable radical scavenger, reduces dinitrobenzene sulfonic acid-induced colitis

Inflammatory bowel disease is characterized by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. The aim of the present study was to examine the effects of tempol, a membrane-permeable radical scavenger, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid. Rats experienced bloody diarrhea and significant loss of body weight. At 4 days after the administration of dinitrobenzene sulfonic acid, the colon injury comprised of large areas of mucosal necrosis. Neutrophil infiltration (measured as increase in myeloperoxidase activity in the mucosa) was associated with up-regulation of ICAM-1 and expression of P-selectin and high levels of malondialdehyde (an indicator of lipid peroxidation). Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) synthetase showed an intense staining in the inflamed colon. Treatment of rats with tempol (15 mg/kg daily i.p.) significantly reduced the appearance of diarrhea and the loss in body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture as well as a significant reduction in the degree of both neutrophil infiltration and lipid peroxidation in the inflamed colon. Tempol also reduced the appearance of nitrotyrosine and poly (ADP-ribose) synthetase immunoreactivity in the colon as well as the up-regulation of ICAM-1 and P-selectin. The results of this study suggest that membrane-permeable radical scavengers, such as tempol, exert beneficial effects in experimental colitis and may, hence, be useful in the treatment of inflammatory bowel disease.