Trinitrobenzene Sulfonic Acid-induced Colitis In Rats Research Articles

Immunohistochemical characterization of transient receptor potential vanilloid types 2 and 1 in a trinitrobenzene sulfonic acid-induced rat colitis model with visceral hypersensitivity

Transient receptor potential vanilloid type 2 (TRPV2) and type 1 (TRPV1) are originally identified as heat-sensitive TRP channels. We compared the expression patterns of TRPV2 and TRPV1 in the rat distal colon and extrinsic primary afferent neurons, and investigated their roles in visceral hypersensitivity in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rats. Both TRPV2 and TRPV1 expressions in the colon, dorsal root ganglion (DRG), and nodose ganglion (NG) were significantly upregulated in the TNBS-induced colitis model. TRPV2 cell bodies co-localized with the intrinsic primary afferent marker NeuN and the inhibitory motor neuronal marker nNOS in the myenteric plexus. TRPV2 expressions were further detected in the resident macrophage marker ED2 in the mucosa. In contrast, no TRPV1-expressing cell bodies were detected in the myenteric plexus. Both TRPV2- and TRPV1-positive cell bodies in the DRG and NG were double-labeled with the neuronal retrograde tracer fluorescent fluorogold. Large- and medium-sized TRPV2-positive neurons were labeled with the A-fiber marker NF200, calcitonin gene-related peptide (CGRP), and substance P (SP) in the DRG while small-sized TRPV1-positive neurons were labeled with the C-fiber markers IB4, CGRP, and SP. TRPV2- and TRPV1-positive NG neurons were labeled with NF200 and IB4. TNBS treatment increased p-ERK1/2-positive cells in TRPV2 and TRPV1 neurons but did not affect the TRPV2 and TRPV1 subpopulations in the DRG and NG. Both TRPV2 and TRPV1 antagonists significantly alleviated visceral hypersensitivity in TNBS-induced colitis model rats. These findings suggest that intrinsic/extrinsic TRPV2- and extrinsic TRPV1-neurons contribute to visceral hypersensitivity in an experimental colitis model.

Histopathological Evaluation of the Effect of Eugenol in a Model of Trinitrobenzenesulfonic Acid-Induced Colitis in Rats

Abstract Introduction: Trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis in animals is a commonly used model of inflammatory bowel disease (IBD). Eugenol (Eug) is a natural phenolic compound possessing promising antioxidant and anti-inflammatory therapeutic properties. Aim: The present study investigated the effects of Eug in a TNBS-induced rat colitis model using criteria for histopathological evaluation of the colonic damage. Materials and methods: Male Wistar rats were divided into 6 experimental groups, each of 10 rats: Control, TNBS, TNBS+Eug1, TNBS+Eug5, TNBS+Eug25, and TNBS+Eug125 group. Eug or the solvent (sunflower oil) was applied orally using an orogastric cannula. The control group and TNBS group were treated only with sunflower oil. Eug groups were treated with corresponding doses of Eug (1, 5, 25 and 125 mg/kg) dissolved in sunflower oil. Colitis was induced by the application of TNBS in the colon. The animal treatment began 6 days before the colitis induction and continued for 8 days after it. At the end of the experiment, colitis severity was evaluated histopathologically regarding epithelium injury, inflammatory cell infiltration, and formation of granulation tissue. Results: In all TNBS+Eug groups, the formation of granulation tissue was enhanced compared to TNBS. In group TNBS+Eug125 the difference was significant compared to the control group (p < 0.05). No significant improvement regarding the scores of epithelium injury and inflammatory cell infiltration was observed in Eug groups compared to TNBS group. Conclusion: Eug did not improve the signs of TNBS-induced epithelial injury and inflammatory cell infiltration, but stimulated the formation of granulation tissue which might be considered as a sign of healing.

Evaluation of glycine-bearing celecoxib derivatives as a colon-specific mutual prodrug acting on nuclear factor-κB, an anti-inflammatory target.

In an inflammatory state where HOCl is generated, glycine readily reacts with HOCl to produce glycine chloramine, an anti-inflammatory oxidant. Colonic delivery of celecoxib elicits anticolitic effects in a trinitrobenzene sulfonic acid-induced rat colitis model. Glycine-bearing celecoxib derivatives were prepared and evaluated as a colon-specific mutual prodrug acting on nuclear factor-κB (NFκB), an anticolitic target. Glycylcelecoxib (GC), N-glycylaspart-1-ylcelecoxib (N-GA1C), and C-glycylaspart-1-ylcelecoxib (C-GA1C) were synthesized and their structures identified using infrared and proton nuclear magnetic resonance spectrometer. The celecoxib derivatives were chemically stable in pH 6.8 and 1.2 buffers. GC and C-GA1C were resistant to degradation in the small intestinal contents, while N-GA1C was substantially cleaved to release celecoxib. In contrast, all the celecoxib derivatives were degraded to liberate celecoxib in the cecal content. These results suggest that GC and C-GA1C could be delivered to and liberate celecoxib and glycine in the large intestine. In human colon carcinoma HCT116 and murine macrophage RAW264.7 cells, combined celecoxib–glycine chloramine treatment additively suppressed the production of proinflammatory NFκB target gene products. Collectively, our data suggest that C-GA1C is a potential colon-specific mutual prodrug acting against NFκB.

Actions of probiotics on trinitrobenzenesulfonic acid-induced colitis in rats.

We investigated the actions of probiotics, Streptococcus faecalis 129 BIO 3B (SF3B), in a trinitrobenzenesulfonic acid- (TNBS-) induced colitis model in rats. After TNBS was administered into the colons of rats for induction of colitis, the rats were divided into two groups: one group was given a control diet and the other group was given a diet containing SF3B for 14 days. There were no apparent differences in body weight, diarrhea period, macroscopic colitis score, and colonic weight/length ratio between the control group and SF3B group, suggesting that induction of colitis was not prevented by SF3B. Next, we investigated whether SF3B-containing diet intake affects the restoration of enteric neurotransmissions being damaged during induction of colitis by TNBS using isolated colonic preparations. Recovery of the nitrergic component was greater in the SF3B group than in the control group. A compensatory appearance of nontachykininergic and noncholinergic excitatory components was less in the SF3B group than in the control group. In conclusion, the present study suggests that SF3B-containing diet intake can partially prevent disruptions of enteric neurotransmissions induced after onset of TNBS-induced colitis, suggesting that SF3B has therapeutic potential.

Investigation of two models of trinitrobenzenesulfonic acid-induced colitis in rats

PURPOSE: Тhe aim of this study was to comparatively evaluate rat experimental colitis resulting from administration of two different doses of trinitrobenzenesulfonic acid (TNBS) dissolved in different volumes of two ethanol concentrations. METHODS: TNBS was applied in the colon by a soft cannula at a depth of about 10 cm from anus. The first group (TNBS20) was treated with 20 mg TNBS dissolved in 0.4 ml of 40% ethanol solution. The second group (TNBS10) was treated with 10 mg TNBS dissolved in 0.25 ml of 50% ethanol solution. Тhe animals were sacrificed 24 hours after the treatment and the colon was examined macroscopically and histopathologically. RESULTS: There were no significant differences regarding the degree of hyperemia, total area of injury and thickening of the colon wall. The higher TNBS dose produced significantly more pronounced shortening of the colon and larger necrotic area. Histopathological evaluation showed similar degree of injuries in the two models. CONCLUSION: The two investigated models of TNBS-induced colitis were comparable according to the overall macroscopic and microscopic criteria for colitis evaluation.

Z eyheria montana Mart. (Bignoniaceae) as source of antioxidant and immunomodulatory compounds with beneficial effects on intestinal inflammation

Zeyheria montana is a medicinal plant used in Brazilian folk medicine for treating skin affections, ulcers, inflammation and diarrhoea, and as an antisyphilitic and antiblenorrhagic agent, but little is known about its mechanisms of action. Herein, a bio-guided assay was carried out to further evaluate its antioxidant and immunomodulatory effects, and the possible benefits on experimental intestinal inflammation. Extracts, partitions, fractions and isolated compounds were tested for inhibition of lipid peroxidation. Isolated compounds were tested in vitro for its antioxidant and immunomodulatory action prior to in-vivo evaluation in trinitrobenzenesulfonic acid-induced rat colitis. Two major compounds were identified in the leaf dichloromethane extract: 3'-hydroxy-5,7,4'-trimethoxyflavone and 6-hydroxy-5,7-dimethoxyflavone, which exhibited an antioxidant activity. The compounds protected the colonic glutathione levels in more than 90% despite the absence of protection against the gross macroscopic colonic damage. In addition, the compounds inhibited IL-1ß secretion by macrophages in 91.5% and 72.7% respectively, whereas both reduced IL-6 secretion in about 44.5%. The major active compounds from Z. montana leaves exerted antioxidant and immunomodulatory effects, endorsing the use of Z. montana in folk medicine as an anti-inflammatory agent. However, further investigation is still needed regarding medicinal plants and the identification of candidate compounds for the treatment of the inflammatory bowel diseases.

Exosomes derived from interleukin-10-treated dendritic cells can inhibit trinitrobenzene sulfonic acid-induced rat colitis

Objective. Inflammatory bowel disease (IBD), which mainly refers to Crohn's disease and ulcerative colitis, is characterized by chronic inflammation of the gastrointestinal tract. Recent reports have demonstrated that exosomes derived from interleukin-10 (IL-10)-treated bone marrow-derived dendritic cells (DCs) can reduce the incidence and severity of established collagen-induced arthritis (CIA) in mice. Based on the essential role of IL-10 in the development of normal mucosal immunity, we investigated whether exosomes derived from DCs treated with IL-10 (known as IL-10-exosomes) can suppress the trinitrobenzene sulfonic acid (TNBS)-induced colitis. Material and Methods. We used the rat TNBS-induced colitis model to address the therapeutic potential of IL-10-exosomes in vivo. More specifically, a rectal enema of TNBS was administered to Wistar rats, and IL-10-exosomes were injected intraperitoneally on Day 3. Results. In the context of a high level of major histocompatibility complex class II (MHC II) and a low level of co-stimulatory molecule and membrane-bound IL-10 expression, IL-10-exosomes treatment substantially reduced all analyzed clinical, macroscopic, and histopathologic parameters of TNBS-induced colitis. The therapeutic effects of IL-10-exosomes were associated with a down-regulation mRNA expression of IL-2, IFN-γ and TNF-α in colon tissues. Importantly, treatment with IL-10-exosomes resulted in a pronounced up-regulation of IL-10mRNA expression in colon tissues and regulatory T cells (Tregs) in Colonic lamina propria. Conclusions. The results suggest that IL-10-exosomes treatment can suppress acute TNBS-induced colitis and may offer a promising new therapeutic strategy for IBD.

Neuronal nitric oxide inhibits intestinal smooth muscle growth

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [(3)H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [(3)H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo.

The Comparison between Laparoscopic vs. Open Surgery for Trinitrobenzene Sulfonic Acid-Induced Rat Colitis

Introduction: The etipathogenesis of inflammatory bowel disease (IBD) includes immunologic, genetic and environmental factors. In addition to the colon, frequent extra-intestinal alterations related to pancreas and hepatobiliary system are evaluated in several studies. A minimally invasive approach was applied to the IBD, with the expectation of earlier recovery, fewer postoperative complications. The aim of the study is to compare the effects of laparoscopic and open techniques applied for the surgical treatment of ulcerative colitis on the gastrointestinal system and pancreas, and related systemic consequences. Methods: Colitis was induced in 20 rats by rectal injection of a 5% 0.25mL 10mg/kg TNBS (Sigma, USA) in 50% ethanol. The rats were randomly divided into two groups: rat control, laparotomy was made for two-hour duration (n=10), a pneumoperitoneum was applied for a two-hour duration (n=10). Following the surgical procedures, samples were drawn for biochemical and histopathologic examinations. TNF-?, IL-6, MDA, NO, MPO and PAP levels were evaluated in serum and in tissues of pancreas and colon. Results: Except the MDA and NO levels in colonic tissues, all the parameters indicating the oxidative injury and inflammation were found significantly lower in a pneumoperitoneum group. There was no difference in histopathologic examinations of the both groups. Discussion: The results revealed that despite the pathophysiologic effects of pneumoperitoneum laparoscopic surgery in patients with ulcerative colitis elicited less oxidative damage and inflammation compared to conventional surgery. Regarding the findings we concluded that laparoscopic procedures are safe in patients with IBD but to comment on the effects of pneumoperitoneum related to the longer surgery duration further studies with variable operation times are required.

DA-6034, a Derivative of Flavonoid, Prevents and Ameliorates Dextran Sulfate Sodium–Induced Colitis and Inhibits Colon Carcinogenesis

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

RETRACTED: Colon-specific, mutual azo prodrug of 5-aminosalicylic acid with l-tryptophan: Synthesis, kinetic studies and evaluation of its mitigating effect in trinitrobenzenesulfonic acid-induced colitis in rats

Mutual azo prodrug of 5-aminosalicylic acid with l-tryptophan was synthesized by coupling l-tryptophan with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure of synthesized prodrug was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in HCl buffer (pH 1.2) showed negligible release of 5-aminosalicylic acid, whereas in phosphate buffer (pH 7.4) 18% release was observed over a period of 7 h. In rat fecal matter, 87.9% of 5-aminosalicylic acid was released with a half-life of 143.6 min, following first order kinetics. The azo conjugate was evaluated for its ulcerogenic potential by Rainsford's cold stress method. The ameliorating effect of the azo conjugate and therapeutic efficacy of the carrier system was evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model. The synthesized prodrug was found to be equally effective in mitigating the colitis in rats as that of sulfasalazine without the ulcerogenicity of 5-aminosalicylic acid.

Bovine Glycomacropeptide Is Anti-Inflammatory in Rats with Hapten-Induced Colitis

Milk kappa-casein-derived glycomacropeptide has immunomodulatory and bacterial toxin binding effects. The intestinal anti-inflammatory activity of glycomacropeptide was assessed in trinitrobenzenesulfonic acid-induced colitis in rats. Rats were administered glycomacropeptide daily starting either 2 d before (pretreatment) or 3 h after (post-treatment) colitis induction. Pretreatment with glycomacropeptide had a dose-dependent anti-inflammatory effect, characterized by lower body weight loss, decreased anorexia (57%), colonic damage (65%), and weight to length ratio (32%), as well as a reduction in colonic alkaline phosphatase activity (42%) and interleukin 1, trefoil factor 3, and inducible nitric oxide synthase mRNA levels (P < 0.05). The mechanism of action of glycomacropeptide is unknown but is consistent with an inhibition of the activation of immune cells. The magnitude of the anti-inflammatory effect was generally comparable to that of sulfasalazine, an established drug used in the treatment of inflammatory bowel disease. Bovine glycomacropeptide may play a role in the management of patients with inflammatory bowel disease.

Establishment of a trinitrobenzene sulfonic acid-induced rat colitis model

OBJECTIVE: To set up a trinitrobenzene sulfonic acid (TNBS)-induced colitis model in the rat and to assess its value in research studies of inflammatory bowel disease (IBD). METHODS: A 0.85-mL enema containing 30 mg TNBS dissolved in 50% ethanol was instilled into the rat colon to induce distal colitis. Control rats were instilled with 50% ethanol or TNBS/saline alone. Macroscopic histological changes of the colon and myeloperoxidase (MPO) activity of the mucosa were evaluated. RESULTS: One week after the TNBS/ethanol enema, hyperemia, edema and ulceration of the colonic mucosa appeared with predominant infiltration of polymorphonuclear leukocytes in the distal colon. Four weeks after TNBS/ethanol enema, ulcers had healed macroscopically and lymphocytes and plasma cells became predominant. The tissue MPO activity increased on the first day after the TNBS/ethanol enema and this increase in MPO activity lasted for 3 weeks, but declined 4 weeks after the TNBS/ethanol enema. In contrast, macroscopic, histological changes and MPO activity returned to normal levels in control rats within the first week. CONCLUSIONS: Trinitrobenzene sulfonic acid/ ethanol-induced rat distal colitis is an ideal model of IBD and may serve as a tool for the investigation of the pathogenesis of and effects of pharmaceuticals on IBD.

Dietary vitamin E supplementation protects the rat large intestine from experimental inflammation.

Vitamin E, the most potent antioxidant in the lipid phase, was tested for antiinflammatory activity in trinitrobenzenesulfonic acid-induced rat colitis. Rats were fed a nonpurified diet (saline and control groups) or a vitamin E supplemented diet (treated group, 300 mg/kg nonpurified diet). Vitamin E supplementation, which resulted in increased colonic vitamin E levels, reduced colonic weight and damage score, prevented lipid peroxidation and diarrhea, reduced interleukin-1 beta levels and preserved glutathione reductase activity and total glutathione levels. However, it did not modify myeloperoxidase levels, which are indicative of neutrophil infiltration in the inflamed colon. Vitamin E protects the rat colon from oxidative stress associated with inflammation.

Bursting pressure in anastomotic healing in experimentally induced colitis in rats.

Experimental studies on healing of colonic anastomosis have been thoroughly investigated. However, clinical parameters of the healing process of anastomosis in the inflamed colon has not yet been reported. In the present study, healing of anastomosis in trinitrobenzene-sulfonic acid-induced colitis in rats was assessed by measuring the bursting pressure and bursting wall tension. On postoperative day 4, bursting pressure and bursting wall tension were significantly lower (P < 0.001) in rats with colitis with or without anastomosis and normal colon with anastomosis, compared with normal colon without anastomosis. On postoperative day 7, bursting pressure and bursting wall tension of normal colon with anastomosis approached that of normal colon without anastomosis. However, bursting pressure and bursting wall tension of rats with colitis with or without anastomosis remained significantly lower (P < 0.001) than the latter. Furthermore, unlike rats without colitis in which perforation occurred mostly at the anastomotic line, the bursting site in colitic rats was predominantly away from the anastomotic line. These results suggest that in surgery for inflammatory bowel disease, it is the adjoining inflamed bowel wall that is vulnerable to be perforated in response to increasing intraluminal pressure rather than the anastomosis that is braced by the sutures.

Oral administration of rutoside can ameliorate inflammatory bowel disease in rats

Rutoside, a flavonoid with antioxidant properties, was tested for acute and chronic antiinflammatory activity in trinitrobenzenesulfonic acid-induced rat colitis. Pretreatment with 10 or 25 mg kg of rutoside by the oral route reduced colonic damage at 2 days. Several mechanisms can be involved in this activity, and one of these may be related to its ability in preventing glutathione depletion of colitic animals, and this could result in mucosal protection against oxidative insult. When rutoside was tested for 1 and 2 weeks after colitis induction, it was able to promote colonic healing. The chronic effect of the flavonoid was also related with its ability to increase colonic glutathione levels and thus reduce the tissue damage derived from intestinal oxidative stress which characterizes inflammatory colitis.