Rosiglitazone, a PPARgamma ligand, modulates signal transduction pathways during the development of acute TNBS-induced colitis in rats

Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPARgamma), a highly nuclear receptor expressed in the colon, may participate in the control of inflammation, especially in regulating the production of immunomodulatory and inflammatory mediators, cellular proliferation and apoptosis. In order to delve into the anti-inflammatory mechanisms and signalling pathways of PPARgamma agonists, we have studied the effects of rosiglitazone, a PPARgamma agonist on the extent and severity of acute ulcerative colitis caused by intracolonic administration of 2,4,6-trinitribenzene sulfonic acid (TNBS) in rats. The inflammatory response was assessed by gross appearance, myeloperoxidase (MPO) activity, tumour necrosis factor alpha (TNF-alpha) levels and a histological study of the lesions. We determined prostaglandin E2 production as well as the cyclooxygenases (COX)-1 and -2 expressions by immunohistochemistry and Western blotting. The nuclear factor kappa (NF-kappaB) p65 and p38 mitogen-activated protein kinase (MAPK) expression levels were also measured by Western blotting. Finally, since PPARgamma agonists modulate apoptosis, we tried to clarify its effects under early acute inflammatory conditions. Inflammation following TNBS induction was characterized by increased colonic wall thickness, edema, diffuse inflammatory cells infiltration, necrosis reaching an ulcer index (UI) of 9.66+/-0.66 cm(2) and increased MPO activity and TNF-alpha colonic levels. Rosiglitazone treatment significantly reduced the morphological alteration associated with TNBS administration and the UI with the highest dose. In addition, the degree of neutrophil infiltration and the cytokine levels were significantly ameliorated. Rosiglitazone significantly reduced the rise in the prostaglandin (PG) E(2) generation compared with TNBS group. The COX-1 levels remained stable throughout the treatment in all groups. The COX-2 expression was elevated in TNBS group; however rosiglitazone administration reduced the COX-2 overexpression. A high expression of NF-kappaB p65 and p38 MAPK proteins appeared in colon mucosa from control TNBS-treated rats; nevertheless, PPARgamma agonist treatment drastically decreased them. There were no significant changes in apoptosis after rosiglitazone treatment when compared to TNBS group. In conclusion, rosiglitazone seems to modulate the acute colitis through NF-kappaB p65 and p38 MAPK signalling pathways.     

Therapeutic Role for Bismuth Compounds in TNBS-Induced Colitis in the Rat

The 2,4,6-trinitrobenzene sulfonic acid (TNBS) -induced model of chronic inflammation of the rat colon was used to determine the efficacy of bismuth subsalicylate (BSS), bismuth subcitrate (CBS), and 5-aminosalicylic acid (5-ASA) administered in enema form. A novel bismuth compound 1, 2-bis[2-(1,3-dithiobismolane)thio]ethane [Bi₂(EDT)₃] was also tested. On day 1 colitis was induced with 50 mg TNBS/50% ethanol in female Sprague-Dawley rats, while controls received a saline enema. On day 3, twice-daily treatment with enemas of either saline, BSS, CBS, Bi₂(EDT)₃, or 5-ASA were initiated in the colitis and control rats. All rats were killed on day 14, and the colons excised, weighed, rated macroscopically, and then fixed for hematoxylin and eosin staining. Blinded microscopic scoring was used to determine injury and healing in all groups. Colon mass and macroscopic scores were increased (P < 0.05) in the group of rats treated with TNBS (N = 16) compared to saline controls (N = 12). Colon mass and macroscopic scores in controls treated with BSS (N = 4), CBS (N = 4), Bi₂(EDT)₃ (N = 4), and 5-ASA (N = 4) alone did not differ from saline control animals. Macroscopic scoring showed a decrease (P < 0.05) in the degree of damage in the group of rats treated with TNBS plus BSS (N = 15), TNBS plus Bi₂(EDT)₃ (N = 10) and TNBS plus CBS (N = 4) compared to the group of rats treated with TNBS plus saline (N = 16). A decrease (P < 0.05) in injury and an increase (P < 0.05, Kruskal-Wallis) in healing was observed in the groups of rats treated with TNBS plus BSS, TNBS plus CBS, and TNBS plus 5-ASA compared to the group of rats treated with TNBS plus saline. It appeared that Bi₂(EDT)₃ was not protective against injury at the microscopic level but that the novel Bi₂(EDT)₃ has an effective healing capacity at the macroscopic level. We conclude that BSS and CBS decrease injury and/or promote healing as effectively as 5-ASA in this model.     

Stress Increases Susceptibility to Oxidative/Nitrosative Mucosal Damage in an Experimental Model of Colitis in Rats

Inflammatory bowel diseases (IBDs) are multifactorial processes. Clinical and animal studies indicate that emotional stress may contribute to the onset and progress of these diseases. On the other hand, enhanced free radical production in mucosal cells has been also implicated in the pathogenesis of IBD. Using an experimental model of colitis induced by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) plus ethanol (vehicle), we sought to determine whether prior exposure to immobilization stress modifies the susceptibility to oxidative damage in colonic mucosa. Several groups of Wistar rats were used: control (C) and stressed (by immobilization of 6 hr every day during 10 days; S) groups and rats receiving a colitis-inducing dose of TNBS on day 5 (30 mg; TNBS30) and a noninflammatory dose of TNBS on day 5 (5 mg; TNBS5) with or without stress (prior exposure, days 0-5, and after, days 5-10). At the 10th day, colonic tissue was dissected and processed for biochemical studies. TNBS30 led to body weight loss, macroscopic colonic ulceration, and inflammation (determined by histological parameters and myeloperoxidase [MPO] activity) and to an increase in inducible nitric oxide synthase (NOS-2) activity and expression. TNBS5-instilled animals' body weight and biochemical inflammatory parameters were not significantly different from those in control animals. Interestingly, while stress did not modify body weight, macroscopic aspect of the mucosa, or NOS activity in animals receiving TNBS30, immobilization increased body weight loss, MPO levels, and malondialdehyde (MDA; an indicator of lipid peroxidation) levels after TNBS5. On the other hand, stress increased NOS-2 activity and immunohistochemical expression after instillation of TNBS5. Moreover, constitutive, Ca2+ -dependent NOS activity decreased in stressed animals instilled with TNBS5 compared with nonstressed animals receiving TNBS5 (-28.5 +/- 6.6%; P < 0.05). These findings indicate that previous exposure to stressful stimuli is a factor in susceptibility to oxidative damage in experimental colitis and support a possible protective effect of treatment of stress before and during the development of inflammation in the colon.     

Enzyme-linked immunosorbent assay (ELISA) with covalently bound protein on glass tubes: 1. Stable antigenicity and binding of IgG as a model antigen after repeated use

A modification of the ELISA procedure is described. The system is based on the covalent binding of protein to glass tubes. Human IgG was used as model antigen. Optimal conditions were tested for the removal of alkaline phosphatase-labeled antibodies from their antigen. Under such optimal conditions, a regenerable system could be created which exhibited many advantages, as compared with conventional ELISAs with antigens absorbed unspecifically to plastic surfaces. The advantages are: 1. A higher density of IgG as model antigen on the solid phase, i.e., 1 molecule IgG per 94 nm2 with glass tubes as compared with 110 nm2 with polystyrene (PS) or 143 nm2 with polyvinylchloride (PVC) microtiter plates. 2. A much smaller unspecific absorption of less than 1% as compared with 39% with PS-plates or 16% with PVC-plates treated under identical conditions. 3. A higher stability of the binding of the model antigen to the solid phase, i.e., a drastically reduced protein loss of less than 6% during the first ELISA procedure (including the regeneration) and of less than 1% during the subsequent ELISA and regeneration cycles with glass tubes as compared with 46% (PS plates) or 55% (PVC plates). 4. A smaller intraday variation coefficient of the ELISAs of 4.8% with glass tubes as opposed to 9.7% or 7.5% with PS or PVC plates respectively. The system with covalently bound antigens on glass tubes could be used in at least 20 consecutive measuring cycles. In five consecutive cycles, a protein loss of less than 5% was observed and the interday variation coefficient of the ELISA reaction was smaller than 5% using the same tubes repeatedly. Our results indicate that covalent linkage of protein can improve ELISA and lead to repeatedly usable systems as long as the antigen is stable against the regeneration procedure. Such an ELISA system may be helpful with highly purified proteins.     

Distribution of calcitonin gene-related peptide, somatostatin, substance P and vasoactive intestinal polypeptide in experimental colitis in rats

Immunohistochemistry was used to examine the distribution of calcitonin gene-related peptide (CGRP), substance P, somatostatin and vasoactive intestinal polypeptide (VIP) in experimental colitis induced with trinitrobenzene sulphonic acid (TNBS) in rats. CGRP immunoreactivity was observed throughout the colonic wall. A significant reduction of CGRP-immunoreactive (IR) nerve fibres was observed in the mucosa after the induction of colitis. After TNBS treatment substance P immunoreactivity was reduced throughout the colon; however, after 7 days there was a marked re-innervation of the circular muscle. Somatostatin immunoreactivity was distributed sparsely within the colonic wall, and was comparatively less affected by colitis. VIP immuno- reactivity was abundantly distributed in the colonic wall and underwent an immediate reduction in the mucosa after TNBS treatment. After 2 days, there was a consistent and progressive increase in the number and density of VIP-IR nerve fibres in the inflamed colon, particularly the circular muscle. This change was associated with a proliferation of nerve fibres within the muscle layers. It was concluded that the early decrease in these neuropeptides was consistent with release from peripheral nerve terminals or the loss of nerves during the initial stages of colonic inflammation, which may be an essential condition for the development of colitis in this model. The observation that the intensity and density of substance P and VIP-IR nerve fibres increased in the circular muscle 7 days after the induction of colitis suggests their possible involvement in tissue repair.     

小鼠骨髓间充质干细胞移植后在炎症性肠病模型的定位

目的 探讨小鼠骨髓间充质干细胞(BMSCs) 移植治疗小鼠炎症性肠病(IBD) 模型后其在结肠的定位情况.方法 将骨片培养法培养的雄性BALB/C 小鼠BMSCs,用羧基荧光素二乙酸盐琥珀酰亚胺酯(CFDA SE) 进行荧光标记后经尾静脉注射于雌性大鼠IBD 模型体内,并设立对照组.分别于移植后第2、5、9 天后取远端结肠组织,一部分制成冰冻切片于荧光显微镜下观察结肠荧光分布情况,另一部分经PCR 检测Y 染色体的性别决定区段(SRY) 基因作为标志,以确定BMSCs 的肠道定位情况.结果 BMSCs 生长迅速、纯度高,可诱导成为脂肪细胞及骨细胞;2,4,6- 三硝基苯磺酸(TNBS) 造成的IBD模型部分肠段缩窄,溃疡形成,黏膜及黏膜下层有大量中性粒细胞、淋巴细胞、巨噬细胞及纤维细胞浸润;荧光显微镜下可见移植BMSCs 的模型组结肠组织存在荧光;SRY 检测显示TNBS-MSCs 移植组及雄性小鼠对照组均能检测到SRY基因.结论 移植后的BMSCs 能在IBD 模型的损伤肠道组织中定位.     

The antihyperalgesic effect of levetiracetam in an inflammatory model of pain in rats: mechanism of action

Background and purpose:

The underlying mechanisms of gastric dysfunction during or after an episode of intestinal inflammation are poorly understood. This study investigated the effects of colitis on the contractile effects of motilin, an important endocrine regulator of gastric motility, in the antrum.

Experimental approach:

Myeloperoxidase (MPO) activity, NF-κB activity and motilin receptor density were determined in the antrum of rabbits 5 days after the induction of 2,4,6-trinitrobenzenesulphonic acid colitis. Smooth muscle and neural responses to motilin were studied in antral smooth muscle strips in vitro.

Key results:

Colitis did not affect MPO activity, but increased NF-κB activity in the antrum. Motilin receptor density in the antrum was not affected. Under control conditions, motilin induced a slowly developing tonic smooth muscle contraction. Five days post-inflammation, tonic contractions to motilin were reduced and preceded by a rapid initial contraction. Other kinases were recruited for the phosphorylation of myosin light chain (MLC) (a multi-functional MLC kinase), and for the inhibition of MLC phosphatase (Rho kinase in addition to protein kinase C) to mediate the motilin-induced contractions during inflammation. Colitis potentiated the cholinergic neural on-contractions in the antrum. This was associated with a hyper-reactivity to motilin and an increased muscle response to ACh.

Conclusions and implications:

Colitis altered the course of the motilin-induced smooth muscle contraction in the antrum. This involved changes in the kinases phosphorylating MLC. Increased cholinergic excitability to motilin in the antrum may play a role in the pathogenesis of inflammation-associated gastric motility disorders.     

Fundamentals of neurogastroenterology: basic science

The focus of neurogastroenterology in Rome II was the enteric nervous system (ENS). To avoid duplication with Rome II, only advances in ENS neurobiology after Rome II are reviewed together with stronger emphasis on interactions of the brain, spinal cord, and the gut in terms of relevance for abdominal pain and disordered gastrointestinal function. A committee with expertise in selective aspects of neurogastroenterology was invited to evaluate the literature and provide a consensus overview of the Fundamentals of Neurogastroenterology textbook as they relate to functional gastrointestinal disorders (FGIDs). This review is an abbreviated version of a fuller account that appears in the forthcoming book, Rome III. This report reviews current basic science understanding of visceral sensation and its modulation by inflammation and stress and advances in the neurophysiology of the ENS. Many of the concepts are derived from animal studies in which the physiologic mechanisms underlying visceral sensitivity and neural control of motility, secretion, and blood flow are examined. Impact of inflammation and stress in experimental models relative to FGIDs is reviewed as is human brain imaging, which provides a means for translating basic science to understanding FGID symptoms. Investigative evidence and emerging concepts implicate dysfunction in the nervous system as a significant factor underlying patient symptoms in FGIDs. Continued focus on neurogastroenterologic factors that underlie the development of symptoms will lead to mechanistic understanding that is expected to directly benefit the large contingent of patients and care-givers who deal with FGIDs.     

Therapeutic action of ghrelin in a mouse model of colitis

BACKGROUND & AIMS: Ghrelin is a novel growth hormone-releasing peptide with potential endogenous anti-inflammatory activities ameliorating some pathologic inflammatory conditions. Crohn's disease is a chronic debilitating disease characterized by severe T helper cell (Th)1-driven inflammation of the colon. The aim of this study was to investigate the therapeutic effect of ghrelin in a murine model of colitis. METHODS: We examined the anti-inflammatory action of ghrelin in the colitis induced by intracolonic administration of trinitrobenzene sulfonic acid. Diverse clinical signs of the disease were evaluated, including weight loss, diarrhea, colitis, and histopathology. We also investigated the mechanisms involved in the potential therapeutic effect of ghrelin, such as inflammatory cytokines and chemokines, Th1-type response, and regulatory factors. RESULTS: Ghrelin ameliorated significantly the clinical and histopathologic severity of the trinitrobenzene sulfonic acid-induced colitis; abrogating body weight loss, diarrhea, and inflammation; and increasing survival. The therapeutic effect was associated with down-regulation of both inflammatory and Th1-driven autoimmune response through the regulation of a wide spectrum of inflammatory mediators. In addition, a partial involvement of interluekin-10/transforming growth factor-beta1-secreting regulatory T cells in this therapeutic effect was demonstrated. Importantly, the ghrelin treatment was therapeutically effective in established colitis and avoided the recurrence of the disease. CONCLUSIONS: Our data demonstrate novel anti-inflammatory actions for ghrelin in the gastrointestinal tract, ie, the capacity to deactivate the intestinal inflammatory response and to restore mucosal immune tolerance at multiple levels. Consequently, ghrelin administration represents a novel possible therapeutic approach for the treatment of Crohn's disease and other Th1-mediated inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis.