Intrarectal Administration Of Trinitrobenzene Sulfonic Acid Research Articles

Curcumin attenuates inflammation through inhibition of TLR-4 receptor in experimental colitis

Curcumin, an active ingredient of Curcumin longa mediates its anti-inflammatory effects through inhibition of NFkB. Several pathways including toll-like receptors (TLR) induce NFkB leading to inflammation. In this study, we investigated the effects of curcumin on the expression of TLR-4 and MyD88, the upstream signaling pathway in experimental colitis induced in the Sprague-Dawley male rats by intra-rectal administration of trinitrobenzenesulfonic acid (TNBS). The animals which received TNBS were divided into two groups: Group 1, received aqueous suspension of curcumin (100 mg/Kg body weight) 2 h prior to inducing colitis, and the treatment was repeated every day for 5 days, and Group 2 and non-colitis (Group 3) animals received phosphate buffered saline (PBS) in a similar fashion. Non-colitis animals (Group 4) received curcumin and served as controls. Animals were sacrificed on day 5 post-TNBS by cervical dislocation, colon was taken out, and cleaned with PBS. Levels of TLR-4, MyD88, and NFkB proteins were measured using ECL Western blot analysis, and TLR-4 mRNA by a competitive RT-PCR method. Colitis was confirmed histologically by measuring myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels in the colonic tissues. TNBS-induced increase in the level of MPO activity and MDA concentrations was reversed by curcumin treatment, whereas the same dose of curcumin did not affect their levels in the non-colitis animals. Increases in the levels of TLR-4, MyD88, and NFkB proteins in inflamed tissue were also suppressed significantly by curcumin treatment. The level of TLR-4 mRNA remained unchanged in the colitis animals. These findings demonstrate that signaling pathway of curcumin-induced inhibition of inflammation involves TLR-4 and MyD88, and therefore may serve as an important therapeutic target in IBD.

Noninvasive Ultrasound Elasticity Imaging (UEI) of Crohn's Disease: Animal Model

Inflammation occurs in episodic flares in Crohn's disease, which are part of the waxing and waning course of the disease. Healing between flares allows the intestine to reconstitute its epithelium, but this healing results in the deposition of fibrotic scar tissue as part of the healing process. Repeated cycles of flares and healing often lead to clinically significant fibrosis and stenosis of the intestine. Patients are treated empirically with steroids, with their many side effects, in the hope that they will respond. Many patients would be better treated with surgery if we could identify which patients truly have intestinal fibrosis. Ultrasound elasticity imaging (UEI) offers the potential to radically improve the diagnosis and management of local tissue elastic property, particularly intestinal fibrosis. This method allows complete characterization of local intestine tissue with high spatial resolution. The feasibility of UEI on Crohn's disease is demonstrated by directly applying this technique to an animal model of inflammatory bowel disease (IBD). Five female Lewis rats (150–180 g) were prepared with phosphate buffered solution (PBS) as a control group and six were prepared with repeated intrarectal administration of trinitrobenzenesulfonic acid (TNBS) as a disease group. Preliminary strain measurements differentiate the diseased colons from the normal colons ( p < 0.0002) and compared well with direct mechanical measurements and histology ( p < 0.0005). UEI provides a simple and accurate assessment of local severity of fibrosis. The preliminary results on an animal model also suggest the feasibility of translating this imaging technique directly to human subjects for both diagnosis and monitoring. (E-mail: kangkim@umich.edu)

Alterations in spontaneous contractions in vitro after repeated inflammation of rat distal colon.

In inflammatory bowel disease, smooth muscle function reportedly varies with disease duration. The aim of these studies was to determine changes in the control of spontaneous contractions in a model of experimental colitis that included reinflammation of the healed area. The amplitude and frequency of spontaneous contractions in circular smooth muscle were determined after intrarectal administration of trinitrobenzenesulfonic acid in rat distal colon. With the use of a novel paradigm, rats were studied 4 h (acute) or 28 days (healed) after the initial inflammation. At 28 days, rats were studied 4 h after a second inflammation (reinflamed) of the colon. Colitis induced transient increases in the amplitude of spontaneous contractions coincident with a loss of nitric oxide synthase activity. The frequency of contractions was controlled by constitutive nitric oxide in controls. Frequency was increased in healed and reinflamed colon and was associated with a shift in the dominance of neural constitutive nitric oxide synthase control to that of inducible nitric oxide synthase (iNOS). The initial colitis induced a remodeling of the neural control of spontaneous contractions reflecting changes in their regulation by constitutive nitric oxide synthase and iNOS.

Molecular basis of altered contractility in experimental colitis: expression of L-type calcium channel.

Colitis in experimental animals or idiopathic inflammatory bowel disease, such as ulcerative colitis or Crohn's disease in humans, is associated with reduced muscle contraction. This is predicted to be due to disturbance of Ca2+ homeostasis in the inflamed muscle cell. However, the underlying molecular mechanism remains to be elucidated. Since the catalytic alpha-1 subunit of the L-type Ca2+ channel regulates Ca2+ influx, levels of the alpha-1 mRNA and protein were examined. Colitis induced by intrarectal administration of trinitrobenzenesulfonic acid was monitored by measuring the myeloperoxidase activity and histology. The levels of mRNA and protein were estimated using RT-PCR and immunoblotting. Myeloperoxidase activity increased in the inflamed colon, and the lamina propria and muscle layers showed infiltration of inflammatory cells and loss of crypts. Two alternatively spliced alpha-1 mRNA isoforms were detected in the colonic muscle. The ratio of unspliced to spliced mRNA isoforms remained unaltered in inflamed muscle. In contrast, the level of corresponding protein isozymes decreased in the colitic animals. Thus colitis-induced reduction in the alpha-1 protein may account for the reduced colonic contractility seen in colitis.

Central interleukin-1 receptors contribute to suppression of feeding after acute colitis in the rat

Experimental colitis, induced in rats by intrarectal administration of trinitrobenzenesulfonic acid (TNB), results in a suppression of eating for 3 days. Because interleukin-1 (IL-1) is elevated within 24 h after TNB treatment, and because chronic administration of IL-1 leads to a pattern of anorexia similar to that seen after TNB, we evaluated the role of endogenous IL-1 in the anorexia observed in the TNB model. Human recombinant IL-1 receptor antagonist (rhIL-1ra) was administered chronically via osmotic minipump either peripherally or centrally after TNB treatment. Peripheral delivery of 40 micrograms/h rhIL-1ra significantly attenuated TNB-induced anorexia. However, 24 micrograms/h rhIL-1ra attenuated TNB-induced anorexia only when delivered centrally, not peripherally. These findings implicate central IL-1 receptors in the suppression of eating during acute experimental colitis but leave open a possible involvement of peripheral IL-1 receptors.

Defective modulation of colonic secretomotor neurons in a rabbit model of colitis.

The present in vitro study was conducted to investigate possible alterations in the control of colonic electrolyte transport in an experimental model of colitis. Intrarectal administration of trinitrobenzenesulfonic acid induced a colitis-like inflammation in the rabbit distal colon. Responses to amiloride and residual short-circuit current after this treatment were unchanged, suggesting that the absorptive and secretory mechanisms remained intact. Electrical field stimulation and vasoactive intestinal polypeptide, a candidate secretomotor neurotransmitter, both elicited similar responses in control and colitic tissue. This suggests that communication at the neuroepithelial junction was unimpaired. In untreated tissue, the effects of prostaglandin E2 (PGE2) and of acetylcholine were attenuated by tetrodotoxin, suggesting, therefore, that both play a role in the modulation of secretomotor neurons. In addition, PGE2 had an appreciable direct epithelial effect. Responses to both of these agonists were absent in colitis. The effects of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate were unchanged in colitis, suggesting that altered PGE2 responsiveness may involve changes in epithelial receptor number, affinity, or in their ability to mediate an increase in adenosine 3',5'-cyclic monophosphate levels. It is concluded that this rabbit model of colitis exhibits 1) defects in the modulation of secretomotor neurons by acetylcholine and PGE2 and 2) an attenuated epithelial response to PGE2.