Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis

Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

IMPLICATION OF TNF-α CONVERTASE (TACE/ADAM17) IN INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION AND INFLAMMATION IN AN EXPERIMENTAL MODEL OF COLITIS

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-α convertase (TACE; ADAM17). TNF-α plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-α levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-α and NO−x levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-α. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-α release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-α release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

A Low Dose of Fermented Soy Germ Alleviates Gut Barrier Injury,Hyperalgesia and Faecal Protease Activity in a Rat Model of Inflammatory Bowel Disease

Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1β and TNF-α predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the anti-inflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD.     

Combination therapy of mesenchymal stromal cells and sulfasalazine attenuates trinitrobenzene sulfonic acid induced colitis in the rat: The S1P pathway

Adipose derived mesenchymal stem cells (ASCs) transplantation is a novel immunomodulatory therapeutic tool to ameliorate the symptom of inflammatory bowel disease (IBD). The objective of this study was to investigate the therapeutic effects of combined sufasalazine and ASCs therapy in a rat model of IBD. After induction of colitis in rats, ASCs were cultured and intraperitoneally injected (3 × 10⁶cells/kg) into the rats on Days 1 and 5 after inducing colitis, in conjunction with daily oral administration of low dose of sulfasalazine (30 mg/kg). The regenerative effects of combination of ASCs and sulfasalazine on ulcerative colitis were assessed by measuring body weight, colonic weight/length ratio, disease activity index, macroscopic scores, histopathological examinations, cytokine, and inflammation markers profiles. In addition, western blot analysis was used to assess the levels of nuclear factor-kappa B (NF-κB) and apoptosis related proteins in colitis tissues. Simultaneous treatment with ASCs and sulfasalazine was associated with significant amelioration of disease activity index, macroscopic and microscopic colitis scores, as well as inhibition of the proinflammatory cytokines in trinitrobenzene sulfonic acid (TNBS)-induced colitis. Moreover, combined ASCs and sulfasalazine therapy effectively inhibited the NF-κB signaling pathway, reduced the expression of Bax and prevented the loss of Bcl-2 proteins in colon tissue of the rats with TNBS-induced colitis. Furthermore, combined treatment with ASCs and sulfasalazine shifted inflammatory M1 to anti-inflammatory M2 macrophages by decreasing the levels of MCP1, CXCL9 and increasing IL-10, Arg-1 levels. In conclusion, combination of ASCs with conventional IBD therapy is potentially a much more powerful strategy to slow the progression of colitis via reducing inflammatory and apoptotic markers than either therapy alone.     

Two functional endothelin receptors in guinea-pig pulmonary arteries

The contractile effects of endothelins (ET-1, ET-2, ET-3) were investigated in pulmonary vessels and trachea from the guinea-pig using a sensitive in vitro method. ET-1 and ET-2 were potent agonists that concentration-dependently contracted pulmonary vessels. ET-3 was also an agonist but was less potent. In contrast, ET-1, ET-2 and ET-3 showed equal potencies in inducing contractions of tracheal segments. Using a pharmacological desensitization technique, evidence was provided for two types of functional endothelin receptors. Putatively, ET-1 and ET-2 act on the same functional receptor in the pulmonary artery whereas ET-3 acts on another receptor. Intraregional differences of the responses to endothelins were noticed when small intrapulmonar resistance arteries were compared to large proximal arteries. These differences are probably due to variations in the distribution of endothelin receptors rather than to receptor heterogeneity.     

Role of tumor necrosis factor receptors in an animal model of acute colitis

TNF-alpha is known to play an important role in inflammatory bowel disease (IBD); however, the pathophysiological role of its receptors is still under study. Acute colitis was induced in rats by intracolonic administration of trinitrobenzene sulfonic acid (TNBS). Control rats received the ethanol vehicle. Rats were sacrificed 72 h later and samples of tissue and fluids were collected. There was a significant increase in the protein levels of sTNF-alpha, sTNFRI, and sTNFRII in the peritoneal fluid (PF) of experimental rats. TNF-alpha, TNFRI, and TNFRII mRNA expression was increased significantly in the colon of experimental animals compared to controls. TRAF3 and TRAF5 expression was also significantly higher, as was that of the adhesion molecules ICAM-1 and E-selectin. The increased expression of TNF-alpha, TNFRs, and the associated signaling factors in the colon of this rat model of IBD provides further evidence for their involvement in the promotion of inflammation and tissue damage. In addition, increased levels of sTNFRs in the PF of experimental rats--particularly sTNFRII--may be involved in the development of colitis by serving as a reservoir of TNF-alpha, and thus provide a novel therapeutic target for IBD.     

Effect of COX-2 inhibitor after TNBS-induced colitis in wistar rats

Inflammatory bowel disease (IBD) is a common chronic gastrointestinal disorder characterized by alternating periods of remission and active intestinal inflammation. Some studies suggest that antiinflammatory drugs are a promising alternative for treatment of the disease. Thus, this study aimed to evaluate the effect of lumiracoxib, a selective-cyclooxygenase-2 (COX-2) inhibitor, on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. Wistar rats (n = 25) were randomized into four groups, as follows: Group (1) Sham group: sham induced-colitis rats; Group (2) TNBS group: nontreated induced-colitis rats; Group (3) Lumiracoxib control group; and Group (4) Lumiracoxib-treated induced-colitis rats. Our results showed that rats from groups 2 and 4 presented similar histopathological damage and macroscopic injury in the distal colon as depicted by significant statistically differences (P < 0.01; P < 0.05) compared to the other two groups. Weak expression of COX-2 mRNA was detected in normal colon cells, while higher levels of COX-2 mRNA were detected in group 2 and group 4. Therapy with lumiracoxib reduced COX-2 expression by 20–30%, but it was still higher and statistically significant compared to data obtained from the lumiracoxib control group. Treatment with the selective COX-2 inhibitor lumiracoxib did not reduce inflammation-associated colonic injury in TNBS-induced experimental colitis. Thus, the use of COX-2 inhibitors for treating IBD should be considered with caution and warrants further experimental investigation to elucidate their applicability.     

Increased colonic apelin production in rodents with experimental colitis and in humans with IBD

Apelin and its receptor, the APJ receptor, are expressed in the gastrointestinal tract. The aims of this study were to examine the effects of sodium dextran sulfate (DSS)-induced experimental colitis in rats and mice and inflammatory bowel disease (IBD) in humans on intestinal apelin production, and the influence of exogenous apelin on colonic epithelial cell proliferation in mice. In rodents with experimental colitis, colonic apelin mRNA levels were elevated during the inflammatory reaction as well as during the tissue repair phase that ensues after DSS withdrawal. Fluctuations in colonic apelin expression were paralleled by similar changes in apelin immunostaining. Apelin immunostaining was increased in the surface epithelium, in epithelial cells along the length of the tubular gland and in the stem cell region at the gland base. In ulcerative colitis (UC) and Crohn's disease patients, apelin immunostaining revealed a pattern of increased intestinal apelin content similar to that observed in rodents with experimental colitis. Administration of synthetic apelin to mice during the recovery phase of DSS-induced colitis stimulated colonic epithelial cell proliferation significantly. Our observations that colonic apelin production is increased during and after DSS exposure indicate that apelin plays multiple roles during the different stages of colitis. Additionally, the stimulatory action of exogenous apelin on colonic epithelial proliferation suggests that the increased apelin production during intestinal recovery stage may contribute to the repair of the intestinal epithelium in experimental rodent models of colitis and in IBD patients.     

MC-12, an Annexin A1-Based Peptide, Is Effective in the Treatment of Experimental Colitis

Annexin A1 (ANXA1) inhibits NF-κB, a key regulator of inflammation, the common pathophysiological mechanism of inflammatory bowel diseases (IBD). MC-12, an ANXA1-based tripeptide, suppresses NF-κB activation. Here, we determined the efficacy of MC-12 in the control of IBD. Mice with colitis induced by dextran sodium sulfate (DSS) or 2,4,6-trinitro benzene sulfonic acid (TNBS) were treated with various doses of MC-12 administered intraperitoneally, orally or intrarectally. We determined colon length and the histological score of colitis, and assayed: in colon tissue the levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 by RT-PCR; prostaglandin E(2) (PGE(2)), cytoplasmic phospholipase A(2) (cPLA(2)) and myeloperoxidase by immunoassay; and COX-2 and NF- κB by immunohistochemistry; and in serum the levels of various cytokines by immunoassay. In both models MC-12: reversed dose-dependently colonic inflammation; inhibited by up to 47% myeloperoxidase activity; had a minimal effect on cytoplasmic phospholipase A(2); reduced significantly the induced levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10, returning them to baseline. DSS and TNBS markedly activated NF-κB in colonic epithelial cells and MC-12 decreased this effect by 85.8% and 72.5%, respectively. MC-12 had a similar effect in cultured NCM460 normal colon epithelial cells. Finally, MC-12 suppressed the induction of COX-2 expression, the level of PGE(2) in the colon and PGE(2) metabolite in serum. In conclusion, MC-12, representing a novel class of short peptide inhibitors of NF-κB, has a strong effect against colitis in two preclinical models recapitulating features of human IBD. Its mechanism of action is complex and includes pronounced inhibition of NF-κB. MC-12 merits further development as an agent for the control of IBD.     

Melatonin reduces inflammatory injury through inhibiting NF-kappaB activation in rats with colitis

Proinflammatory mediators are important in the pathogenesis of IBD, which are regulated by activation of NF-kappaB. The aim of this study was to investigate whether melatonin reduces inflammatory injury and inhibits proinflammatory molecule and NF-kappaB in rats with colitis. Rat colitis model was established by TNBS enema. NF-kappaB p65, TNF-alpha, ICAM-1, and IkappaBalpha in colon tissue were examined by immunohistochemistry, EMSA, RT-PCR, and Western blot analysis. Expression of proinflammatory molecule and activation of NF-kappaB were upregulated and IkappaB level decreased in rats with colitis. Melatonin reduces colonic inflammatory injury through downregulating proinflammatory molecule mediated by NF-kappaB inhibition and blockade of IkappaBalpha degradation.