ABSTRACT
AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn's disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry. RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time. CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation.
Subject(s)
Colitis/pathology , Colon/ultrastructure , Crohn Disease/pathology , Intestinal Obstruction/pathology , Animals , Apoptosis , Blotting, Western , Colitis/chemically induced , Colitis/genetics , Colitis/metabolism , Colon/metabolism , Constriction, Pathologic , Crohn Disease/chemically induced , Crohn Disease/genetics , Crohn Disease/metabolism , Disease Models, Animal , Gene Expression Regulation , Immunohistochemistry , Intestinal Obstruction/genetics , Intestinal Obstruction/metabolism , Male , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Microscopy, Electron, Transmission , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Time Factors , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Transforming Growth Factor beta2/genetics , Transforming Growth Factor beta2/metabolism , Transforming Growth Factor beta3/genetics , Transforming Growth Factor beta3/metabolism , Trinitrobenzenesulfonic AcidABSTRACT
C-150 a Mannich-type curcumin derivative, exhibited pronounced cytotoxic effects against eight glioma cell lines at micromolar concentrations. Inhibition of cell proliferation by C-150 was mediated by affecting multiple targets as confirmed at transcription and protein level. C-150 effectively reduced the transcription activation of NFkB, inhibited PKC-alpha which are constitutively over-expressed in glioblastoma. The effects of C-150 on the Akt/ Notch signaling were also demonstrated in a Drosophila tumorigenesis model. C-150 reduced the number of tumors in Drosophila with similar efficacy to mitoxantrone. In an in vivo orthotopic glioma model, C-150 significantly increased the median survival of treated nude rats compared to control animals. The multi-target action of C-150, and its preliminary in vivo efficacy would render this curcumin analogue as a potent clinical candidate against glioblastoma.
Subject(s)
Acrylamides/chemistry , Brain Neoplasms/drug therapy , Curcumin/analogs & derivatives , Curcumin/chemistry , Glioblastoma/drug therapy , NF-kappa B/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptors, Notch/antagonists & inhibitors , Unfolded Protein Response/drug effects , Animals , Antineoplastic Agents/chemistry , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Drosophila melanogaster , Drug Screening Assays, Antitumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Humans , Inhibitory Concentration 50 , Melanoma, Experimental , Mice , Neoplasm Transplantation , Rats , Rats, Nude , Receptors, Notch/metabolism , Signal Transduction , Transcription, GeneticABSTRACT
AIM: To establish a rat model suitable to investigate the repetitive relapsing inflammations (RRI) characteristic to Crohn's disease. METHODS: Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). RRI were mimicked by repeating administrations of TNBS. Tissue samples were taken from control, once, twice and three times treated rats from the inflamed and adjacent non-inflamed colonic segments at different timepoints during the acute intestinal inflammation. The means of the ulcerated area were measured to evaluate the macroscopic mucosal damage. The density of myenteric neurons was determined on whole mounts by HuC/HuD immunohistochemistry. Heme oxygenase-1 (HO-1) expression was evaluated by molecular biological techniques. RESULTS: TNBS-treated rats displayed severe colitis, but the mortality was negligible, and an increase of body weight was characteristic throughout the experimental period. The widespread loss of myenteric neurons, and marked but transient HO-1 up-regulation were demonstrated after the first TNBS administration. After repeated doses the length of the recovery time and extent of the ulcerous colonic segments were markedly decreased, and the neuronal loss was on a smaller scale and was limited to the inflamed area. HO-1 mRNA level was notably greater than after a single dose and overexpression was sustained throughout the timepoints examined. Nevertheless, the HO-1 protein up-regulation after the second TNBS treatment proved to be transient. Following the third treatment HO-1 protein expression could not be detected. CONCLUSION: Experimentally provoked RRI may exert a protective preconditioning effect against the mucosal and neuronal damage. The persistent up-regulation of HO-1 mRNA expression may correlate with this.
