Hieber, F. (Fabian) (2017) “Newfound hope in an old enemy” Intestinal targeting of carbon monoxide for the treatment of IBD; an in vitro proof of principle study. thesis, Medicine.
Full text available on request.Abstract
Background: The incidence of Inflammatory bowel disease (IBD), e.g. Crohn’s disease and ulcerative colitis, like many other non-communicable diseases, is steadily rising. Currently, there is no cure for IBD and treatment revolves around suppression of inflammatory flares in the digestive tract. Carbon monoxide (CO) has been shown to exert potent anti-inflammatory and anti-apoptotic effects when applied in low dosages, therefore being also a promising new agent in the treatment of IBD. CO, however, lacks specificity when applied as a gas and can be toxic when doses are too high. CO-releasing molecules (CORMs) are a novel way to deliver therapeutic amounts of CO to a target tissue thereby preventing potential toxic effects elsewhere in the body. A new generation of CORMs uses bile acids as targeting vehicles to drive specific accumulation in enterocytes, where it only releases CO upon enzymatic cleavage of the pro-drug (bile acid-esterase-triggered CORMs; BA-ET-CORMs). The aim of this study is to establish uptake of the BA-ET-CORM into enterocytes and verify CO release inside these cells. Furthermore, we aim to establish anti-inflammatory and cyto-protective effects of BA-ET-CORM in enterocytes. Material and Methods: This study uses the DLD-1 cell line as a model for human intestinal epithelium to demonstrate the therapeutic potential of BA-ET-CORMs. The fluorescent COP-1 probe was used to verify uptake of the BA-ET-CORM and release of CO inside cells. Anti-inflammatory properties of BA-ET-CORM treatment was analyzed by measuring inducible nitric oxide synthase (iNOS) expression after exposure to the CORM. Cytoprotective effects were analyzed by quantifying Caspase-3 activity (apoptosis), LDH leakage and Sytox green staining (necrosis). Metabolic activity was analyzed by WST-1 assays. Results: Exposure of DLD-1 cells to BA-E-CORM lead to cellular release of CO as observed by the COP-1 probe. Stimulation of DLD-1 cells with a cytokine mixture (CM: TNFα, INFγ and IL-1β) induced high levels of both iNOS expression (700-fold compared to untreated controls) and Caspase-3 activity (13-fold). Doses of up to 10 μM of the BA-ET-CORM did not show any cytotoxicity (caspase-3 activity, LDH leakage) or inflammatory signaling in (iNOS expression) in DLD-1 cells. Co-treatment of CM-exposed DLD-1 cells with BA-ET-CORM reduced iNOS expression (>2-fold reduction compared to CM-treated cells) while dose-dependently inducing cell-protective HO-1 expression (15-fold). BA-ET-CORM did not prevent CM-induced Caspase-3 activity or LDH leakage in DLD-1 cells. Conclusion: BA-ET-CORM is taken up of DLD-1 intestinal epithelial cells and demonstrates anti-inflammatory properties in these cells, while no cytotoxic effects were observed at the applied concentrations. This opens a novel avenue for targeted treatment of ileal and colonic inflammation in IBD.
| Item Type: | Thesis (Thesis) |
|---|---|
| Supervisor name: | Supervisors and Mposhi, Archibold and Dijkstra, Prof. Dr. Gerard and Faber, Prof. Dr. Klaas Nico and Department for Gastroenterology and Liver diseases and Universitair medisch centrum Groningen |
| Faculty: | Medical Sciences |
| Date Deposited: | 25 Jun 2020 11:07 |
| Last Modified: | 25 Jun 2020 11:07 |
| URI: | https://umcg.studenttheses.ub.rug.nl/id/eprint/2662 |
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