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P020. Selective glucocorticoid receptor agonists (SEGRAs) in intestinal disease – promising effects for inflammation and intestinal tissue repair in vitro

K.C. Reuter1, S.M. Loitsch1, W. Eberhardt2, D. Steinhilber1, J. Stein1,3

1Goethe University Frankfurt, Institute for Pharmaceutical Chemistry, Frankfurt/Main, Germany; 2Pharmazentrum Frankfurt, Institute for Pharmacology and Toxicology, Goethe-University hospital, Frankfurt/Main, Germany; 3Katharina-Kasper-Kliniken, Internal medicine, Frankfurt/Main, Germany

Background and Aim: Despite their excellent anti-inflammatory and immunsuppressive action, treatment of inflammatory bowel disease (IBD) with Glucocorticoids (GC) still carries significant risks in terms of frequently occurring side effects, such as inhibition of intestinal tissue repair. Therefore, several selective Glucocorticoid Receptor (GR) Agonists (SEGRAs) have been introduced, offering anti-inflammatory action with fewer side effects. We investigated the effect of the non-steroidal SEGRAs, Compound A (CpdA) and ZK216348 in in vitro models of IBD and intestinal wound healing.

Materials and Methods: Caco-2, IEC-6 and HEK293T cells were cultured in the presence of various concentrations of Dex, CpdA, ZK216348 or specific inhibitors and, where required, were stimulated with TNF-α/IL-1β [0.5 nM] from 15 min – 24 h. GR translocation was shown by indirect immunfluorescence. NF-κB/p65 activity was detected by EMSA. Western Blot analysis was used for detection of p65, IκB-α, GR, Annexin-1 expression and evaluation of TGF-β and EGF-pathways. IL-8 and TGF-β1 protein level in cell culture supernatants were measured by ELISA technique, mRNA levels of GR and MKP-1 by qPCR. Reporter gene assays were used for trans-activation potency and investigation of TGF-β signalling pathway. Cell migration was determined using an in vitro wound healing model, cell proliferation by BrdU incorporation assay. Apoptosis of intestinal epithelial cells was assessed using Annexin V/7 AAD – FACS analysis.

Results: Both Dex and SEGRAs induced the nuclear translocation of GR in Caco-2 cells and revealed no apoptotic potential in used concentrations. However in contrast to SEGRAs, Dex significantly decreased GR expression, mRNA-levels and -stability over time. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activation and reduced IL-8 secretion of Caco-2 cells, but showed no trans-activation potency in pGRE-Luc reporter gene assay. Furthermore, Dex, but not SEGRAs, caused a dose-dependent inhibition of IEC-6 intestinal epithelial cell migration with no effect on cell proliferation in relevant concentrations. The addition of TGF-β1 or EGF partially reversed this effect. TGF-β1 and MAPK signalling pathways important for intestinal wound healing were not affected by SEGRAs but Dex, shown by decreased TGF-β1 peptide levels, smad-expression and decreased pSBE4-Luc reporter gene activity. Additionally, Dex inhibited EGFR phoshorylation and ERK1/2 phosphorylation by trans-activation of MKP-1 transcription and Annexin-1 expression, respectively.

Conclusion: Our results illustrate an anti-inflammatory action of CpdA and ZK216348 comparable to Dex with fewer side-effects in respect to intestinal wound healing. We suggest that SEGRAs might offer a new therapeutic option for inflammatory disorders as IBD.