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P004. Involvement of the endocannabinoid system in Crohn's disease fibrogenesis

A. Di Sabatino1, L. Rovedatti1, P. Biancheri2, M. Guerci1, T.T. MacDonald2, G.R. Corazza1

1First Department of Medicine, Fondazione IRCCS Policlinico S.Matteo, University of Pavia, Pavia, Italy; 2Centre for Immunology and Infectious Disease, Blizard Institute of Cell and Molecular Science, Bart's and The London School of Medicine and Dentistry, London, United Kingdom

Background and Aims: There is evidence that the endocannabinoid system is involved in liver fibrosis [1]. To elucidate the role of endogenous cannabinoids in the fibrogenic process in Crohn's disease (CD), we explored the in vitro effects of methanandamide, the synthetic analogue of the major cannabinoid ligand anandamide [2], on collagen production and migration by intestinal myofibroblasts isolated from CD strictures.

Materials and Methods: Mucosal myofibroblasts were isolated from surgical specimens collected from colonic strictured and unstrictured areas of 12 patients affected by fibrostenosing CD. Colonies of myofibroblasts were seeded in 25 cm2 flasks with Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal calf serum and antibiotics, and splitted until passage 3. Subconfluent monolayers of myofibroblasts were harvested for 24 h in serum-free DMEM in 12-well plates then incubated for 24 h with methanandamide or medium only. Soluble total collagen was detected on myofibroblast supernatants using Sircol collagen assay. A wound healing scratch assay [3] was performed on myofibroblast monolayers grown in cell culture dishes with 2-mm grids in the presence or absence of methanandamide for 48 h. Photographs of the cells in each grid along the induced wound were taken at 0, 2, 4, 8, 16, and 24 h using a digital camera attached to a light microscope.

Results: We observed that methanandamide significantly down-regulated collagen production by CD strictured myofibroblasts (from mean 343.7±48.2 μg/ml to 155.5±16.9 μg/ml; p < 0.01). The same effect was observed on unstrictured CD myofibroblasts (from mean 181.2±15.5 μg/ml to 64.7±8.4 μg/ml; p < 0.01). As expected, in unstimulated conditions CD strictured myofibroblasts showed a significantly (p < 0.001) higher collagen production in comparison to unstrictured CD myofibrobalsts. Moreover, methanandamide significantly (p < 0.005) improved migration of CD strictured myofibroblast after 24 h-culture (27.8%) in comparison to medium only (15.0%). The same effect was observed on unstrictured CD myofibroblasts (from 36.4% to 18.1%). As expected, in unstimulated conditions CD strictured myofibroblasts showed a significantly (p < 0.001) lower migration capacity in comparison to unstrictured CD myofibrobalsts.

Conclusions: Our results provide evidence for an anti-fibrogenic role of anandamide in CD. Targeting the endogenous cannabinoid system by synthetic cannabinoid receptor agonists might represent a promising therapeutic strategy in counteracting intestinal fibrogenesis in CD.