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P025. Phosphatidylcholine regulates intestinal inflammation and modifies in vivo and in vitro extracellular matrix regulation

A.C. Chew1, F. Lloyd1, I.C. Lawrance1

1University of Western Australia, Centre for Inflammatory Bowel Diseases, School of Medicine and Pharmacology, Fremantle, Australia

Background: Ulcerative colitis (UC) is associated with a lack of phosphatidylcholines (PC) and this may produce a defective mucosal barrier and allow for the translocation of bacteria into the mucosa promoting inflammation. This study investigated PC on inflammation and extracellular matrix (ECM) regulation in the murine model of 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-induced intestinal inflammation and fibrosis, and ECM gene expression in human intestinal fibroblasts.

Methods: C57bl6 mice received TNBS enemas weekly for 2 or 6 weeks, ± PC (200 mg/kg/day/orally), ± indomethacin (Indo) (0.2 mg/kg/day/orally), or a combination of PC/Indo. Inflammation and fibrosis was assessed histologically. The human colonic fibroblast cell line, CCD-18co, was stimulated with PMA (10 ng/µl) for 24hrs prior to treatment with Indo (50 µM) or two isoforms of PC, dilineoyl-PC (DLPC) (50 µM) and linoleoyl-palmitoyl-PC (LPPC) (50 µM) ± Indo. mRNA was isolated and collagen 1 (Col1), MMP‑1, TIMP‑1 and TGF‑β1 expression assessed by RT-qPCR.

Results: At 2 weeks compared to TNBS alone, mice treated with Indo/TNBS had significantly more inflammation and fibrosis (p = 0.004 and 0.00004), those receiving PC/TNBS had significantly less inflammation (p = 0.009), while those treated with PC/Indo/TNBS had less inflammation and fibrosis (p = 0.001 and 0.03). At 6 weeks compared to TNBS alone, inflammation and fibrosis was not different in the Indo/TNBS and PC/Indo/TNBS-treated animals, but PC/TNBS-treated animals had less inflammation (p = 0.01) and fibrosis (p = 0.006). The addition of PC to Indo/TNBS at 2 weeks significantly decreased inflammation and fibrosis compared to Indo/TNBS alone (p = 0.03 and 0.00001), however at 6 weeks, this addition had no effect on Indo/TNBS-induced inflammation or fibrosis. In CCD-18co cells the addition of DLPC increased MMP‑1 and TIMP‑1 mRNA expression (p = 0.03 and 0.01), with no effect on Col1 or TGF‑β1 suggesting that DLPC may decrease ECM deposition. By contrast, LPPC increased Col1 (p = 0.0008) and TIMP‑1 (p = 0.03) and decreased MMP‑1 expression (p = 0.003) with no effect on TGF‑β1 suggesting that LPPC may increase ECM deposition. DLPC/Indo and LPPC/Indo treatment mirrored DLPC and LPPC treatment alone.

Conclusions: The findings demonstrate that PC can reduce inflammation and fibrosis but may require a higher dose to completely abolish the effects of Indo/TNBS in vivo. The PC isoform appears to be important in vitro as DLPC may decrease and LPPC increase ECM deposition by isolated fibroblasts.