Mo1823 Involvement of MDR1 in Intestinal Epithelial Injury Caused by Acetylsalicylic Acid

Abstract

Introduction Intestinal fibrosis (IF) is a major complication of chronic inflammation of the intestinal tissue in inflammatory bowel disease (IBD). IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. Currently, adequate models are lacking to study the mechanism of intestinal fibrosis in IBD. Our aim is to develop an ex vivo model for IF by using mouse precision-cut intestinal slice (PCIS). In addition, regional differences in the onset of fibrosis in the bowel will be studied. In PCIS all cell types are present in their original tissue-matrix environment. As fibrosis is a multicellular phenomenon, PCIS can be used as a model to study the early onset of intestinal fibrosis. Methods Mouse jejunum, ileum and colon were excised and prepared as segments embedded in agarose. PCIS (estimated 300-400 μm) were prepared and incubated up to 48 hr. ATP content of the PCIS was used to assess the general viability. To study the early onset of fibrosis, the gene expression of different fibrosis markers: Pro-Collagen 1 A1 (COL1A1), Heat Shock Protein 47 (HSP47), alpha-Smooth Muscle Actin (SMA) and Fibronectin (FN1) was determined in PCIS after 48 hr of incubation. Results Mouse PCIS from the different regions of the bowel were viable up to 48 hr in culture. After 48 hr of incubation of PCIS, HSP47 and FN1 gene expression was significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.9 and 3.6 fold, respectively) and in ileum (4.5 and 4.9 fold, respectively). Gene expression of Col1A1 (0.7 fold) was only down-regulated in PCIS from the colon. SMA gene expression was down-regulated in PCIS from all the regions of the intestine. PCIS from jejunum cultured in the presence of 5ng/mL Transforming-Growth Factor (TGF)β1 for 48 hr, HSP47 (1.2 fold), FN1 (4.6 fold) and COL1A1 (2.0 fold) were significantly upregulated compared to control PCIS after 48 hr of incubation. In addition, in PCIS from Ileum incubated with 5ng/mL TGF-β1, the gene expression of HSP47 (1.9 fold) and FN1 (3.9 fold) was significantly increased. In PCIS from the colon cultured for 48 hr in the presence of 5ng/ml TGF-β1, the gene expression of fibrosis markers was not affected. Conclusion Mouse PCIS from different regions of the bowel can successfully be cultured for 48 hr. Fibrosis markers are induced by incubation up to 48 hr and by addition of TGFβ1 in jejunum and ileum PCIS, but not in colon PCIS. We are the first to show that regional differences in the onset of intestinal fibrosis can be measured using PCIS.