Figure 2.

Milk fat globule-epidermal growth factor 8 (MFGE8) exerts antifibrotic properties in chronic dextran sodium sulfate (DSS)-induced colitis. (A–G) Chronic DSS colitis was induced in Balb/C mice by two cycles of 3.5% DSS administration and recovery. 3600 ng or recombinant mouse MFGE8 or vehicle control was applied as enema every 4 days starting from the first day of DSS administration. The severity of DSS-induced colitis was evaluated by measuring (A) body weight loss and (B) calculating the clinical score consisting of blood in stool, weight loss and stool consistency. MFGE8 and DSS-treated animals have reduced weight loss and lower clinical score. (C) Colon length was less reduced in MFGE8-treated and DSS-exposed mice compared with DSS alone. (D) Representative images from mouse colon sections stained withH&E, Masson’s trichrome (MT), sirius red (SR), collagen I (COLI), fibronectin (FN) or α-smooth muscle actin (α-SMA). Slides are representative of n=5–8 per group. Arrows point towards the area of fibrosis. (E) Inflammation score was determined by an IBD pathologist in a blinded fashion using H&E sections. There was no difference in DSS-treated animal irrespective of exposure to MFGE8 or not. Fibrosis score as determined by an IBD pathologist in a blinded fashion using MT sections and automatic quantification using SR sections was analysed. MFGE8 reduced the fibrosis score per cent sirius red area in DSS-exposed animals. (F) MFGE8 reduced the per cent positive area for FN and COLI and showed a trend of reduction of α-SMA. (G) MFGE8 reduced the thickness of the submucosa, muscularis mucosa and muscularis propria in DSS-exposed animals. Data are presented as mean±SEM (n=8 per group from two independent experiments). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. DSS, dextran sodium sulfate; MFGE8, milk fat globule-epidermal growth factor 8; PBS, phosphate-buffered saline; α-SMA, α-smooth muscle actin; FN, fibronectin; COLI, collagen I.