Effects of MTX and TGF-β on enterocyte proliferation (A) and apoptosis (B).

<p>Values are mean ± SEM. CONTR-control, MTX-methotrexate, TGF-β- transforming growth factor beta. *P<0.05 MTX and CONTR-TGF-β versus control, ^†P<0.05 MTX-TGF-β versus MTX rats. (<b>C</b>) Representative slides of cell proliferation and apoptosis following TGF-β2 administration during MTX-induced mucositis. MTX-treated animals showed a significant decrease in cell proliferation rates and concomitant increase in cell apoptosis compared to control animals. TGF-β2 administration in MTX rats resulted in a significant increase in cell proliferation and decrease in cell apoptosis compared to MTX animals.</p

Effects of MTX on body weight changes (A), bowel and mucosal weight (B) and mucosal DNA and protein (C).

<p>Values are mean ± SEM. CONTR- control, MTX-methotrexate. * P<0.05 MTX-2 and MTX-4 vs CONTR rats, † P<0.05 MTX-4 vs MTX-2 rats.</p

Effect of TGF-β on intestinal injury score and microscopic intestinal appearance following MTX-induced mucositis.

<p>The degree of intestinal tissue injury was evaluated on a grading scale from 0 to 8 as described previously by Park (A). The villus height and crypt depth were measured using the Image Pro plus 4 image analysis software (B). (<b>C</b>) Representative slides of MTX-induced intestinal injury. The histopathology analysis of the tissue sections from MTX-treated animals showed a significant epithelial atrophy, blunting of the villi and signs of crypt remodeling that was accompanied by marked cellularity mainly by mononuclear cells in the lamina propria, the presence of the flattened and vacuolated cells, and an increased number of blood vessels in the stroma. TGF-β2 administration resulted in less significant epithelial atrophy and crypt remodeling compared to MTX rats. Values are mean ± SEM. CONTR-control, MTX-methotrexate, TGF-β- transforming growth factor beta. *P<0.05 MTX and MTX-TGF-β versus control, ^†P<0.05 MTX-TGF-β versus MTX rats. CONTR-control,</p

Effects of MTX on crypt cell proliferation (A) and apoptosis (B).

<p>The number of labeled cells in 10 well-oriented, longitudinal crypts per section from each rat was determined using light microscopy (B). The apoptotic index is expressed as the percentage of apoptotic cells per 10 villi. Values are mean ± SEM. CONTR- control, MTX-methotrexate. * P<0.05 MTX-2 and MTX-4 vs CONTR rats, † P<0.05 MTX-4 vs MTX-2 rats.</p

Effect of MTX and TGF-β on bowel and mucosal weight.

<p>Values are mean ± SEM. CONTR-control, MTX-methotrexate, TGF-β - transforming growth factor beta. *P<0.05 MTX and CONTR-TGF-β versus control rats. ^†P<0.05 MTX-TGF-β versus MTX rats.</p

Effect of TGF-β on MTX-induced apoptosis in Caco-2 cells.

<p>Treatment of Caco-2 cells with MTX resulted in a significant increase in cell apoptosis compared to non-treated cells. Incubation of MTX-pre-treated Caco-2 cells with TGF-β (0.1 and 0.5 ng/ml) resulted in two-fold decrease in MTX induced cell apoptosis. Values are mean ± SEM. CONTR- control, MTX-methotrexate, TGF-β-transforming growth factor beta. *P<0.05 versus non-treated cells, ^†P<0.05 MTX-TGF-β 0.1 ng/ml and MTX- TGF-β 0.5 ng/ml versus MTX pretreated cells.</p

Effect of MTX on Wnt/β-catenin related genes (β-catenin, FRZ-2, Wnt 3A Wnt 5A, c-myc and Cyclin D1 mRNA) expression in gut mucosa following methotrexate induced intestinal damage.

<p>Values are mean ± SEM. CONTR- control, MTX-methotrexate. * P<0.05 MTX-2 and MTX-4 vs CONTR rats, † P<0.05 MTX-4 vs MTX-2 rats.</p

Effect of TGF-β and MTX on cell viability of Caco-2 cells (Alamar Blue reduction test).

<p>Cells were cultured in 96-well plates at a density of 4×10⁴ cells per well up to asubconfluent monolayer (80%), and then they were treated with 0.1 and 0.5 ng/ml TGF-β or control medium for 48 h,and then treated with 0.1 and 0.5 ng/ml TGF-β or MTX 125 nM, or 0.1 ng/ml and 0.5 ng/ml TGF-β with MTX 125 nM or control medium for 72 h. After the treatment, 20% of Alamar Blue was added to each well, and cells were incubated at 37°C for 3 h. Optical density was measured spectrophotometrically at 570 and 630 nm. Cell viability was calculated as percentage of the difference between the reductions of Alamar Blue in treated versus control. Results are presented as percentage of controls, mean ± SEM. CONTR-control, MTX- methotrexate, TGF-β- transforming growth factor beta. *P<0.05 MTX versus control rats, ^†P<0.05 MTX-TGF-β 0.1 ng/ml and MTX-TGF-β 0.5 ng/ml versus MTX rats.</p

Effect of TGF-β on the mucosal DNA and protein content following MTX-induced mucositis.

<p>Values are mean±SEM. CONTR-control, MTX-methotrexate, TGF-β- transforming growth factor beta. *P<0.05 MTX and MTX-TGF-β versus control, ^†P<0.05 MTX-TGF-β versus MTX rats.</p

Effect of TGF-β on Bax mRNA (A) and Bcl-2 mRNA (B) expression in gut mucosa following MTX-induced intestinal mucositis.

<p>Results are expressed as the ratio of the investigated mRNA over 18S mRNA expression. Values are mean ± SEM. CONTR-control, MTX- methotrexate, TGF-β -transforming growth factor beta. *P<0.05 MTX, MTX-TGF-β and CONTR-TGF-β versus control rats, ^†P<0.05 MTX- TGF-β versus MTX rats.</p