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

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 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 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

Effects of MTX and TGF-β on TGF-βR protein.

<p>Relative expression of TGF-βR protein in jejunum, as determined by Western blot analysis, of rats following MTX induced mucositis (A). Values are mean ± SEM. CONTR–control, MTX-methotrexate, TGF-β-transforming growth factor beta. *P<0.05 MTX-TGF-β and CONTR-TGF-β versus control rats, ^†P<0.05 MTX-TGF-β versus MTX rats. ERK was detected using anti-ERK antibody to verify equal protein loading. (<b>B</b>) Immunohistochemistry showing the expression of the type II TGF-β receptor along the crypt-villus axis in jejunum. Sections were incubated with an affinity purified rabbit anti-rat type II TGF-β receptor polyclonal antibody which is non-reactive with the type I receptor. Color was developed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045221#s2" target="_blank">Materials and methods</a>. There was no change in the expression of the receptor along the villus-crypt axis (arrows-expression in villus, arrowheads- expression in crypt) in control rats. Treatment with MTX resulted in a lower type II TGF-β2 receptor expression compared to control animals. Treatment of control and MTX animals with TGF-β2 resulted in a more significant receptor expression compared to control and MTX-non-treated animals, both in the crypt region and along the entire villus, especially at the basolateral side of enterocytes. The staining was more prominent in differentiated villus tip cells compared to crypt cells. The changes in distribution of Type II TGF-β2 receptor was correlated with the protein levels observed in the Western blot analysis.</p

Grade of intestinal mucosal injury and microscopic intestinal appearance after administration of MTX.

<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