Preparation Confluent MDCK.2 cells were washed twice with ice-cold PBS and detached using a rubber policeman in 1 ml of the hypotonic buffer A,

d with our in vivo study and with data from the literature. Interestingly, the expression of the pro-apoptotic gene bax was also down-regulated in TGF-b rats compared to control animals. We believe that the decreased bcl-2 expression drives increased cell apoptosis, while the down-regulation of bax mRNA creates a higher resistance of enterocytes to apoptosis. The other explanation of this phenomenon may be the different rates of apoptosis at multiple time points following development of mucositis. Consistent with this DCC-2036 concept is the observation that in the first few days MTX may down-regulate the bcl-2 gene expression that drives elevated cell apoptosis. After three days, the bulk of apoptotic enterocytes appear, leading to down-regulation of bax mRNA in attempt to decrease cell death, while the bcl-2 gene still remains decreased. These findings suggest an important role for the differential regulation of apoptosis related genes as coordinators of the early increase in cell apoptosis after MTX-induced damage. Consistent with our previous experiments, MTX-induced mucositis in the current study resulted in apparent intestinal damage. This conclusion is supported by the observed increase in the Park injury score compared to control animals. MTX rats also showed severe villous atrophy, epithelial flattening, and extensive crypt loss. In addition, treatment with MTX resulted in significant mucosal hypoplasia. A decrease in bowel and mucosal weight, a decrease in mucosal DNA and protein, and decrease in villus height and crypt depth support this conclusion. Parallel decreases in mucosal DNA and protein indicate that the smaller mucosal mass of MTX animals can be attributed to cellular hypoplasia. Histologically, villus height and crypt depth decreased in response to MTX administration, suggesting decreased absorptive surface area. We also observed strong inhibitory effects of MTX on enterocyte proliferation, which may be considered as a major mechanism responsible for decreased intestinal cell mass and mucosal hypoplasia. As a folic acid analogue, the action of MTX primarily inhibits DNA synthesis by binding to the enzyme dihydrofolate reductase. This leads to an inhibition of proliferation in the crypts of the small intestine. These changes were in agreement with previous findings that demonstrated marked damage in the crypt epithelium at days 1 and 2 after MTX administration, while days 3 and 4 represented a phase of prominent damage to the villous epithelium, marked by reduced cell and villous heights, and villous atrophy. A decreased cell proliferation rate in MTX animals was accompanied by decreased levels PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/2221058 of p-ERK protein levels. The transmission of extracellular proliferation and differentiation signals into their intracellular targets is mediated by a signaling cascade culminating in mitogenactivated protein kinase. One of the MAPK signaling pathways triggered by cytokines or growth factors is the extracellular signal-related kinase pathway. The relationship between MTX and MAPK pathway has been described previously in various experimental models and clinical trials. TGF-b2 Reduces MTX Induced Intestinal Injury treated rats showed more preserved architecture as well as the presence of newly formed crypts and regeneration. 80% of rats showed a significant decrease in intestinal mucosal injury grade compared to MTX animals, suggesting lesser degrees of intestinal damage. In addition, exposure to enteral TGFb2 accelerated intestinal muco