title: Dietary transforming growth factor-beta 2 (TGF-β2) supplementation reduces methotrexate-induced intestinal mucosal injury in a rat.
abstract: OBJECTIVE
  Dietary supplementation with transforming growth factor-beta (TGF-β) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-β2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model.
  
  
  METHODS
  Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-β2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-β rats were treated with diet enriched with TGF-β2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-β rats were treated with diet enriched with TGF-β2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, β-catenin, IL-1B and bax protein expression.
  
  
  RESULTS
  Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-β2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-β2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-β2 also led to increased pERK, IL-1B and β-catenin protein levels in intestinal mucosa.
  
  
  CONCLUSIONS
  Treatment with TGF-β2 prevents mucosal-injury, enhances p-ERK and β-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.
authors.affiliation: Laboratory of Intestinal Adaptation and Recovery, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Shani
authors.initials: S
authors.lastname: Ben-Lulu
authors.forename: Yulia
authors.initials: Y
authors.lastname: Pollak
authors.forename: Jorge
authors.initials: J
authors.lastname: Mogilner
authors.forename: Jacob
authors.initials: J
authors.lastname: Bejar
authors.forename: Arnold
authors.initials: A
authors.lastname: G Coran
authors.forename: Igor
authors.initials: I
authors.lastname: Sukhotnik
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001835:Body Weight / Q000187:drug effects; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D019587:Dietary Supplements; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008727:Methotrexate / Q000633:toxicity*; D052016:Mucositis / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053781:Transforming Growth Factor beta2 / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D053583:Interleukin-1beta; D019253:Proto-Oncogene Proteins c-bcl-2; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D053781:Transforming Growth Factor beta2; D051028:bcl-2-Associated X Protein; D051176:beta Catenin; D008727:Methotrexate