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Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease
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Disruption of ZO-1/claudin-4 interaction in relation to inflammatory responses in methotrexate-induced intestinal

Kazuma Hamada1, Naoko Kakigawa, Shuichi Sekine

  • 1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan.

Cancer Chemotherapy and Pharmacology
|August 22, 2013
PubMed
Abstract

Insights

Methotrexate (MTX) treatment elevates intestinal inflammatory mediators, disrupting tight junction protein interactions. This leads to compromised intestinal barrier function, potentially explaining MTX-induced mucositis.

Area of Science:

  • Gastroenterology
  • Pharmacology
  • Molecular Biology

Background:

  • Methotrexate (MTX) treatment can cause intestinal mucositis, limiting its clinical use.
  • MTX-induced reactive oxygen species initiate neutrophil migration and intestinal barrier dysfunction.
  • Alterations in zonula occludens (ZO)-1, a tight junction protein, contribute to this dysfunction.

Purpose of the Study:

  • To identify inflammatory mediators in the intestine of MTX-treated rats.
  • To evaluate MTX-induced alterations in tight junction proteins beyond ZO-1, including occludin and claudins.

Main Methods:

  • Male Wistar rats received daily oral MTX (15 mg/kg) for 4 days.
  • Real-time RT-PCR measured mRNA levels of TNF-α, IL-1β, MIP-2, TLR4, and occludin.
  • Western blotting, immunohistochemistry, and immunoprecipitation assessed TJ protein expression, distribution, and interactions.

Main Results:

  • MTX increased small intestinal mRNA levels of TNF-α, IL-1β, MIP-2, and TLR4.
  • Claudin-2 protein expression and immunostaining increased, while claudin-4 immunostaining decreased.
  • Occludin mRNA decreased, but protein levels and ZO-1 interaction remained unchanged; ZO-1/claudin-4 interaction was compromised.

Conclusions:

  • Elevated intestinal inflammatory cytokines and chemokines in MTX-treated rats contribute to inhibited ZO-1/claudin-4 binding.
  • Compromised ZO-1/claudin-4 binding may lead to reduced claudin-4 expression, impacting intestinal barrier function.

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