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Updated: Jun 20, 2026
Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease
Published on: February 9, 2016
Oxidative stress and enhanced paracellular permeability in the small intestine of methotrexate-treated rats
Tomoko Maeda1, Yuko Miyazono, Kousei Ito
1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan.
Purpose:
We previously demonstrated the increase of reactive oxygen species (ROS) production and myeloperoxidase (MPO) activity in the small intestine of methotrexate (MTX)-treated rats. In the present study, we investigated the role of ROS modulating intestinal mucosal permeability in this damage.
Method:
MTX (20 mg/kg body weight) was administered to rats intravenously. N-Acetylcysteine (NAC; 80 mg/kg body wt), an antioxidant and a precursor of glutathione (GSH) was administered to rats intraperitoneally to investigate the contribution of ROS to the intestinal permeability enhancement. Intestinal permeability was evaluated by determining that of a poorly absorbable marker, fluorescein isothiocyanate-labeled dextran (FD-4; average molecular mass, 4.4 kDa) using the in vitro everted intestine technique. The occurrence of oxidative stress in the small intestine was assayed by measuring chemiluminescence and thiobarbituric acid reactive substances (TBARS) productions in mucosal homogenates of the small intestine.
Results:
The mucosal permeability of FD-4 significantly (p < 0.01) increased in MTX-treated rats compared with control rats, as demonstrated by a twofold increase of FD-4 permeation clearance. This suggests an increase in paracellular permeability. Interestingly, the ROS production was observed preceding the increase of paracellular permeability. Treatment with NAC prevented the MTX-induced ROS production and the increase of paracellular permeability.
Conclusions:
NAC protected the small intestine of rats from MTX-induced change in paracellular permeability, suggesting that ROS played an important role in the enhanced paracellular permeability.
Insights
Reactive oxygen species (ROS) increase intestinal permeability after methotrexate (MTX) treatment. N-Acetylcysteine (NAC) treatment prevented this damage, indicating ROS
Area of Science:
- Gastroenterology
- Toxicology
- Biochemistry
Background:
- Methotrexate (MTX) is a chemotherapy agent known to cause gastrointestinal toxicity.
- Previous studies indicated increased reactive oxygen species (ROS) and myeloperoxidase (MPO) activity in the small intestine of MTX-treated rats.
Purpose of the Study:
- To investigate the role of ROS in modulating intestinal mucosal permeability following MTX-induced damage.
- To assess the protective effects of N-Acetylcysteine (NAC) against MTX-induced intestinal permeability changes.
Main Methods:
- Rats were administered MTX (20 mg/kg) intravenously.
- N-Acetylcysteine (NAC; 80 mg/kg), an antioxidant, was administered intraperitoneally.
- Intestinal permeability was assessed using fluorescein isothiocyanate-labeled dextran (FD-4) and the in vitro everted intestine technique.
- Oxidative stress was measured by chemiluminescence and thiobarbituric acid reactive substances (TBARS) production.
Main Results:
- MTX treatment significantly increased intestinal mucosal permeability to FD-4, indicating enhanced paracellular permeability (p < 0.01).
- ROS production was observed to precede the increase in paracellular permeability.
- NAC administration prevented both the MTX-induced ROS production and the subsequent increase in paracellular permeability.
Conclusions:
- ROS play a significant role in the enhanced paracellular permeability of the small intestine observed after MTX treatment.
- NAC demonstrates a protective effect against MTX-induced intestinal damage by mitigating ROS production and preserving intestinal barrier function.
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