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Hypoxia-Induced Intestinal Epithelial Injury Disrupts Tight Junction Integrity and Activates Proinflammatory Signaling: Evidence From In Vitro and Mouse Models

Aditya Pratama Wijaya, MD, PhD1; Dewi Kartika Sari, MSc, PhD2; Rizky Maulana Putra, MD, PhD2; Nur Aisyah Rahmawati, MD, PhD1,3 

1Department of Biomedical Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya,
Indonesia.
2Department of Anatomical Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya,
Indonesia.
3Laboratory of Molecular Medicine and Stem Cell Research, Faculty of Medicine, Universitas
Airlangga, Surabaya, Indonesia.

DOI: 10.18081/2378-5225/11.1 
Cited by 0

 Article history: Received 22 December 2021 · Revised 01 February 2022 · Accepted 11 March 2022 · Published 30 March 2022

© 2022 Bennett et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0). 

CC BY 4.0                                                                                                   

Abstract

Background: Intestinal epithelial hypoxia is a major component of ischemia, sepsis, shock, and inflammatory gastrointestinal disorders and can compromise mucosal barrier integrity. However, the relationship among sustained hypoxic stress, tight junction disruption, oxidative injury, and proinflammatory signaling remains incompletely defined. This study investigated the molecular mechanisms linking hypoxia to intestinal epithelial barrier dysfunction using complementary in vitro and in vivo models.

Methods:Differentiated Caco-2 intestinal epithelial monolayers were exposed to normoxia or 1% O₂ for 6, 12, or 24 hours. Barrier integrity was evaluated by transepithelial electrical resistance (TEER), FITC-dextran permeability, and expression and localization of ZO-1, occludin, and claudin-1. HIF-1α accumulation, reactive oxygen species (ROS), mitochondrial membrane potential, cellular ATP, NF-κB/p38/JNK signaling, TNF-α, IL-1β, IL-6, and apoptotic markers were assessed. In parallel, intestinal injury was examined in a mouse model of superior mesenteric artery ischemia followed by reperfusion using histopathology, tight junction analysis, oxidative-stress indices, inflammatory signaling, cytokine measurements, and TUNEL analysis.

Results: Sustained hypoxia produced a time-dependent deterioration of Caco-2 epithelial barrier function, characterized by decreased TEER, increased paracellular permeability, and progressive loss and redistribution of ZO-1, occludin, and claudin-1. These changes were accompanied by HIF-1α stabilization, ROS accumulation, mitochondrial depolarization, ATP depletion, and activation of NF-κB, p38 MAPK, and JNK signaling. Hypoxic exposure also increased TNF-α, IL-1β, and IL-6 production and promoted apoptotic signaling characterized by increased Bax and cleaved caspase-3 and reduced Bcl-2. Intestinal ischemia–reperfusion reproduced the principal molecular phenotype observed in vitro, including oxidative stress, tight junction disruption, NF-κB/MAPK activation, increased inflammatory cytokines, epithelial apoptosis, and marked mucosal structural injury.

Conclusion: Sustained hypoxic stress is associated with intestinal epithelial barrier failure through an integrated process involving mitochondrial and oxidative dysfunction, disruption of tight junction architecture, activation of NF-κB/MAPK-dependent inflammatory signaling, and epithelial apoptosis. Concordant findings across Caco-2 and mouse intestinal ischemia–reperfusion models support a mechanistic framework linking hypoxia to progressive mucosal injury. Preservation of mitochondrial function and epithelial tight junction integrity and modulation of excessive inflammatory signaling may represent potential therapeutic strategies for hypoxia-associated intestinal injury.

Keywords: Intestinal epithelial injury; hypoxia; HIF-1α; tight junctions; ZO-1; occludin; claudin-1; oxidative stress; NF-κB

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BM-Publisher · Pathophysiology of Cell Injury Journal (PCIJ) · E-ISSN 2378-5225 · DOI Prefix 10.18081/pcij/2378-5225

Pathophysiology of Cell Injury Journal (PCIJ)
E-ISSN 2378-5225 · Biannual
BM-Publisher (London, UK)
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Vol 11, Issue 1 (March 2022), pp. 40–72

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Wijaya AP, Sari DK, Putra RM, Rahmawati NA. Hypoxia-Induced Intestinal Epithelial Injury Disrupts Tight Junction Integrity and Activates Proinflammatory Signaling: Evidence From In Vitro and Mouse Models. Pathophysiology of Cell Injury Journal (PCIJ). 2022;11(1):40–72. doi: 10.18081/2378-5225/11.40.

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