Abstract
Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.
Highlights
The intestinal epithelium acts as a barrier between the internal and external environments and plays an important role in absorbing nutrient substances into the circulation and limiting the infiltration of harmful substances
Well-established intestinal epithelial co-culture model should have a tight barrier, produce mucus and express mucin-related genes, so the optimal combination of Caco-2 and HT-29 cells was determined by measuring epithelial monolayer integrity and mucus production
trans-epithelial electrical resistance (TEER) values of individual groups were summarized as areas under the curve (AUC) and this measure was used for statistical analysis (Fig. 1B)
Summary
The intestinal epithelium acts as a barrier between the internal and external environments and plays an important role in absorbing nutrient substances into the circulation and limiting the infiltration of harmful substances. Intestinal epithelial integrity is dependent on the organization of the cell–cell junctional complexes, including tight junctions (TJs), adherens junctions (AJs) and d esmosomes[2]. Enterocytes that make up more than 80% of all intestinal cells highly express TJs/AJs proteins, forming a tight epithelial layer[7, 8]. Goblet cells protect the epithelium from the luminal contents by secreting mucins and forming a mucous buffer layer[9]. Both the junctional network and the mucous layer protect the intestinal epithelial integrity. Any factor that adversely affects the expression or localization of intercellular junctions or the mucosal barrier may lead to loss of epithelial integrity, which is possibly involved in gastro-intestinal (GI) d isorders[9]. When hypoxia occurs simultaneously with a heat stimulus, synergic deleterious effects on gut integrity may occur, knowledge about this topic is scarce
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