Effects of hyperoxia on the permeability of 16HBE14o– cell monolayers – the protective role of antioxidant vitamins E and C

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Abstract

The use of hyperoxia for critically ill patients is associated with adverse impacts resulting in lung injury accompanied by inflammation. The aim of this study was to evaluate aspects of mechanisms that contribute to hyperoxia-induced disruption of the epithelial permeability barrier, and also the protective effects of the antioxidants α-tocopherol and ascorbate. 16HBE14o– cells were cultured as monolayers at an air–liquid interface for 6 days, after which transepithelial electrical resistance reached 251.2 ± 4.1 Ω.cm2 (mean ± standard error of the mean). They were then exposed for 24 h to normoxia (21% O2, 5% CO2), hyperoxia (95% O2, 5% CO2), hyperoxia with 10−7 m α-tocopherol, hyperoxia with 10−7 m ascorbate, hyperoxia with 10−6 m ascorbate, and hyperoxia with a combination of α-tocopherol and ascorbate (10−7 m and 10−6 m, respectively). Significant reductions (P < 0.05) in transepithelial electrical resistance seen after hyperoxia (with or without antioxidants) were associated with reductions in the levels of zona occludens-1 (ZO-1) observed by immunohistochemistry, and downregulation of ZO-1 expression (P < 0.01) as compared with normoxia. In contrast, the expression levels of interleukin (IL)-8, IL-6 and tumour necrosis factor-α (TNF-α) were increased after hyperoxia (P < 0.01), and marked increases in the levels of these cytokines (ELISA) were seen in the medium (P < 0.001) as compared with normoxia. The antioxidant vitamins E and C had a partial protective effect against the hyperoxia-induced reduction in ZO-1 levels and the increase in levels of the proinflammatory cytokines IL-8, IL-6, and TNF-α. In conclusion, hyperoxia-induced epithelial disruption is associated with tight junction weakening, and induction of a proinflammatory environment.

16HBE14o- cell monolayers were exposed for 24 h to normoxia, hyperoxia and hyperoxia with α-tocopherol and/or ascorbate. Decreased TER seen after hyperoxia was associated with decreased ZO-1 (immunohistochemistry, see figure), and decreased ZO-1 expression. Expression of IL-8, IL-6 and TNF-α were increased after hyperoxia and these cytokines increased in the medium. Antioxidant vitamins had a partial protective effect.

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