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Triamcinolone acetonide prevents oxidative stress-induced tight junction disruption of retinal pigment epithelial cells

  • Basic Science
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Abstract

Purpose

Oxidative stress is known to disrupt the integrity of retinal pigment epithelium (RPE) tight junctions. The goal of this study is to evaluate the effect of triamcinolone acetonide (TA) on the junctional integrity of RPE under oxidative stress and to identify the underlying mechanisms.

Methods

Second passage porcine RPE cells were cultured on 6-well membrane inserts until 4 weeks after reaching confluence. Cells were incubated with TA (10−5 M) for 30 min. FITC-containing medium was added to the upper chamber (cell’s apical side). The cells were then challenged with 1 mM Hydrogen Peroxide (H2O2). After 5 h, the fluorescence intensity of the medium from lower chamber (cell’s basolateral side) was measured using a fluorescence spectrofluorophotometer. This transepithelial flux of FITC-dextran was measured until the 21st day. The immunolocalization of occludin and F-actin was examined with fluorescence microscope. Reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio was determined by a colorimetric assay kit.

Results

Non-lethal oxidative stress by H2O2 increased transepithelial flux of FITC-dextran significantly. TA inhibited this increase and preserved the lower flux through the whole experimental period. This permeability change by H2O2 was reversible and recovered to the normal level within 3 weeks. In immunohistological study, H2O2 reduced linear occludin staining at the cell border and increased actin stress fibers. TA prevented H2O2-induced disruption of junctional assembly of occludin and F-actin. Glutathione assay demonstrated that intracellular GSH/GSSG ratio decreased significantly with H2O2, while TA preserved this ratio by up-regulating GSH synthesis.

Conclusions

TA has a protective effect against oxidative stress-induced disruption of RPE tight junction by preserving cellular redox state.

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Acknowledgment

We would like to thank Dr. Alexa Klettner for helpful discussion.

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  1. Department of Ophthalmology, University Hospital Schleswig-Holstein, Campus Kiel, Hegewischstrasse 2, 24105, Kiel, Germany

    Yoko Miura & Johann Roider

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Miura, Y., Roider, J. Triamcinolone acetonide prevents oxidative stress-induced tight junction disruption of retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol 247, 641–649 (2009). https://doi.org/10.1007/s00417-009-1041-6

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