ABSTRACT
OBJECTIVE: To investigate the effects of hydrogen peroxide (H2O2) on the barrier function and expression of tight junction protein in human retinal pigment epithelium (RPE) cells. METHODS: Experimental study. The human RPE cell line (D407) were cultured and treated with (H2O2 treated group) or without H2O2 (normal control group). The effect of H2O2 on cell viability of RPE cells was determined by MTT test. After treated with low concentration of H2O2 for 24 h to 72 h, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudin-1 to -4 were determined by real-time polymerase chain reaction and Western blot analysis.t-text and one-way ANOVA were used to assess statistical significance between H2O2 treated and normal control groups. RESULTS: H2O2 at 0.2 mmol/L showed no decrease of cell viability of D407 cells, and this concentration was selected for the present study. The TER of D407 cells gradually increased, peaking at day 8 and then remained stable for 1 week. As compared to the control group, a reduction in the TER was first evident after 3 hours of treatment. Continuous culturing of cells for longer periods further reduced the TER, with a maximum effect after 24 hours of treatment and was maintained to 72 hours (24 h: 11.86 ± 1.19 vs. 24.13 ± 1.26, t = 12.260, P = 0.000; 72 h: 11.56 ± 1.47 vs. 24.33 ± 1.52, t = 10.460, P = 0.000). At any time point after adding sodium fluorescein, the permeability values of cells after treated with H2O2 for 24 hours were significantly higher than those of cells without H2O2 treatment (20 min: 25% ± 3% vs. 12% ± 4%, t = -4.50, P = 0.011; 40 min: 36% ± 4% vs. 16% ± 5%, t = -5.41, P = 0.006; 60 min: 51% ± 5% vs. 29% ± 6%, t = -4.88, P = 0.008). The expression of mRNA and protein in claudin-1, -3, and -4 were all downregulated in D407 cells treated with H2O2, whereas the expression of claudin-2 was upregulated (claudin-1 mRNA: 0.98 ± 0.18 vs. 0.28 ± 0.12, t = 5.60, P = 0.005, claudin-1 protein, 48 ± 10 vs. 100 ± 12, t = 5.77, P = 0.004; claudin-3 mRNA: 0.37 ± 0.12 vs.1.03 ± 0.15, t = 5.95, P = 0.004; claudin-3 protein: 63 ± 13 vs. 100 ± 15, t = 3.23, P = 0.032; claudin-4 mRNA: 0.38 ± 0.11 vs.0.99 ± 0.17, t = 5.22, P = 0.002, claudin-4 protein, 57 ± 12 vs. 100 ± 13, t = 4.21, P = 0.014). However, the expression of these occluding did not differ between cells treated with and without H2O2 (mRNA:1.30 ± 0.21 vs. 1.02 ± 0.16, t = -1.84, P = 0.140; protein: 109 ± 15 vs. 100 ± 14, t = -0.76, P = 0.490). CONCLUSION: Oxidative stress causes increase in the paracellular permeability of RPE cells in vitro, which may depends on the changes in expression of certain transmembrane proteins associated with the tight junction.
