PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression.

Abstract

To investigate the protective function of pigment epithelium-derived factor (PEDF) against oxidative stress (OS) in ARPE-19 cells, ARPE-19 cells were divided into different OS groups and treated with various concentrations of H2O2 (0, 75, 150 and 200 µmol/l) for 24 h. To establish the protective group, 200 ng/ml of PEDF was administered to ARPE-19 cells. Cell Counting Kit-8 assays and cell growth curve experiments were performed to determine levels of cell viability; lactate dehydrogenase and propidium iodide (PI) staining assays were also performed. The expression levels of genes associated with apoptosis as well as uncoupling protein 2 (UCP2) were detected by reverse transcription-quantitative, or semi-quantitative polymerase chain reaction. Furthermore, an OS injury animal model was established in both C57BL/6 and BALB/c mice via injection of 5 µg of PEDF in the vitreous cavity and subsequent injection of 150 µM H2O2 following a 24 h time interval. Hematoxylin and eosin (H&E) staining, as well as UCP2 immunofluorescent labeling were also performed. One-way analysis of variance was used to determine statistically significant differences, followed by multiple comparison analysis using the Newman Keuls method. The results of cell viability assays demonstrated that the numbers of apoptotic cells were increased following treatment with H2O2 in a dose-dependent manner; however, this effect was reversed following treatment with PEDF. The expression levels of caspase 3 and B cell lymphoma (Bcl2) associated X genes associated with apoptosis were inhibited, whereas levels of the anti-apoptotic gene Bcl2 were enhanced following treatment with PEDF in different passages of ARPE-19 cells. Significant differences were demonstrated in the levels of UCP2 gene expression between the PEDF+ H2O2 treated group and cells treated with H2O2 alone. Labeling of the UCP2 detector in the confocal images demonstrated decreased UCP2 protein staining in the retinal pigment epithelium (RPE) cells and RPE layers following H2O2 injury; however, this effect was inhibited following treatment with PEDF. H&E staining was performed to investigate the thickness of the RPE layers, and the results revealed that thicknesses were significantly increased in sections treated with PEDF during OS, due to increased numbers of RPE cells. Furthermore, PEDF was demonstrated to increase UCP2 gene expression in ARPE-19 cells and animal RPE layers under OS, which suggested that PEDF may protect RPE cells and tissues during oxidative injury.