Down regulation of UCP2 expression in retinal pigment epithelium cells under oxidative stress: an in vitro study

To evaluate the expression of uncoupling protein 2 (UCP2) in a retinal pigment epithelium cell line (ARPE-19), under oxidative stress (OS).

ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide (H₂O₂; 0, 150, 300, 500, 700, and 900 µmol/L) for 24h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H₂O₂ was investigated by reverse transcription-polymerase chain reaction (RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry (FCM). Further, UCP2-siRNA treated cultures were exposed to H₂O₂ (0, 75, 150, and 300 µmol/L) for 2h and cell viability determined by MTT assay.

Cells treated with higher concentrations of H₂O₂ appeared shrunken; their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H₂O₂. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 µmol/L H₂O₂ (P<0.05). Cell metabolic activity was decreased with increased concentrations of H₂O₂ as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H₂O₂ alone (P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H₂O₂-treated groups compared with controls (P<0.05). FCM analysis showed that cell reactive oxygen species (ROS) levels were increased in H₂O₂-treated groups and further upregulated by UCP2-siRNA treatment (P<0.05).

Expression levels of UCP2 are decreased in ARPE-19 cells treated with H₂O₂. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H₂O₂ alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.