PRDX1 exerts a photoprotection effect by inhibiting oxidative stress and regulating MAPK signaling on retinal pigment epithelium

Abstract

Background: The purpose of this study was to investigate the photoprotection effect of peroxiredoxin 1 (PRDX1) protein in ultraviolet B (UVB) irradiation-induced damage of retinal pigment epithelium (RPE) and its possible molecular mechanism.

Methods: ARPE-19 cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the PRDX1 expression. The corresponding kits were employed to measure the levels or activities of lactate dehydrogenase (LDH), 8-hydroxy-2-deoxyguanosine (8-OHdG), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD). Western blotting was applied to examine PRDX1 expression and mitogen-activated protein kinase (MAPK) signaling pathway-related proteins.

Results: After exposure to 20 mJ/cm² intensity of UVB irradiation for 24 h, ARPE-19 cells viability was decreased, the leakage degree of LDH and 8-OHdG were increased, and cell apoptosis was elevated. The expression of PRDX1 was significantly down-regulated in UVB-induced ARPE-19 cells. The low expression of PRDX1 was involved in high irradiation intensity. Overexpression of PRDX1 increased cell activity, decreased cell apoptosis, and LDH as well as 8-OHdG leakage in UVB-induced ARPE-19 cells. In addition to alleviating UVB-induced cell damage, PRDX1 overexpression also inhibited UVB-induced oxidative stress (down-regulation of ROS and MDA levels, up-regulation of GSH-Px and SOD activities) and the activation of MAPK signaling pathway in ARPE-19 cells.

Conclusion: PRDX1 exerts a photoprotection effect on RPE by attenuating UVB-induced cell damage and inhibiting oxidative stress, which can be attributed to the inhibition of MAPK signaling pathway activation.

Keywords: Mitogen-activated protein kinase signaling pathway; Peroxiredoxin 1; Photoprotection; Retinal pigment epithelium.

Fig. 1

PRDX1 expression is down-regulated in UVB-induced ARPE-19 cells. A–D, MTT, biochemical kits, qRT-PCR and western blotting to analyze the effects of UVB irradiation with different intensities (5, 10, 15 and 20 mJ/cm²) on ARPE-19 cell viability (A), levels of LDH in cell culture supernatant (B), the relative mRNA levels of PRDX1 in cells (C) and protein expression levels of PRDX1 (D), **P < 0.01. PRDX1, peroxiredoxin 1; UVB: ultraviolet B; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; LDH: lactate dehydrogenase

Fig. 2

PRDX1 overexpression attenuates UVB-induced damage in ARPE-19 cells. A–E, qRT-PCR, western blotting, MTT and biochemical kits to determine the relative mRNA levels of PRDX1 (A), relative protein expression levels of PRDX1 (B), cell viability (C), cell morphology (D), and the levels of LDH (E) and 8-OHdG (F) in cell culture supernatant in ARPE-19 cells in the Control group, the UVB group, the UVB + vector group and the UVB + PRDX1 group, respectively. (G/H) Flow cytometry to evaluate the effect of UVB irradiation on apoptosis of ARPE19 cells that were untransfected or transfected with vector or PRDX1. **P < 0.01. PRDX1, peroxiredoxin 1; UVB: ultraviolet B; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; LDH, lactate dehydrogenase; 8-OHdG: 8-hydroxy-2-deoxyguanosine

Fig. 3

PRDX1 overexpression inhibits UVB-induced oxidative stress in ARPE-19 cells. A–D, the corresponding biochemical kits to detect the levels of ROS (A) and MDA (B), GSH-Px activity (C) and SOD activity (D) in the Control group, the UVB group, the UVB + vector group and the UVB + PRDX1 group, respectively, **P < 0.01. PRDX1, peroxiredoxin 1; UVB: ultraviolet B; ROS: reactive oxygen species; MDA, malondialdehyde; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase

Fig. 4

PRDX1 overexpression inhibits UVB-induced MAPK signaling activity. A–D, western blotting to measure the protein expression of ERK1/2, p-ERK1/2, JNK, p-JNK, p-38, and p-p38 in ARPE-19 cells (A) in the Control group, the UVB group, the UVB + vector group and the UVB + PRDX1 group, as well as the ratios of p-ERK1/2/ERK1/2 (B), p-JNK/JNK (C) and p-p38/p38 (D) in each group of cells, **P < 0.01. PRDX1, peroxiredoxin 1; UVB: ultraviolet B; MAPK: mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase; p-ERK1/2, phosphorylated-ERK1/2; JNK, c-Jun N-terminal kinase