Biological Effect of Mycosporine-Gly-Ser (Shinorine) Against Bis-Retinoid N-Retinyl- N-Retinylidene Ethanolamine- and Blue-Light-Induced Retinal Pigment Epithelium Cell Damage

Abstract

Shinorine is a mycosporine-like amino acid isolated from laver (Porphyra dentata), and interest in its functionality has increased recently due to increased production using yeast. There have been few reports on the pharmacological activity of shinorine, and we sought to find the pharmacological significance of shinorine. In the present study, we investigated the pharmacological effects of shinorine purified from Porphyra dentata on ARPE-19 cells. First, when ARPE-19 cells were treated with bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) and blue light (BL), cytotoxicity increased, and apoptosis was observed. We investigated the effects of shinorine on A2E- and BL-induced cytotoxicity and changes in apoptotic factors, inflammation, and carbonyl stress. A2E and BL exposure increased ARPE-19 cell apoptosis, but this increase was attenuated by shinorine in a concentration-dependent manner. Treatment with A2E and BL induced ARPE-19 cell apoptosis, but treatment with shinorine decreased the apoptotic factors, such as MAPKs. Shinorine reduced p-JNK and p-P38, which were increased by A2E and BL. In addition, shinorine was found to regulate inflammatory proteins and proteins associated with carbonyl stress. In conclusion, shinorine may suppress cell damage caused by A2E treatment and BL exposure at the cellular level by regulating various cell death and inflammatory response pathways.

Keywords: ARPE-19; Porphyra dentata; black paper; eye health; mycosporine-like amino acid.

Figure 1

Cytotoxicity of shinorine on ARPE-19. (A) ARPE-19 cells treated with lutein (10 µM) and shinorine (1.25–5 µM); (B) shinorin attenuated A2E- and BL-induced cytotoxicity. Mean ± S.D. (n = 3) triplicate. * p < 0.05 vs. CON; ^# p < 0.05 vs. A2E; ^$ p < 0.05 vs. Lutein; ^&& p < 0.001 vs. 1.25 µM; ^@ p < 0.05 vs. 2.5 µM.

Figure 2

Shinorine reduces apoptosis induced by A2E and BL. (A) Annexin V/PI staining and flow cytometry. Q1: Necroptotic cells; Q2: Late apoptotic cells; Q3: Early apoptosis; Q4: Live cells. (B) TUNEL results indicating a decrease in apoptosis with an increase in shinorine concentration. α-tubulin (green) and DAPI (blue) were visualized using a confocal microscope. Magnification 10×; Scale bar, 50 μm.

Figure 3

Expression of Bcl family induced by A2E plus BL. (A) Western blot of Bcl proteins. (B) Densitometry (Image J 1.54 software) of expressed Bcl proteins. Mean ± S.D. (n = 3), triplicate. * p < 0.05 vs. CON; ^# p < 0.05 vs. A2E; ^$ p < 0.05 vs. Lutein; ^& p < 0.05 vs. 1.25 µM; ^@ p < 0.05 vs. 2.5 µM.

Figure 4

Expression of of MAPKs induced by A2E plus BL. (A) Western blots of p-JNK, JNK, p-P38, P38, and GAPDH. (B) Densitometry (Image J software) of expressed MAPKs. Mean ± S.D (n = 3). Triplicate, * p < 0.05 vs. CON; ^# p < 0.05 vs. A2E; ^$ p < 0.05 vs. Lutein; ^& p < 0.05 vs. 1.25 µm; ^@ p < 0.05 vs. 2.5 µM.

Figure 5

Shinorine has anti-inflammatory effect in ARPE-19. (A) Expression of inflammatory biomarkers; (B) densitometry (Image J software) of expressed inflammatory biomarkers; (C) immunofluorescence images of p-NF-κB and COX-2. DAPI (blue) was visualized using a confocal microscope. Magnification 10×; Scale bar, 50 μm; (D) ELISA assay of pro-inflammatory cytokines. Mean ± S.D. (n = 3). * p < 0.05 vs. CON; ** p < 0.001 vs. CON; ^# p < 0.05 vs. A2E; ^## p < 0.001 vs. A2E; ^$ p < 0.05 vs. Lutein; ^$$ p < 0.001 vs. Lutein; ^& p < 0.05 vs. 1.25 µM; ^@ p < 0.05 vs. 2.5 µM; ^@@ p < 0.001 vs. 2.5 µM.

Figure 6

Shinorine decreases carbonyl stress induced by BL plus A2E. (A) Expression of 4-HNE, MDA, and GAPDH; (B) densitometry (Image J software) of 4-HNE, MDA. Mean ± S.D. (n = 3). * p < 0.05 vs. CON; ^# p < 0.05 vs. A2E; ^$ p < 0.05 vs. Lutein; ^& p < 0.05 vs. 1.25 µM; ^@ p < 0.05 vs. 2.5 µM.