Temporary alleviation of MAPK by arbutin alleviates oxidative damage in the retina and ARPE-19 cells

Abstract

Dry age-related macular degeneration (AMD) is one of the main diseases that causes blindness in humans, and the number of cases is increasing yearly. However, effective treatments are unavailable, and arbutin (ARB) has been reported to have antioxidant, anti-inflammatory, and anti-aging effects in other age-related diseases. However, whether ARB can be used to treat dry AMD remains unknown. To explore the therapeutic potential and molecular mechanism of arbutin in the treatment of dry AMD. MTT assays, reactive oxygen species (ROS) production assays, flow cytometry assays, qPCR and western blotting were used to assess the impact of ARB on human RPECs induced by H₂O₂. A transcriptome sequencing assay was used to further explore how ARB acts on human RPECs treated with H₂O₂. Hematoxylin and eosin (H&E) staining and total antioxidant capacity (T-AOC) assays were used to observe the impact of ARB on mouse retina induced by sodium iodate. ARB counteracted the H₂O₂-induced reduction in human RPECs viability, ARB reversed H₂O₂-induced cellular ROS production by increasing the expression of antioxidant-related genes and proteins, ARB also reversed H₂O₂-induced cell apoptosis by altering the expression of apoptosis-related genes and proteins. Transcriptome sequencing and western blotting showed that ARB reduced ERK1/2 and P-38 phosphorylation to prevent H₂O₂-induced oxidation damage. The in vivo experiments demonstrated that ARB protected against retinal morphology injury in mice, increased serum T-AOC levels and increased antioxidant oxidase gene expression levels in the mouse retina induced by sodium iodate. We concluded that ARB reversed the H₂O₂-induced decrease in human RPECs viability through the inhibition of ROS production and apoptosis. The ERK1/2 and P38 MAPK signaling pathways may mediate this process. ARB maintained retinal morphology, increased serum T-AOC level and improved the expression of antioxidant oxidase genes in mice.

Keywords: ARB; Dry AMD; MAPK signaling pathway; Oxidative damage; RPECs; Retinal thickness.

Fig. 1

ARB reverses the decrease in human RPEC viability induced by H₂O₂. A: MTT assays were used to detect the viability of human RPECs treated with 0–1000 μM ARB for 24, 48 and 72 h. B: MTT assays were used to detect the viability of human RPECs treated with different concentrations of 0–700 μM H₂O₂ for 24 h. C: MTT assays were used to detect the viability of human RPECs treated with 600 μM H₂O₂ with or without ARB. One-way ANOVA was used for comparisons among multiple groups, and Tukey's test was used for comparisons between two groups. ((^nsP>0.05, *P < 0.05, ****P < 0.0001).

Fig. 2

ARB decreases H₂O₂-induced ROS production by increasing the levels of antioxidant enzymes. A: ROS production was measured by ROS assay (scale bar = 100 μm). B: qPCR assay was used to measure CAT, FoxO1, and PRDX4 gene expression. C: Western blot assay was used to measure CAT and PRDX4 protein expression. (^nsP>0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 3

ARB reduces H₂O₂-induced apoptosis in human RPECs. A: The apoptosis rate of each group was assessed by flow cytometry (apoptosis rate = Q2+Q3). B: The expression of the apoptosis-related genes Bax and Bcl-2 was measured by qPCR. C: The expression of the apoptosis-related proteins Bax, Bcl-2, cleaved-caspase 3 and pro-caspase 3 was measured by Western blot assay. (^nsP>0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 4

ARB alleviates H₂O₂-induced oxidative damage in Human RPECs through the ERK1/2 and P38 MAPK signaling pathways. A: A differential gene clustering heatmap was used to visualize gene expression between the ARB + H₂O₂ group and the H₂O₂ group. B: Transcriptome sequencing results were combined with the KEGG database for pathway enrichment analysis. C: Western blotting was used to measure the expression of the ERK1/2, P-ERK1/2, P38 and P–P38 proteins in each group. D: MTT assays were used to detect the viability of human RPECs pretreated with FR 180204 or SB203580 for 1 h, treated with or without ARB for 24 h, and finally stimulated with H2O2 for an additional 24 h (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 5

ARB protects retinal morphology from SI-induced injury in mice. A: Schematic diagram of the experimental design in mice. B: H&E staining was used to observe retinal morphology, retinal thickness, INL thickness and ONL thickness. C: T-AOC results for mouse serum from each group (red arrow and green arrow: drusen-like change). D: qPCR assay was used to measure CAT and SOD2 gene expression. (^nsP>0.05, *P < 0.05, **P < 0.01, ***P < 0.001).