Protective effect of diphlorethohydroxycarmalol against oxidative stress-induced DNA damage and apoptosis in retinal pigment epithelial cells

Abstract

Purpose: Reactive oxygen species (ROS) contribute to the onset and progression of disease pathogenesis in a variety of organs, including age-related macular degeneration (AMD). Diphlorethohydroxycarmalol (DPHC), a phlorotannin compound, is one of the major components of the brown alga Ishige okamurae Yendo, and has been shown to have strong antioxidant capacity. The purpose of this study was to evaluate the protective effects of DPHC against oxidative stress (hydrogen peroxide, H₂O₂)-induced DNA damage and apoptosis in cultured ARPE19 retinal pigment epithelial (RPE) cells.

Materials and methods: Cell viability was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay. Intracellular ROS generation was measured by flow cytometer using 2′,7′-dichlorofluorescin diacetate. The magnitude of apoptosis was measured by flow cytometry using the annexin V/propidium iodide double staining. DNA damage was evaluated by DNA fragmentation assay, comet assay and 8-hydroxy-2′-deoxyguanosine (8-OHdG) analysis. To observe the mitochondrial membrane potential, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide staining was performed. In order to identify the underling mechanism of DPHC against H₂O₂-induced cellular alteration, we performed immune blotting.

Results: The results of this study showed that the decreased survival rate brought about by H₂O₂ could be attributed to the induction of DNA damage and apoptosis accompanied by the increased production of ROS, which was remarkably reversed by DPHC. In addition, the loss of H₂O₂-induced mitochondrial membrane potential was significantly attenuated in the presence of DPHC. The inhibitory effect of DPHC on H₂O₂-induced apoptosis was associated with a reduced Bax/Bcl-2 ratio, the protection of the activation of caspase-9 and -3 and the inhibition of poly (ADP-ribose) polymerase cleavage, which was associated with the blockage of cytochrome c release to the cytoplasm.

Conclusions: Our data proved that DPHC protects ARPE19 cells against H₂O₂-induced DNA damage and apoptosis by scavenging ROS and thus suppressing the mitochondrial-dependent apoptosis pathway. Therefore, this study suggests that DPHC has the therapeutic potential to prevent AMD by inhibiting oxidative stress-induced injury in RPE cells.

Keywords:

• Diphlorethohydroxycarmalol
• ARPE19 retinal pigment epithelial cells
• oxidative stress
• DNA damage
• apoptosis
• age-related macular degeneration

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was supported by BGN Eye Clinic and the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (2017R1D1A1B03032689) and the International Science and Business Belt Program through the Ministry of Science, ICT and Future Planning (2017K000490).