Hinokitiol Protects RPE cells from Oxidative and Autophagic Dysfunction: Implications for AMD Therapy

Abstract

Age-related macular degeneration (AMD) is a leading cause of vision loss in the elderly, driven by dysfunction of retinal pigment epithelial (RPE) cells. Oxidative stress-induced reactive oxygen species (ROS) play a critical role in AMD progression, although the underlying mechanisms remain unclear. Autophagy is essential for maintaining retinal homeostasis by clearing damaged organelles and misfolded proteins through lysosomal degradation. However, excessive ROS can disrupt autophagy balance, leading to the excessive degradation of cell components and ultimately triggering autophagy dysfunction-induced cell death. Hinokitiol, a natural compound derived from the heartwood of Cupressaceae plants, possesses potent antioxidant properties. This study aimed to investigate its roles against oxidative damage in RPE cells exposed to H₂O₂-induced ROS generation. Cell viability was assessed using MTT and crystal violet staining. ROS were measured using H₂DCFDA and MitoSOX probes, while catalase activity was evaluated as indicator of antioxidant capacity. DNA damage was assessed by γ-H2AX immunocytochemistry and comet assay. Mitochondrial membrane potential (MMP) was analyzed using JC-1, and autophagy markers were examined by Western blotting. Hinokitiol significantly enhanced RPE cell viability, reduced ROS by increasing catalase activity, preserved mitochondrial function, and mitigated DNA damage. Furthermore, it restored autolysosome fusion impaired by H₂O₂, thereby maintaining cellular homeostasis. These findings suggest that hinokitiol may be a promising therapeutic candidate for AMD treatment.

Keywords: AMD; Hinokitiol; Oxidative stress; RPE.