The Protective Effect of Hispidin against Hydrogen Peroxide-Induced Oxidative Stress in ARPE-19 Cells via Nrf2 Signaling Pathway.

Huang Huang,Chau Chau,Chiu Chiu,Chang Chang

doi:10.3390/biom9080380

Abstract

Hispidin, a polyphenol compound isolated from Phellinus linteus, has been reported to possess antioxidant activities. In this study, we aimed to investigate the mechanisms underlying the protective effect of hispidin against hydrogen peroxide (H2O2)-induced oxidative stress on Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells. Hispidin was not cytotoxic to ARPE-19 cells at concentrations of less than 50 μM. The levels of intracellular reactive oxygen species (ROS) were analyzed by dichlorofluorescin diacetate (DCFDA) staining. Hispidin significantly restored H2O2-induced cell death and reduced the levels of intracellular ROS. The expression levels of antioxidant enzymes, such as NAD(P)H:Quinine oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM) were examined using real-time PCR and Western blotting. Our results showed that hispidin markedly enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), HO-1, NQO-1, GCLM, and GCLC in a dose-dependent manner. Furthermore, knockdown experiments revealed that transfection with Nrf2 siRNA successfully suppresses the hispidin activated Nrf2 signaling in ARPE-19 cells. Moreover, activation of the c-Jun N-terminal kinase (JNK) pathway is involved in mediating the protective effects of hispidin on the ARPE-19 cells. Thus, the present study demonstrated that hispidin provides protection against H2O2-induced damage in ARPE-19 cells via activation of Nrf2 signaling and up-regulation of its downstream targets, including Phase II enzymes, which might be associated with the activation of the JNK pathway.

Highlights

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the developed world [1]
The MTT assays revealed that there was no significant change in the viability of Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells after treated with hispidin at concentrations ranging from 2.5–50 μM (Figure 1A)
Using H2 O2 to explore the protective effect against oxidative stress in retinal pigment epithelial (RPE) cells is a well-known model [22,23]

Summary

Introduction

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the developed world [1]. The most common type of AMD, known as the dry-type or atrophic. AMD, is initiated by changes in the pigmentation of the retinal pigment epithelial (RPE) cells and the sub-retinal deposits between the RPE and Bruch’s membrane. These progressions eventually result in RPE cell death, photoreceptors degeneration, and eventually loss of vision [2,3]. There is no specific treatment available for dry/atrophic AMD. Oxidative stress is characterized by the generation of reactive oxygen species (ROS), which plays a key role in the pathogenesis of AMD [4]. Long-term accumulation of oxidative damage leads to dysfunction in RPE cells and increases their susceptibility to oxidative stress. Several dietary supplements of antioxidants, including lutein, β-carotene, and vitamin

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