Protective Effects of Myricetin and Related Flavonols Against A2E and Light Mediated Cell Death in Bovine Retinal Primary Cell Culture

Abstract

Purpose:: A2E and its isomer, iso-A2E are the major lipofuscin fluorophores in human retinal pigment epithelium (RPE). Accumulation of fluorescent lipofuscin granules in RPE cells increases the risk of developing age-related macular degeneration (AMD). Light exposure has also been attributed to the incidence of AMD. In this study, we investigated the effect of flavonols against A2E or light-induced photoreceptors death in primary retinal cell cultures and studied the structure-activity relationships of myricetin with two others flavonols.

Methods:: Primary retinal cell cultures were prepared from bovine retinas. Fourteen day-old cultures were pretreated with different concentrations of myricetin, quercetin or kaempferol (1-40 µM) for 24 hours, then cells were treated with 30 µM of A2E or exposed to blue-actinic light (500 lux, 420 nm, 17.4 W/m²) for 20 hours. Green nucleic acid stain assay was used to evaluate cell death. Photoreceptor and bipolar cells were immunolabeled with specific antibodies and were counted using automated microscope imaging and image-based cell counting software.

Results:: Primary retinal cell cultures prepared contained a mixture of retinal cells enriched in photoreceptors, bipolar cells and Müller cells. Twenty hours exposure to blue light induced 70% death of photoreceptors in bovine retinal cell cultures. Myricetin protected 100% of photoreceptors against blue-light-mediated damage with an EC₅₀ of 9 ± 0.7 µM. Fourty µM quercetin resulted in a maximum of 15 % protection against light damage, and kaempferol was inactive. A2E induced photoreceptor and bipolar cell death in a concentration-dependent manner with an EC₅₀ of 23 µM for photoreceptors and 30 µM for bipolar cells. Myricetin, quercetin and kaempferol protected photoreceptors and bipolar cells from A2E damage with EC₅₀ values of 2 ± 0.3 µM, 2 ± 0.3 µM, 5 ± 0.09 µM and 0.8 ± 0.07 µM, 0.44 ± 0.06 µM, 1 ± 0.4 µM, respectively.

Conclusions:: These results suggest that myricetin function as potent and effective neuroprotective agents for photoreceptor cells against A2E and light damage. Flavonols structurally related to myricetin could become leads for the development of a new generation of molecules for clinical application in retinal diseases associated with photoreceptor cell death.

Keywords: age-related macular degeneration • neuroprotection • apoptosis/cell death