Abstract
Purpose :
Ferroptosis is a programmed cell death characterized by iron-dependent lipid peroxidation. Retinal pigment epithelium(RPE) is particularly prone to lipid peroxidation since it’s persistent phagocytosis of photoreceptor outer segments which highly enriched in polyunsaturated fatty acids (PUFAs). Here, we aimed to elucidate whether ferroptosis involved in light exposed murine RPE.
Methods :
To induced excessive phagocytosis and lipid peroxidation in murine RPE, six- to eight-week-old C57/6J mice were exposed to irradiation at 10000 lux for 20 h with a high-pressure mercury lamp followed by a dark period of 4 h. Before irradiation, mice were treated with or without iron chelator deferiprone (DFP) in the drinking water for 2 weeks or a single intraperitoneal injection with ferroptosis inhibitor Liproxstatin(Lip-1) or intravitreal injection with pan-caspase inhibitor Z-VAD-FMK. After sacrifice, murine RPE was dissected and purified, followed by qPCR as well as western blot for identifying ferroptosis associated factors SLC7A11, GPX4, and ACSL4. The reactive oxygen species (ROS) level was measured using DCFH-DA; lipid peroxidation was measured with BODIPY staining. Flatmount RPE samples were immunostained with anti-Scone opsin and anti-rhodopsin antibody for evaluating excessive phagocytosis. Transmission electron microscopy(TEM) was used to verify the ultrastructure of light exposed murine RPE. Histology was performed for morphologic analysis. For in vivo imaging, funduscopy and spectral-domain optical coherence tomography (SD-OCT) were used.
Results :
Light exposure elevated ROS and triggered lipid peroxidation in murine RPE where phagosomes were observed by TEM. Immunostaining further confirmed excessive phagocytosis in light exposed RPE. Ferroptosis associated factors SLC7A11, and ACSL4 level upregulated while GPX4 level downregulated after light exposure. Systemic administration of iron chelator DFP and ferroptosis inhibitor Lip-1 both ameliorated reduction of outer nuclear layer(ONL) thickness observed in histology and SD-OCT, whereas pan-caspase inhibitor Z-VAD-FMK did not achieve equivalent effect.
Conclusions :
Our results demonstrate for the first time that ferroptosis involves in excessive light induced damage of murine RPE, which rescued by iron chelator and ferroptosis inhibitor but not by apoptosis inhibitor.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.