Melatonin Suppressed Photoreceptor Cell Ferroptosis In Dry Age-related Macular Degeneration Via Nrf2 Nuclear Translocation

Objective:Age-related macular degeneration（AMD）is a degenerative disease of the outer retina that leads to irreversible vision loss.There are two main types of AMD:dry（non-exudative）and wet（exudative）.Dry AMD is characterized by subchoroidal drusen deposition and the formation of geographic atrophy（GA）.In dry AMD,the retinal pigment epithelial cells（RPE）and the photoreceptors on the surface of the fovea are atrophic,which leads to vision loss.Current there is no effective treatment for dry AMD.Sodium iodate（Na IO3）,a chemical oxidant,has been shown to induce replicable retinal degeneration and has become a method for modeling dry AMD.Ferroptosis is a regulatory necrotic cell dependent on lipid peroxidation induced by iron ions and reactive oxygen species.Melatonin（MLT）is an endogenous hormone secreted by the pineal gland and retina and has strong antioxidant properties.There are MLT receptors in the retina,cornea,ciliary body,lens,choroid,and sclera,indicating that there are MLT targets in these tissues.Nuclear factor erythroid2-related factor 2（Nrf2）is a transcription factor that regulates cell response to oxidative stress by expressing antioxidant genes.The purpose of this study is to investigate whether there is ferroptosis in photoreceptor cells during the pathogenesis of dry AMD,and previous studies of our group have proved that the oxidative damage of RPE can be reduced by targeting Nrf2 signaling pathway.Therefore,this study further explores whether melatonin can affect the development process of ferroptosis in dry AMD by regulating Nrf2 signaling pathway.It provides a new idea for the drug treatment of dry AMD.Methods:1.To establish a sodium iodate-induced oxidative stress model and verify the occurrence of ferroptosis in photoreceptor cells:In vivo:the 6-8 week-old C57BL/6 mice were injected with 50mg/kg Na IO₃through the caudal vein once,and the control group was injected with 1X PBS through the caudal vein.In vitro:mouse cone line 661W cells were treated with different concentrations of sodium iodate and ferroptosis positive control Erastin for 24 h.The cell activity was detected by CCK-8 assay.The experimental group（Na IO₃group）was selected when the cell activity was reduced to about 50%.The normal 661W cells were used as the Control group.The m RNA and protein expressions of Tf R1,SLC40A1,SLC7A11,GPX4 and FTH1 were detected by q PCR and western blot.Apoptosis inhibitor ZVF,necrosis inhibitor Nec-1,ferroptosis inhibitor Fer-1 were pretreated,and CCK-8 were used to detect cell activity.Ferro Orange staining,ROS,and MDA kit were used to detect the changes in intracellular ferrous ions and lipid peroxidation levels.2.Melatonin inhibited ferroptosis in photoreceptor cells:In vivo and in vitro models,different concentrations of melatonin were treated,and CCK-8 was used to detect cell viability after treatment.Western blot was used to detect the expression of Tf R1,SLC40A1,SLC7A11,GPX4,FTH1.Ferro Orange staining and MDA kit were used to detect the changes in intracellular ferrous ions and lipid peroxidation.3.Nrf2 mediated melatonin inhibition of ferroptosis in photoreceptor cells:si RNA Nrf2or si RNA NC was transfected into 661W cells,and Na IO₃was treated 24-48h later.Systemic Nrf2 knockout mice were injected with Na IO₃through a single caudal vein.The expression level and location of Nrf2 were detected by immunofluorescence staining,and the expression changes of ferroptosis-related proteins,intracellular ferrous ions,and lipid peroxidation levels were detected.4.Melatonin inhibited ferroptosis of photoreceptor cells by regulating Nrf2 nuclear translocation:661W cells transfected with si Nrf2 and Nrf2-KO mice were treated with Na IO₃and melatonin,and the expression level and location of Nrf2 were detected by immunofluorescence staining.The protein levels of Nrf2,Keap1,GSK-3β,and Fyn were detected by western blot.Cytoplasmic nucleoprotein was extracted to detect the change of Nrf2 expression in the nucleus.Results:1.In vivo:the mitochondria in photoreceptor layer of mouse eye retina showed ferroptosis-like changes under the electron microscope,indicating ferroptosis in the photoreceptor cells.In vitro:Fer-1,an inhibitor of ferroptosis,had an obvious salvage effect on Na IO₃injury,which proved that ferroptosis was the main phenotype.Compared with the control group,Tf R1 expression was increased in Na IO₃and Erastin groups,while the expressions of SLC40A1,SLC7A11,GPX4,and FTH1 were decreased,and the expressions of intracellular ferrous ions,reactive oxygen species,and lipid peroxidation were increased.2.After treatment with different concentrations of melatonin,the cell activity increased,the expression of Tf R1 decreased,and the expressions of SLC40A1,SLC7A11,GPX4,and FTH1 increased.The changes were proportional to the melatonin concentration,and all of them could be inhibited by melatonin receptor inhibitors Luzindole（Luz）.The expression of intracellular ferrous ions and lipid peroxidation decreased.3.After the expression of Nrf2 is silenced in the established in vivo and in vitro oxidative stress models,the expression level of Nrf2 was significantly decreased,the therapeutic effect of melatonin on ferroptosis of photoreceptor cells was weakened,cell vitality was lost,and GPX4 expression level was decreased.4.Immunofluorescence staining showed that Na IO3 promoted the nucleation of Nrf2,decreased the expression in the nucleus and increased the expression in the cytoplasm.Upstream GSK-3βand Fyn phosphorylation regulate Nrf2 nuclear translocation,melatonin treatment significantly improves the Nrf2 level in the nucleus,and may initiate downstream factors to inhibit ferroptosis.Conclusions:Melatonin can suppress the development of ferroptosis in photoreceptor cells induced by sodium iodate.By interfering the expression of Nrf2,it was found that melatonin may play a role in inhibiting ferroptosis by regulating Nrf2 nuclear translocation.Melatonin may inhibit ferroptosis of photoreceptor cells by promoting Nrf2 nuclear translocation via GSK-3β/Fyn signaling pathway,providing a new idea for drug treatment of dry AMD.