| ObjectivesAge-related macular degeneration(AMD),an age-related chronic degenerative ophthalmopathy,is the third leading cause of blindness ranked behind cataract and glaucoma.The global prevalence of AMD was 8.7%.AMD could be divided into two subtypes:the non-neovascular type,also called the dry AMD(dAMD),and the neovascular type,also called the wet AMD(wAMD).dAMD accounts for 85%?90%of AMD.There is no effective treatment for dAMD.Naringenin(NAR),a kind of dihydro-flavonoid,is rich in fruits such as grapefruit and citrus.We previously reported that NAR protected 661W cells(cone cells)from oxidant damage.One percent;of NAR eye drop had preventive effect on N-methyl-N-nitrosourea-induced photoreceptor death in rats.We also found that NAR arrived at the retina within 5 min after a single topical administration.Loss of photoreceptor cells is the pathological basis of vision loss.Thus,NAR may be a potential candidate for dAMD.Therefore,in this study,retinopathy models induced by white light in rats and by sodium iodate in mice,and spontaneous dAMD in cynomolgus monkeys were used to evaluate the effect of NAR eye drop on dAMD.Moreover,ARPE-19 cells injuries induced by blue light and by sodium iodate were applied to investigate its underlying mechanisms.Methods1.Effect of NAR eye drop on dAMDIn this part,retinopathy models induced by white light in rats and by sodium iodate in mice,and spontaneous dAMD in cynomolgus monkeys were used to evaluate the effect of NAR eye drop on dAMD.1.1 Effect of NAR eye drop on white light-induced retinopathy in rats1.1.1 Establishment of white light-induced retinopathy model in ratsThis study was designed to investigate the factors including light exposure during the daytime and nighttime,gender,the pupil size and light duration on white light-induced retinopathy.(1)Light exposure(LE)during daytime and nighttimeSD rats were randomly divided into three groups:normal group,LE during daytime group,and LE during nighttime group.After dark adaptation,rats of LE group were exposed to white light for 6 h.Flash eletroretinogram(FERG)was recorded 1 d after LE.(2)GenderRats were randomly divided into four groups:normal male group,normal female group,LE+male group and LE+female group.After dark adaptation,rats of LE groups were exposed to white light for 8 h.FERG was recorded 1 d after LE.(3)Pupil sizeRats were randomly divided into three groups:normal group,LE+tropicaine group and LE group.After dark adaptation,rats of LE+tropicaine group were topically administered with a drop of tropicamide,while rats of LE group without receiving any treatment.All rats except normal rats exposed to white light for 8 h.FERG was recorded 1,3 and 7 d after LE.(4)Duration of LERats were randomly divided into seven groups:normal group,4 h,8 h,12 h,16 h,20 h and 24 h LE group.After dark adaptation,rats were exposed to light according to preset time points.FERG was recorded 7,14 and 21 d after LE.Hematoxylin and eosin(HE)staining of retinas was also carried out.1.1.2 Effect of NAR eye drop on white light-induced retinopathy in ratsRats were randomly divided into five groups:normal group,model group,0.5%NAR group,1%NAR group and Stulln group.After dark adaptation,all rats except normal ones exposed to white light for 8 h.Rats of normal and model group were topically administered with one drop of vehicle,while rats of other groups were administered with 0.5%NAR,1%NAR or Stulln eye drop,respectively.Treatment was taken triple daily and maintained for 21 consecutive days.FERG was recorded 7,14 and 21 d after LE.Histological analysis was performed after final FERG recording.Thickness of outer nuclear layer(ONL)and number of retinal pigment epithelial(RPE)cells were measured.1.2 Effect of NAR eye drop on sodium iodate-induced retinopathy in mice1.2.1 Establishment of sodium iodate-induced retinopathy in miceKM mice were randomly divided into three groups:normal group,low and high dose of sodium iodate(25,30 mg/kg).Normal mice were intraperitoneally injected with saline solution,while other mice were injected with the corresponding dose of sodium iodate.FERG was recorded 1,3 and 5 d after injection.1.2.2 Effect of NAR eye drop on sodium iodate-induced retinopathy in miceMice were randomly divided into five groups:normal group,model group,0.5%NAR group,1%NAR group and Stulln group.Normal mice was intraperitoneally injected with saline,while others were injected with a single dose of 25 mg/kg sodium iodate.Both eyes of all mice were instilled with one drop of vehicle,NAR or Stulln eye drop triple daily from one day before to 5 d after injection.Five minutes after the last administration,FERG recording and histological analysis were performed.ONL thickness and RPE cell numbers were measured.1.3 Effect of NAR eye drop on drusen in spontaneous dAMD cynomolgus monkeysSpontaneous dAMD cynomolgus monkeys with two eyes manifested with drusen invovled in macula lutea were applied.Left eyes were instilled with 1%NAR eye drop,while other eyes were instilled with Stulln eye drop triple daily for three consecutive months.Fundus photos were collected before and 1,2 and 3 months after treatment.The effect was evaluated by the number of drusen.2.Underlying mechanisms of NAR against dAMDIn this part,retinopathy models induced by sodium iodate in mice,ARPE-19 cells injuries induced by blue light and by sodium iodate were applied to investigate the underlying mechanisms of NAR.2.1 Protective mechanisms of NAR eye drop on sodium iodate-induced retinopathy in miceMice were randomly divided into four groups:normal group,model group,0.5%NAR group and 1%NAR group.Normal mice were intraperitoneally injected with saline,while others were injected with a single dose of 25 mg/kg sodium iodate.Both eyes of all mice were instilled with one drop of vehicle or NAR eye drops triple daily from 1 d before to 5 d after injection.Five minutes after the last administration,all eyes were removed and fixed with fixed solution.The protein expressions of SIRT1,Nrf2 and HO-1 were analyzed with immunochemical staining.Mice were randomly divided into three groups:normal group,model group and 1%NAR group.Normal mice were intraperitoneally injected with saline,while others were injected with a single dose of 25 mg/kg sodium iodate.Both eyes of mice were instilled with one drop of vehicle or 1%NAR eye drop triple daily from 1 d before to 10 d after injection.Five and ten days after injection,the eyes were removed and the retinas were collected.Protein expressions of SIRT1,Nrf2 and HO-1 were detected with Western Blot.2.2 Effect of NAR on blue light-induced injury in ARPE-19 cellsNormal ARPE-19 cells were treated with different concentrations of NAR for 24 h.Cell viability was determined with CCK-8 method.Cells treated with blue light for 1 h,2 h,3 h,4 h or 5 h and cell viability was then assayed.After irradiation with blue light,cells were incubated with different concentrations of NAR for 24 h.Cell viability was assayed.2.3 Effect of NAR on sodium iodate-induced injury in ARPE-19 cellsCells were treated with sodium iodate or combination of sodium iodate and NAR.Cell viability was assayed.Cytotoxicity was evaluated by the release of lactate dehydrogenase(LDH).Cellular death was observed using propidium iodide(PI)dye.Levels of intracellular reactive oxygen species(ROS)and carbonyl protein were measured using the 2',7'-dichlorodihydrofluorescein and immunocytochemical method,respectively.The protein expressions of SIRT1,total Nrf2,HO-1 and nuclear Nrf2 were determined using Western Blot analysis.SIRT1 protein expression was also analyzed using the immunofluorescence method.ATP level was detected using the chemiluminescence method.Levels of NAD+and NADH were assayed with WST-8 method,and the ratio of NAD+/NADH was then calculated.In addition,SIRT1 agonist SRT1720,SIRT1 inhibitor EX527 and HO-1 inhibitor Zn Protoporphyrin IX(ZnPP)were applied to investigate the anti-dAMD mechanism of NAR.Results1.Effect of NAR eye drop on dAMD1.1 Effect of NAR eye drop on white light-induced retinopathy in rats1.1.1 Establishment of white light-induced retinopathy model in rats(1)LE during daytime and nighttimeCompared with normal group,the amplitudes of a-and b-wave were remarkably decreased in rats exposed to white light during nighttime,while there were no changes in rats received irradiation during daytime.(2)GenderThere were no difference on the amplitudes of a-and b-wave between normal male and female rats.After LE,the amplitudes of a-and b-wave of male and female rats were all significantly reduced.However,there was no difference on amplitudes of a-and b-wave between the male and female rats exposed to light.(3)Pupil sizeAn remakable reduction on the amplitudes of a-and b-wave was appeared in rats exposed to light.But there was no difference on the amplitudes between LE+tropicaine group and LE group.(4)Duration of LECompared with normal rats,7,14 and 21 d after LE,the amplitudes of a-and b-wave were significantly reduced in rats exposed to light for 4,8,12,16,20 and 24 h.There was no significant difference on the reduction of amplitudes among rats exposed to light from 4 h to 20 h.However,visual function of rat was almost completely lose after exposed to 24 h irradiation.Results of histological analysis showed that normal retina consisted of several well organized layers,in which cells were tightly arranged.Twenty-one days after irradiation,retinal layers of rat for 4 h exposure were well arrange,accompanied with 15.8%reduction in outer nuclear layer(ONL).Fourteen and twenty-one days after irradiation,retinal layers were also well arranged in rats exposed to light for 8,12,16 and 20 h.Inner nuclear layer was slightly thin and the thickness of ONL were decreased by about 40.0%.Retinal cells in INL and ONL were obviously disappeared in rats received 24 h irradiation.Moreover,ONL thickness was reduced by about 80%and the neural retina laye tightly adhered to RPE layer.These results indicate that the retinopathy induced by white light could be established in SD rats without considering gender and pupil size.The retinal functional and structural damage are appropriate when rats received 8 h irradiation during the nighttime and were raised for another 14-21 d.1.1.2 Effect of NAR eye drop on white light-induced retinopathy in ratsAs compared with model group,the amplitudes of a-and b-wave were significantly increased with 14 and 21 d treatment of 1%NAR eye drop.Histological analysis results showed that 1%NAR eye drop remarkably increased ONL thickness and RPE cell numbers.1.2 Effect of NAR eye drop on sodium iodate-induced retinopathy in mice1.2.1 Establishment of sodium iodate-induced retinopathy in miceRemarkable reductions on the amplitudes of a-and b-wave were observed in mice 5 d after injection of sodium iodate at the doses of 25 and 30 mg/kg.The amplitudes of a-wave were dropped by 39.9%and 79.3%,respectively,and the amplitudes of b-wave were reduced by 56.4%and 87.8%,respectively.Therefore,a dose of 25 mg/kg was chose for the following research.1.2.2 Effect of NAR eye drop on sodium iodate-induced retinopathy in miceAs compared with model group,the amplitudes of a-and b-wave in 1%NAR group were significantly increased.Histological analysis results showed that 1%NAR eye drop remarkably increased ONL thickness and RPE cell numbers.1.3 Effect of NAR eye drop on drusen in spontaneous dAMD cynomolgus monkeysMany yellow round puncta,called drusen,were appeared at macula lutea in spontaneous dAMD cynomolgus monkeys.After three months treatment,both 1%NAR eye drop and Stulln eye drop reduced the number of drusen.2.Underlying mechanisms of NAR against dAMD2.1 Mechanisms of NAR eye drop on sodium iodate-induced retinopathy in miceImmunostaining results showed that SIRT1 immunolabeling was low in normal retinal ganglion cell layer(GCL),INL,ONL and RPE cell,while Nrf2 and HO-1 immunolabelings were low in normal retinal GCL,inner plexiform layer,outer plexiform layer,photoreceptors inner segment(PIS),photoreceptors outer segment(POS)and RPE cell.Five days after injection of sodium iodate,SIRT1 immunolabeling was increased in morphologically abnormal GCL,INL,ONL and RPE cell,while Nrf2 and HO-1 immunolabeling were increased in retinal layers mentioned above.Low immunolabelings of SIRT1,Nrf2 and HO-1 were observed in 1%NAR-treated retinas after 5 d injection of sodium iodate..Results of Western Blot analysis showed that the protein expressions of SIRT1,Nrf2 and HO-1 in retinas were significantly increased 10 d after injection of sodium iodate.One percent of NAR eye drop markedly decreased the protein expressions mentioned above.2.2 Effect of NAR on blue light-induced injury in ARPE-19 cellsNAR at concentrations from 0.1 to 100 ?M had no influence on viability of normal ARPE-19 cells.Blue light exposure at 3000 Lux for 1,2,3,4 and 5 h inhibited cell viability in a time-dependent manner,by 10.9%,19.4%,21.0%,35.8%and 56.5%,respectively.NAR at concentrations from 0.1 to 10 ?M significantly promoted cell viability after 4 h irradiation.2.3 Effect of NAR on sodium iodate-induced injury in ARPE-19 cellsCompared with normal group,10 mM sodium iodate treatment for 24 h markedly inhibited cell viability,increased the release of LDH,as well as increased PI-positive cells.Sodium iodate also reduced level of ATP and the radio of NAD+/NADH.Compared with model group,NAR significantly decreased the release of LDH and PI-positive cells,and increased ATP level and NAD+/NADH radio.Levels of intracellular ROS and carbonyl protein were remarkably increased in cells incubated with 10 mM sodium iodate for 24 h.Protein expression of SIRT1 was also up-regulated.Compar ed with model group,SRT1720 significantly increased cell viability and decreased ROS level,while EX527 enhanced cytotoxicity of sodium iodate.NAR increased cell viability,decreased levels of ROS and carbonyl protein,as well as promoted SIRT1 protein expression.As compared with NAR group,cells co-treated with EX527 and NAR displayed a reduction of cell viability and an increase of ROS level.Since cell damage induced by 10 mM sodium iodate was approximate to acute injury,4 mM sodium iodate mimicked a chronic injury was applied to investigate SIRT1 protein expression and the effect of NAR.Compared to normal group,cell viability was decreased and protein expression of SIRT1 was up-regulated in cells treated with 4 mM sodium iodate for 24 h and 72 h.NAR up-regulated SIRT1 protein expression after 24 h treatment,but down-regulated after 72 h treatment.Moreover,NAR treated for 72 h markedly promoted cell viability by 24.2%.10 mM sodium iodate treated for 3-24 h markedly increased Nrf2 nuclear content.However,compared with model group,NAR treated for 1-3 h remarkably promoted Nrf2 translating into nuclear and decreased Nrf2 nuclear content after 24 h treatment.As compared with NAR group,cells co-treated with ZnPP and NAR displayed a reduction of cell viability.Conclusion1.Topical treatment with NAR eye drop not only prevented retinal function and morphology,maintained photoreceptor cells and RPE cells from white light and sodium iodate-induced retinopathy in rodents,but also reduced drusen in dAMD cynomolgus monkeys.The protective effect based on three animal models indicates that NAR eye drop had good preventive and treatment effect on dAMD.2.NAR eye drop restored the retinal protein expressions of SIRT1,Nrf2 and HO-1 towards normal level in mice exposed to sodium iodate.NAR inhibited sodium iodate-induced ARPE-19 cells death,decreased levels of intracellular ROS and carbonyl protein,and increased ATP level as well.Moreover,NAR up-regulated SIRT1 protein expression and activated SIRT1 and Nrf2.These results indicate that NAR protects RPE cells and retina from oxidative damage via up-regulating SIRT1 and activating Nrf2 pathway.The antioxidative activity may be the important mechanism involved in the protective effect of NAR eye drop on dAMD. |
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