| Background:Age-related macular degeneration(AMD)is a common cause of irreversible vision loss among elderly people,which affects the macula.As aging population increases in our country,the patients of AMD are growing and an increased number of people lose their eyesight.Although the pathogenic mechanism of AMD is poorly understood,it is a disease related to genetic factor and environmental factor.Studies have shown that oxidative stress has an important role in the pathogenic mechanism of AMD,and confirmed that reactive oxygen species from retina would lead to damage in retinal pigment epithelial(RPE)cells.RPE cells has many important physiological functions,it is located in the outermost layer of retina with lots of metabolic activity.Alpha-mangosteen,is a quinonoid constituent isolated from mangosteen(Garcinia mangostana Linn),which have important biological activity such as anti-oxidant,anti-inflammatory,anti-bacterial and anti-cancer.In this study,we established model of AMD,observed the treatment effect of ?-mangostin in AMD through in vivo and in vitro.Methods:(1)Experiment of in vitro:RPE cells were randomly divided into normal control group,oxidative damage group(H2O2 induced group)and a-Mangostin treatment groups.And a-Mangostin treatment groups were divided into subgroups according to the different concentrations of a-Mangostin.CCK8 method was used to detect Cell viability changes in each group.The expression of Reactive Oxygen Species(ROS)level was detected by flow cytometry(FCM)and the expression of transcription factor nuclear factor-Erythroid-2-related factor 2(Nrf2),Heme oxygenase-1(HO-1),NADH Dehydrogenase,Quinone 1(NQO1)and nuclear factor-kappaB(NF-?B)Protein was detected by Western blot analysis.(2)Experiment of in vivo:Totally 30 Balb/c mice,aged 6-8 weeks,were randomly divided into the control group,light-exposure group and ?-mangostin group.Every group contained 10 mice.Mice from ?-mangostin group were treated with alpha-mangostin at the dose of 30mg/kg body weight by intragastric administration daily for 7 days,and then exposed to white light at the fifth day.The light-exposure group and ?-mangostin group were exposed to 5000±200Lux white light-emmiting diodes(LEDs)for continuously 1 hour to establish the mice model of retinal light damage.Flash-electroretinograme was recorded 72 hours after light exposure.The changes in retinal morphology of mice were observed by light microscopy.Compared the thinning of outer nuclear layer of three groups.Retinas were extracted to detect the activity of caspase-3,Superoxide Dismutase(SOD),Glutathione peroxidase(Gpx),and the content of malondialdhyde(MDA)and glutathione in the retinal homogenate.Immunofluorescence was used to determine the distribution of Nrf2 and the nuclear translocation of Nrf2.Western blot was used to detect the expression of HO-1 in total protein of retina in three groups,and the expression of Nrf2 in nuclear protein of retina in three groups.Results:(1)Experiment of in vitro:CCK8 examination results showed that:within 0-12?M,?-Mangostin had no damage effects on cell activity compared with normal control group.After H2O2 induced,the activity of RPE cells in oxidative damage group significantly reduced compared with normal control group(p<0.05).And compared with oxidative damage group,cell viability of ?-Mangostin treatment groups gradually increased when the concentrations of ?-Mangostin were within 0 to 12?M.ROS results showed:compared with normal control group,the expression of ROS level significantly increased in oxidative damage group(p<0.05)and the expression of ROS level in 12?M ?-Mangostin treatment group decreased compared with oxidative damage group(p<0.05).Western blot results showed that oxidative damage group showed a positive expression of Nrf2,HO-1,NQO1,NF-kB protein compared with normal control group(p<0.05).Compared with oxidative damage group,the expression of Nrf2,HO-1,NQO1,NF-kB protein in 12?M ?-Mangostin treatment group increased(p<0.05).(2)Experiment of in vivo:Flash-electroretinogram showed that retinal dysfunction was less severe in ?-mangostin group than in light-exposure group(p<0.05).Light microscopy test showed that retina structural damage was less severe in ?-mangostin group than in light-exposure group(p<0.05).Under the fluorescence microscope,we observed that t apoptotic cells were mainly distributed in outer nuclear layer.The number of apoptotic cells in light-exposure group was more than ?-mangostin group and normal group(P<0.05).The level of apoptic markers caspase-3 was markedly increased in light-exposure group.However,alpha-mangostin significantly prevented caspase-3 activation induced by light damage(P<0.05).Light exposure to retina produced excessive lipid peroxides.Daily administration of alpha-mangostin(30mg/kg)significantly blocked the light-induced generation of MDA(P<0.05).The activity of SOD,Gpx and the content of GSH in light-exposure group reduced dramatically versus the normal group(P<0.05).?-mangostin supplement suppressed the reduction induced by light exposure(P<0.05).The expression of HO-1 and Nrf2 increased after light exposure.The expression of HO-1 and Nrf2 in ?-mangostin group was significantly higher than that in normal group.Conclution:?-mangostin through regulated expression of Nrf2,activate the downstream endogenous antioxidant defense system,include SOD,Gpx,GSH and HO-1.It also can suppress lipid peroxidation and protect retina.?-mangostin can inhibits photoreceptor cells apoptosis induced by intensive light and improve abnormal histologic changes and functional decrease in retina.It is indicated that?-mangostin have an antioxidant effect that could be a new approach to treat AMD in clinic. |
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