The Protective Mechanisms And Effects Of Resveratrol On Mitochondrial Oxidative Stress-mediated Retinal Injury

BackgroundRetinal degenerative diseases(RDD)such as age-related macular degeneration(AMD)and retinitis pigmentosa(RP)cause vision damage and eventually irreversible blindness.Light injury and retinal ischemia are considered major contributors to these diseases.Photoreceptor cell death is the basic pathological feature of RDD.The mechanisms that cause RDD may be complex and involve multiple genetic and environmental factors.Although hundreds of causative gene mutations have been discovered,the exact molecular pathways leading to photoreceptor death are not fully understood.The retina is particularly liable to oxidative stress due to its high oxygen consumption and photosensitivity.Mitochondria regulate oxidative stress within a certain range,but when reactive oxygen species(ROS)exceeds the antioxidant defense capacity,programmed cell death will be triggered,also called apoptosis.The caspase-dependent pathway is a classical apoptotic pathway,which activate the series of caspases.It works by mitochondria-mediated endogenous pathways or by receptor-mediated exogenous pathways.In addition to the classical caspase-dependent pathway,Parthanatos is a unique and highly choreographed form of cell death.Parthanatos act through overactivation of the PARP-1.It does not require caspases to execute,but it relies on PARP-1-mediated accumulation of PAR and subsequent nuclear translocation of apoptosis-inducing factor(AIF)leading to cell death.Resveratrol(RSV)is a polyphenolic compound found in many plants,such as red grapes,peanuts,and mulberry trees.RSV has a variety of pharmacological effects,such as antioxidant,anti-aging,anti-inflammatory,anti-cancer,as well as Protection of the nervous and cardiovascular system.RSV is an activator of SIRT1.It is a ribozyme that is a member of the mammalian sirtuins(SIRT 1-7)family.It also an NAD~+-dependent deacetylase.SIRT1 plays an important role in many biological processes,including metabolism,development,cell survival,and aging.Until now,there is no good clinical strategy to prevent RDD and restore visual function.Therefore,it is very meaningful to investigate a treatment which delays or even block the loss of photoreceptor cells.In this study,661 W cells were subjected to glucose deprivation and light exposure in vitro to simulate RDD.We investigated the effect and mechanisms of RSV on 661 W under these two different injury conditions.The light injury mouse model was also established in vitro to explore the protective effect and mechanism of RSV on the retina.Methods1.Detecting the effect of RSV on retinal photoreceptor cells under glucose deprivation and light injuryMMT assay was performed on 661 W under normal culture conditions,and the dose-response curve and IC50 of RSV were observed.Real-time cell impedance assay was used to monitor the effect of different concentrations of RSV on the cell activity of 661 W under GD and Lt.PI/Hoechst staining was used to observe the effects of RSV on apoptosis under GD and Lt.2.Detecting the effects of RSV on oxidative stress and mitochondria under glucose deprivation and light injuryOxidative stress produces excess reactive oxygen species.The level of intracellular ROS was measured using a DCFH-DA probe under glucose deprivation and light injury.Reduced glutathione(GSH)is one of the most important scavengersof ROS,and its ratio with oxidized glutathione(GSSG)has been regarded as a marker of oxidative stress.GSH/GSSG were detected by using GSH and GSSG kits to observe the redox state of cells under glucose deprivation and light injury.HO-1 is a ubiquitous and redox-sensitive inducible stress protein that can exert indirect,yet potent,anti-oxidative e ects by degrading heme to carbon monoxide(CO),iron,and biliverdin.Western blot was used to detect the changes of HO-1protein level under glucose deprivation and light injury.Oxidative stress affects mitochondrial morphology and function.Mito-tracker Green fluorescent staining was used to detect mitochondrial morphological changes under glucose deprivation and light injury.The changes of mitochondrial membrane potential were observed by JC-1staining to evaluate the mitochondrial function under glucose deprivation and light injury.3.Detecting the effect of RSV on SIRT1 under glucose deprivation and light injuryRSV is the activator of SIRT1.Using the SIRT1 inhibitor Ex-527 and PI/Hoechst staining was used to observe cell apoptosis under glucose deprivation and light injury.Western blot was used to detect the changes of SIRT1 protein level under glucose deprivation and light injury.SIRT1 is a ribozymeSIRT1 activity/inhibition assay kit was used to detect the changes of SIRT1 enzyme activity under glucose deprivation and light injury.4.Detecting the protective mechanism of RSV against GD and Lt-induced cell deathThe caspase pathway is a classic apoptosis pathway.Using the caspase inhibitor z-VAD-fmk and PI/Hoechst staining was used to observe cell apoptosis under glucose deprivation and light injury.Parthanatos does not require caspases for its execution but is dependent on the PARP-1-mediated accumulation of PAR(poly ADP-ribose)and the subsequent nuclear translocation of the apoptosis-inducing factor(AIF).Using the caspase inhibitor z-VAD-fmk and PI/Hoechst staining was used to observe cell apoptosis under glucose deprivation and light injury.The lentivirus expressing PARP-1-targeted short hairpin RNA(shRNA)was transfected to661 Wand PI/Hoechst staining was used to observe cell apoptosis under light injury.Western blot was used to detect the changes of caspase-3,caspase-9,AIF and Parp-1 protein level under glucose deprivation and light injury.5.Detecting the effect of RSV on the retina of mice under light injuryThe retinal function in mice under light injury was detected by ERG.HE staining was used to observe the changes of the retinal ONL structure in mice under light injury.Western blot was used to detect the changes of AIF and PARP-1 protein levels under light injury.Results1.The dose-response curve of RSV showed a typical“hormetic effect”,and IC50 of RSV was 209.6?M.Under both glucose deprivation and light injury,10?M,50?M,100?M RSV had the protective effect in a dose-dependent.2.RSV decrease the production of ROS induced by glucose deprivation and light injury,increase the intracellular GSH/GSSG ratio and the protein expression level of HO-1,improve mitochondrial morphology and mitochondrial membrane potential.3.SIRT1 inhibitor inhibit the protective effect of RSV under glucose deprivation and light injury;RSV improve the expression level of SIRT1 protein,and improve the activity of SIRT1 enzyme in the nucleus.4.Caspase inhibitor reduce the protective effect of RSV on GD-induced cell death,but have no effect on Lt-induced cell death;RSV reduce the protein expression level of GD-induced caspase-3 and caspase-9;The number of cells death knocked out by PARP-1 was significantly reduced under light injury,and RSV reduce the protein expression level of Lt-induced AIF and PARP-1.5.RSV improve the ONL thinning caused by light injury,increase the amplitude of a wave and b wave,and increase the protein expression level of AIF and PARP-1 in the retina.Conlusionglucose deprivation andlight injury induced oxidative stress,mitochondrial structural changes,and mitochondrial dysfunction,and eventually cell death.RSV decreased the production of ROS,improve the ratio of GSH/GSSG,mitochondrial morphology,and mitochondrial membrane potential,as well as reduced cell death.Caspase inhibitor and lentivirus-mediated shRNA knockout of parp-1 suggest that GD-and Lt-induced cell death via different pathways.RSV protects GD-induced cell death by inhibiting the caspase-dependent pathway,while protects Lt-induced cell death by inhibiting the parp-dependent pathway.In addition,RSV increases the protein expression of SIRT1 and the activity of SIRT1 enzyme.SIRT1 inhibitor reduced the protective effect of RSV on glucose deprivation and light injury,suggesting that the protective effect of RSV is related to the activation of SIRT1.RSV protects light injury-induced ONL thinning and improves the retinal function through parp-1 dependent pathway.In summary,RSV has neuroprotective effects on glucose deprivation and light injury.