The Role Of Oxidative Stress In Age-related Macular Degeneration

Age-related macular degeneration （AMD） is a sight threatening eye diseasethat affects millions of humans over the age of50years.It is considered to be themajor cause of irreversible blindness in the elderly population in the developedworld. AMD is characterized by a progressive loss of central vision attributable todegenerative and neovascular changes that occur in the interface between theneural retina and the underlying choroid.This location contains retinalphotoreceptors,retinal pigmented epithelium,basement membrane complexknown as Bruch’s membrane and a network of choroidal capillaries.AMD isincreasingly recognized as a complex genetic disorder where one or more genescontribute to an individual’s susceptibility to development of the condition,whilethe prevailing view is that the disease stems from the interaction of multiplegenetic and environmental factors.Although it has been proposed that a thresh-old event occurs during normal aging,the sequelae of biochemical,cellular,andmolecular events leading to AMD are not fully understood.Oxditive Stress isconsidered to play an important role in the pathological mechanism of AMD. Oxidative stress is subjected to a variety of harmful stimuli, produced too muchhighly reactive molecules such as reactive oxygen species （ROS） and reactivenitrogen species （RNS）. Oxidation and antioxidant system is in balancein vivo,when the balance of oxidation and antioxidant is impaired,tissue damagemay take place. Under normal condition, production and decompositionof reactive oxygen species is in a state of homeostasis. And a variety ofendogenous and exogenous noxious stimuli can break this homeostasis ofoxidation and antioxidant,generating a large number of reactive oxygenspecies, causing oxidative DNA damage and abnormal expression of protei-ns, which have cytotoxic effects on the body and cause irreversible damageeventually,namely the formation of oxidative stress.Growth, aging,and manyother pathophysiological processes of cells in the body play important roles totrigger apoptosis except for oxidative damage.The study found that reactiveoxygen species-induced apoptosis caused by oxidative stress is an important partin this course.Polyunsaturated fatty acid is rich in retinal photoreceptor outer segm-ent disk membranes, so it is highly vulnerable to oxidative damage,and thenthe chain of cell lysis occurres. In the early AMD,RPE （retinal pigmentepithelium,RPE） cells, with the burden of age,RPE phagocytosis of photore-ceptor occurred during lysosomalenzyme degradation fragmentation, the degra-dation is not fully digest the form of insoluble aggregates,which is knownas lipid deposited in RPE has not been completely degraded in lysosomes of p-hotoreceptor outer segment disk membrane residues, such as lipofuscin accumul-ation in the cell, which can interference RPE cell’s metabolism,and causemetabolites Bruch builds up in the membrane,lead to oxygen diffusion block,induce angiogenesis.Formation of CNV secondary to up-regulation of VEGF,which mainly produced by RPE cells,RPE is located between the ne-ural retina and choroid, as the outer blood retinal barrier, controlling thechemical composition of the subretinal space.RPE cells transfer nutritionaland metabolic wastes between sensory retina and choroidal capillary.The surfaceof RPE cell membrane and the extracellular matrix is linked together.In theearly embryodevelopment process,RPE spontaneous transdifferentiation intoneural retina.In the vicinity photoreceptors,RPE plays an important role ofproducing a large number of reactive oxygen species in the development andmaintenance in lighting conditions.A mixture of different lipid molecules,including the retina induced-complexes, some of which have light induc-ed nature,can enhance the oxidative stress.The retinal has developed a range of protective mechanisms to defend theoxidatie stress damage from ROS damage.Antioxidant enzymes,such as SOD,GST and GSH, playing an important role in the process of anti-oxidation.Based on the backgrounds indicated above,we collected the samples ofblood of AMD patients in this study.The levels of3antioxidant enzymes, SOD,GST and GSH in the blood were investigated.Furthermore,we simulatedoxidative stress state in RPE in vitro by H₂O₂,the level of three antioxidantenzymes,SOD,GST and GSH in REP cells were observed after oxidative damageby H₂O₂.At last,we assayed the possible protection mechanism of RPE cells inthe oxidative condition.The main results are as follow:1.The level of three antioxidant enzyme of AMD patient:AMD group hadsignificantly higher plasma GST （76.96U/L） compared with that in control group（62.74U/L）,the difference was statistical significance（Z=﹣2.082,P=0.037）;Thelevel of GSH in AMD group（61.38mgGSH/L）was significantly lower than that in control group （81.33mgGSH/L）,the difference was statistical significance （Z=﹣2.900，P=0.004）;The level of SOD was not significantly different between AMDgroup（64.47U/L） and control group （62.53U/L）（Z=﹣0.090，P=0.725）.2. Morphological changes of RPE under oxidative stress:RPE cells treatedwith0.6μM H₂O₂for24h demonstrated substantial condensed nuclei,DNAfragmentation, characteristic of dead cells,as indicated by Hoechst33258staining,whereas RPE cells under normal conditions seldom displayed apoptotic nuclei.3.The change of RPE cell viability:Treatment of RPE cells with differentH₂O₂concentrations,the cell viability was significantly decreased,400μmol/L ormore concentration of H2O2stimulatedcells compared with the control group wasstatistically significant （P<0.05）;RPE was treated with different concentrationsof Vit C,cell viability increased significantly,each concentration group com-pared with the control group were statistically significant （P<0.05）;differentconcentrations of Vit C pretreatment RPE4h, a single concentration of H2O2treatment RPE24h, compared with the control group, found that after150μmol/L or more concentrations of cells treated with Vit C,its activity wassignificantly higher than the untreated cell activity, with statistical significance （P<0.05）; single concentration of Vit C-treated cells after4h,differentconcentrations of H2O2stimulation RPE24h,compared with the control group,theviability of the cells treated with different concentrations of Vit Cincreased apparently.4. The change of intracellular antioxidant enzyme activity:H₂O₂treatment（0.6μmol/L,24h） caused significant alterations in all of these antioxidantbiomarkers.Aftre treatment with H₂O₂, the SOD,GSTand GSH level were reduced.However, with H₂O₂exposure, pretreatment with Vit C for4h significantlyprotected against all these changes. 5. RPE cell protection by Vit C is dependent on increased activity ofERK1/ERK2,Western blot assay was performed for phospho-ERK1/ERK2afterH₂O₂challenge for2h. Vit C and H₂O₂treatment independently （as well ascombined） significantly increased the level of phospho-ERK1/ERK2.Thisincreased level of phospho-ERK1/ERK2was locked by PD98059,which is aninhibitor of ERK1/ERK2phosphorylation.In short,this study shown that the levels of antioxidant enzymes GST and SODin AMD patients’ plasma are increased,indicating that the patients are in theoxidative stress state,AMD patients with reduced GSH levels are morevulnerable to have oxidative stress attack.Activity of SOD,GSTand GSH decreasein RPE cells under oxidative stress state,after the cells treated with theantioxidant Vit C,the intracellular antioxidant enzymes activity increase,whichcan effectively protect RPE cells from oxidative stress induced the apoptosis.Meanwhile,the results are inconsistent with the plasma levels of GSH, suggestingthat in oxidative stress state,GSH only play a role in ocular part,of course,itneeds more extensive research to confirm.Based on the experimental study,thefurther research focus on more sample of research,including looking for itspossible protection mechanism and reason,for clinical treatment AMD with newways and direction.