The Mechanism Of Klotho Protein Alleviating Retinal Oxidative Stress Injury By Decreasing Autophagy

Objective:Age related macular degeneration（AMD）is a leading cause of vision loss and blindness in the elderly over 60 years of age.It is characterized by dysfunction of Retinal Pigment Epithelium（RPE）cells.There are two types of AMD clinically.One is neovascular AMD,also known as wet AMD.The other is atrophic AMD,also known as dry AMD.At present,the pathogenesis of dry AMD is still unclear,and there is no effective treatment.Studies have shown that oxidative stress and autophagy play important roles in the occurrence and development of AMD,and Klotho can play an antioxidant role by inhibiting oxidative stress.However,the specific mechanism by which Klotho protects RPE cells and retinal tissue through autophagy is still unclear.The aim of this study is to investigate the protective mechanism of Klotho protein attenuating oxidative stress injury in Adult Retinal Pigment Epithetial-19 Cells（ARPE-19）and retinal tissues of C57BL/6J mice by decreasing autophagy.This will provide a theoretical basis for new therapeutic strategies for AMD in clinic.Methods:The ARPE-19 cell line was used as the cell model,and C57BL/6J male mice were used as the animal model.The protective mechanism of Klotho protein in alleviating retinal oxidative stress injury by decreasing autophagy was explored from the cellular level and animal level.1.In cell experiments,ARPE-19 cells were induced with Hydrogen Peroxide（H₂O₂）to establish oxidative stress cell model,and pretreated with Klotho protein.Cell viability of each group was determined using CCK-8 assay.The number of surviving cells in each group was observed microscopically.Flow cytometry was used to detect the proportion of apoptotic cells in each group.The proteins of Bcl-2,Bax and C-Caspase-3,which are related molecular markers of apoptosis in each group,and the proteins of LC3 â…¡,LC3 â… ,p62,Beclin-1,which are related molecular markers of autophagy in each group,as well as the expression levels of Akt,p-Akt,mTOR,p-mTOR proteins in the PI3K/Akt/mTOR autophagy signaling pathway,were detected by Western Blot assay.In addition,the expression of p62 protein,an intracellular autophagy binding protein,was verified by immunofluorescence.2.In animal experiments,C57BL/6J mice were induced to establish an oxidative stress animal model by tail vein injection of Sodium Iodate（NaIO₃）,and treated by intraperitoneal injection of Klotho protein.Hematoxylin-Eosin Staining（HE）and Optical Coherence Tomography（OCT）were used to observe the morphological changes of the retina of mice in each group.Electroretinography（ERG）was used to detect the electrophysiological function of mice retina.Western Blot method was used to detect the expression levels of autophagy-related molecular markers LC3 â…¡,LC3 â… ,p62,Beclin-1,including the protein expression of Akt,p-Akt,mTOR,p-mTOR in PI3K/Akt/mTOR autophagy signaling pathway in mice retina tissues.Results:1.In cell experiments:（1）The treatment of ARPE-19 cells with H₂O₂resulted in decreased cell viability,decreased number of surviving cells,increased proportion of apoptotic cells,decreased expression of anti-apoptotic protein Bcl-2 and increased expression of pro-apoptotic proteins Bax and C-Caspase-3.However,pretreatment with Klotho protein could restore cell viability,reduce cell damage and apoptosis,and reverse the changes of apoptosis-related proteins caused by H₂O₂.（2）The ratio of LC3 â…¡/â…  and the protein expression of p62 and Beclin-1 in ARPE-19 cells increased after H₂O₂treatment.Klotho protein pretreatment could reverse the changes of autophagy-related proteins induced by H₂O₂.（3）Treatment of ARPE-19 cells with H₂O₂increased the ratio of p-Akt/Akt and p-mTOR/mTOR in PI3K/Akt/mTOR autophagy signaling pathway.After pretreatment with Klotho protein,the ratio of p-Akt/Akt and p-mTOR/mTOR further increased,which promoted the expression of phosphorylated proteins in this pathway.（4）PI3K inhibitor LY294002 was added to ARPE-19 cells before Klotho protein pretreatment,which counteracted the effect of Klotho on the expression of PI3K/Akt/mTOR pathway proteins,and decreased the number of surviving cells and cell viability.2.In animal experiments:（1）The results of HE staining of mice retinal tissue showed that NaIO₃treatment could cause structural disorder of the mice retina,the thickness of the retina became thinner,the number of Outer nuclear Layer（ONL）cells decreased,and the RPE layer appeared black and round deposits.However,Klotho protein treatment could improve the retinal histomorphological changes caused by NaIO₃.（2）The results of mice retinal OCT showed that NaIO₃treatment could lead to the thinning of retinal thickness,the structure of each layer was fuzzy and disordered,and the RPE layer appeared continuous high reflective protrusions.However,Klotho protein treatment could improve the retinal histomorphological changes caused by NaIO₃.（3）The ERG results of mice retina indicated that NaIO₃treatment could lead to a decrease in the amplitude of a and b wave of mice electroretinogram,and the latency was prolonged.However,Klotho protein treatment could improve the NaIO₃induced ERG changes.（4）Western Blot analysis of mice retinal tissue indicated that NaIO₃treatment could increase the ratio of autophagy marker protein LC3 â…¡/â…  and the expressions of p62 and Beclin-1.The ratio of p-Akt/Akt and p-mTOR/mTOR in PI3K/Akt/mTOR autophagy signaling pathway was decreased.However,Klotho protein treatment could reverse the changes of autophagy-related proteins caused by NaIO₃.Conclusion:This research show that Klotho protein may reduce autophagy by activating the PI3K/Akt/mTOR signaling pathway.It alleviates oxidative stress damage of cells,thus enhancing RPE cell viability,reducing cell apoptosis,and finally playing a protective role on retina.