The Mechanism Of Pretreatment Of Lactate Alleviates Oxidative Stress Injury In ARPE-19 Cells And Retina By Regulating Autophagy

Objective:In this study,we want to investigate the protective effects of lactate by upregulating autophagy in oxidative injury in ARPE-19 cells and retina.Methods:1.CCK-8 assay was used to detect the damage concentration and time of H₂O₂on ARPE-19 cells,and to screen the protective concentration and time of lactate pretreatment.Western blot method was used to detect the expression of LC3II、LC3I protein in ARPE-19 cells treated with different concentrations of H₂O₂ and combined with lactate preconditioning.2.3-MA inhibited autophagy induced by lactate preconditioning,and Western blot method was used to detect the expression of LC3,p62 protein level;the formation of autophagosome was observed by transfecting EGFP-LC3B plasmid;the proliferation activity of cells in each group was detected by CCK-8 method.Level of reactive oxygen species and mitochondrial membrane potential were detected by flow cytometry;Detection of ARPE-19 cell death by Calcein-AM/PI fluorescence double staining.Combined with the previous experimental results,we can explore the role of lactate-induced autophagy in oxidative stress in ARPE-19 cells.3.Pretreatment was carried out by injecting lactate or lactate+3-MA into the vitreous cavity of C57/BL6 mice,then the model of oxidative stress was induced by intraperitoneal injection of sodium iodate.The changes of retinal tissue structure were detected by HE staining,and the apoptosis of retinal tissue was detected by TUNEL fluorescence.Results:1.ARPE-19 cells treated with low concentration of H₂O₂ increased autophagy,while the level of autophagy decreased gradually in high concentration treatment,and the cell viability decreased with the increase of concentration;After pretreatment with lactate,autophagy level and cell viability were increased,ROS level and membrane potential damage caused by H₂O₂ were decreased.2.3-MA blocked the increase of autophagy level,ROS production and decrease of mitochondrial membrane potential by lactate.3.The results of HE staining showed that after sodium iodate treatment,the continuity of retinal pigment epithelium disappeared,the arrangement of cells in outer nuclear layer was disordered,the thickness of retina became thinner.Lactate pretreatment reduced the damage of retinal structure caused by sodium iodate and the structural continuity of retinal pigment epithelium was slightly better than that in sodium iodate group.TUNEL results showed that sodium iodate treatment increased apoptosis in RPE layer but lactate preconditioning reduced retinal cell apoptosis induced by sodium iodate.Conclusion:1.Lactate pretreatment of ARPE-19 cells can increase autophagy to reduce the injury of H₂O₂ on cell viability,and reduce ROS production,mitochondrial membrane potential disruption and cell death.2.Lactate pretreatment can reduce the oxidative stress injury of retina induced by sodium iodate and reduce the degree of retinal tissue damage and cell apoptosis.After3-MA blocked autophagy induced by lactate preconditioning,the protective effect of lactate preconditioning on retina was reduced.