Luteolin Inhibits The Retinal Pigment Epithelial Cells And Retinal Damage Induced By Sodium Iodate By Improving Autophagy Flow

ObjectiveTo investigate the effect and mechanism of luteolin on autophagy to protect retinal pigment epithelial cells and retinal injury induced by sodium iodateMethodsIn vitro,Arpe-19 cells were selected and divided into control group,sodium iodate group and luteolin group.MTT assay was used to detect the effect of luteolin on the viability of ARPE-19 cells,and the optimal treatment concentration of luteolin was obtained.Respectively to extract 3 h,6 h,12 h,24 h ARPE-19 proteins in cells,using western blot detection mignonette element of autophagy protein Beclin1,LC3BⅡ/Ⅰratio and the quantity of P62 expression;Ad-m Cherry-GFP-LC3 B was used to detect the effect of luteolin on autophagy flow in RPE cells.Western blot was used to detect the effects of luteolin on the expression of Akt,p-Akt,m TOR and p-m TOR.Furthermore,m TOR inhibitors were used to further prove whether luteolin affects autophagy by Akt /m TOR signaling pathway.In vivo experiment,male C57BL/6J mice were randomly divided into control group,sodium iodate model group and luteolin treatment group.Luteolin treatment group was intraperitoneally injected with luteolin(50mg/kg)daily for pre-protection for 1 week,and sodium iodate group and luteolin treatment group were injected with sodium iodate(25 mg/kg)once through tail vein.SI within 24 h after treatment retina extract protein,using western blot detection LC3 B Ⅱ / Ⅰ ratio and the quantity of P62 protein expression;The luteolin treatment group was continued to receive luteolin,and the retinal thickness was measured by OCT after 4w.After paraffin embedment,the eyeballs of mice were sected and the structural changes of retina were observed by HE staining.The apoptosis of retina cells was detected by TUNEL assay.ResultsIn vitro experiment: MTT indicated that luteolin significantly inhibited the damage of retinal epithelial pigment cells induced by sodium iodate.Group compared with control group,sodium iodate in 3 h,6 h,12 h and 24 h Beclin1 protein involved in autophagy,LC3BⅡ/Ⅰand P62 is significantly higher than the control group,treatment group is lower than sodium iodate and mignonette element groups.After transfection with Ad-m Cherry-GFP-LC3 B recombinant adenovirus,the yellow fluorescence(GFP and m Cherry fusion)of the sodium iodate group was enhanced,while the red spot fluorescence(autophagosome-lysosome fusion)of the luteolin group was stronger than that of the sodium iodate group,suggesting that sodium iodate enhanced autophagy and blocked autophagy flow.Luteolin inhibits autophagy and increases autophagy flux.Further detection of Akt and m TOR proteins showed that the phosphorylated proteins of Akt and m TOR were decreased in the sodium iodate group and increased in the luteolin group.After treatment with rapamycin,autophagy related proteins were decreased in luteolin group.It is suggested that luteolin can inhibit autophagy through the Akt /m TOR pathway,improve autophagy flow,and protect the retinal pigment epithelial cell injury induced by sodium iodate.n vivo experiment: 24 hours after sodium iodate treatment,autophagy related protein LC3 B Ⅱ / Ⅰ and P62 were detected in the control group,the sodium iodate group and the luteolin treatment group.The results showed that the sodium iodate group was higher than the control group,while the luteolin group was lower than the sodium iodate group.OCT and HE results showed that the retina of the sodium ioate group became thinner,and the outer nucleus layer disorder was obvious,while the relative thickness of the retina of the luteolin group increased,and the outer nucleus layer disorder degree decreased.TUNEL staining showed that the apoptotic cells in the sodium iodate group were significantly higher than those in the control group,and those in the luteolin treatment group were lower than those in the sodium iodate group,suggesting that luteolin could protect the retinal injury induced by sodium iodate by inhibiting autophagy.ConclusionsBy activating the Akt /m TOR pathway,luteolin can improve autophagy flow to protect the retinal pigment epithelial cells and retinal damage induced by sodium iodate,providing a potential new target for the treatment of dry AMD.