Quercetin Prevents The Loss Of Outer Blood-retina Barrier Proteins By Regulation Of Autophagy After Oxidative Stress Injury

Objective:The tight junctions of human retinal pigment epithelial cells（ARPE-19 cells）not only constitute outer blood-retina barrier（OBRB）,but also play an important role on occurrence and development of Age-related macular degeneration.Quercetin is a ubiquitous flavonoid compound,which acts as a strong free radical scavenger.Previous research has confirmed that quercetin plays a role in mitigating oxidative stress-related retinal disease.However,it remains unclear that whether it plays an important role in preventing the loss of tight junction proteins after human retinal pigment epithelial cells oxidative stress injury.The present study is aimed to investigate the effect of quercetin on tight junction proteins of ARPE-19 cells,and explore the autophagy-associated mechanism.Methods:In order to select the optimal concentration of quercetin and H₂O₂,MTS assay was used to evaluate the change of cell viability.ARPE-19 cells were pretreated with different concentrations of quercetin before H₂O₂ exposure.The expression and distribution of tight junction proteins occluding,claudin-1 were corroborated by Western Blot and immunostaining.Expression levels and distribution of autophagy-associated proteins Beclin-1,LC-3 and P62 were assessed by Western Blot and immunostaining,respectively.Then ARPE-19 cells were pretreated with 5m M 3-MA,and the previous Western Blot and immunostaining steps were repeated.Results:Among the different H₂O₂ concentration groups（0-1000μM）,the cell viability of ARPE-19 cells all decreased compared with control group（p<0.05）.The cell viability of ARPE-19 cells in quercetin group was higher than that in H₂O₂ group.It was observed that the expression levels of occludin、claudin-1 were decreased in H₂O₂group（p<0.05）.Quercetin treatment significantly enhanced the expression levels of occludin、claudin-1 as compared to H₂O₂ group（p<0.05）.The location of ZO-1 was examined by immunofluorescence experiments.H₂O₂ alone could strongly decrease the ZO-1 expression in the cytomembrane and induce the cytoplasm translocation.Quercetin supplementation significantly enhanced the accumulation of ZO-1 in ARPE-19 cells membranes yet.Compared with control group,the expression levels of autophagy-associated proteins beclin-1 was slightly up-regulated in H₂O₂ group.And the expression levels of beclin-1、LC3-II were significantly increased and that of P62 was decreased in quercetin protection group（p<0.05）.The appearance of LC-3,which examined by immunofluorescence experiments,were enhance in quercetin protection group as compared with control group.Compared with quercetin protection group,the expression levels of beclin-1,LC3-II were obviously increased and that of P62 was decreased in autophagy-inhibited group.Otherwise,the expression levels of occludin、claudin-1 were also decreased（p<0.05）.The fluorescein of LC-3,and ZO-1 were reduced in autophagy-inhibited group as compared with quercetin protection group.Conclusion:Quercetin may prevent the loss of tight junction proteins by up-regulation of autophagy after H₂O₂-induced oxidative stress in human retinal pigment epithelial cells.