Protective Effect Of Quercetin Against Oxidative Stress In Human Retinal Pigment Epithelial Cells And The Possible Molecular Mechanism

Background and Objective:Quercetin is a ubiquitous flavonoid compound,which acts as a strong free radical scavenger and has great application prospects.So far,it remains unclear that whether quercetin plays a role in mitigating oxidative stress-related retinal disease.The present study is aimed to investigate the protective effects of quercetin on human retinal pigment epithelial cells（ARPE-19 cells）and explore the possible molecular mechanism.Methods:MTS assay was used to evaluate the effects of quercetin on cell viability.H₂O₂ was used to establish oxidative stress model of ARPE-19 cells. ARPE-19 cells were pretreated with different concentrations of quercetin before H₂O₂ exposure.Cell viability was assessed by MTS assay and cell apoptosis was measured by flow cytometry.Reactive oxygen species （ROS）were determined by DCFH-DA assay.The importance of the NF-E2-related factor 2（Nrf2）signaling pathway was corroborated by Western blot and immunostaining.Expression levels of endoplasmic reticulum（ER）stress responsive genes and apoptotic markers were assessed by Western blot.Results:（1）Among the different quercetin concentration groups（0-80μM）,differences of cell viability have no significance（p>0.05）.（2）The cell viability of ARPE-19 cells decreased in a dose-dependent manner under H₂O₂ stimulation.The cell viability of ARPE-19 cells in quercetin group was higher than that in H₂O₂ group.The percentage of apoptotic and necrotic cells was decreased in quercetin compared with H₂O₂ group（p<0.05）.（3）The level of ROS decreased in quercetin group as compared to H₂O₂group（p<0.05）.（4）It was observed that 20μM quercetin administration induced expression of Nrf2 and NQO1 in a time-dependent manner（p<0.05）.In H₂O₂group,the expression levels of Nrf2,NQO1 and HO-1 were slightly increased.Quercetin supplementation significantly enhanced the expression levels of Nrf2 and NQO1 as compared to H₂O₂（p<0.05）.The location of Nrf2 was examined by immunofluorescence experiments.H₂O₂alone could only slightly enhance the Nrf2 expression in the cytoplasm,whereas,quercetin treatment significantly stimulated the expression and accumulation of Nrf2 and induced the nuclear translocation of Nrf2 in ARPE-19 cells.（5）Compared with control group,the expression levels of ER stress markers-Bip and CHOP were higher with e IF2αphosphorylation increase in H₂O₂ group.Compared with H₂O₂ group,the expression levels of ER stress markers-Bip and CHOP were decreased with e IF2αphosphorylation reduction in quercetin group（p<0.05）.（6）Compared with control group,the expression level of Bax was increased and that of Bcl-2 was decreased in H₂O₂ group.Cell apoptosis-related protein levels were altered with the reduction of Bax and increase of Bcl-2 in quercetin group（p<0.05）.Conclusion:（1）Quercetin had no obvious toxicity on ARPE-19 cells.（2）Quercetin protected ARPE-19 cells from H₂O₂-induced cell death and had anti-apoptosis function.（3）Quercetin exerted its protective effect against H₂O₂-induced oxidative stress by activating Nrf2 pathway.（4）Quercetin could regulate the expression levels of endoplasmic reticulum stress markers-Bip,CHOP,e IF2αand P-e IF2α.It alleviated H₂O₂-induced cytotoxicity by inhibiting ER stress and up-regulated Bcl-2/Bax.