PEDF Protected Human Retinal Pigment Epithelial Cells Against Oxidative Stress By Upregulating Expression Of UCP2

ObjectiveTo investigate the function of Pigment epithelium-derived factor（PEDF）against oidative stress in ARPE-19 cells.MethodsThe ARPE-19 cells were divided into oxidative groups,using different concentrations of H₂O₂（0,75,150,200umol/L）to injury respectively for 24 h.And protective groups,200ng/ml PEDF was applied to these cells.CCK-8 assay,LDH assay,PI staining and cell growth curve experiments were used for cell viability.The apoptotic genes and the mRNA levels of UCP2 gene expression were determined by RT-PCR and Real-time PCR.We also selected 40 one week old C57BL/6 mice and BALB/c mice respectively,and induced an oxidative stress injury animal model in both C57BL/6 mice and BALB/c mice by injecting5μg of PEDF pretreatment in the vitreous cavity and injecting 150μM H₂O₂ after 24h.HE staining and UCP2 immunofluorescent labeling were conducted.One-way analysis of variance（ANOVA）test was used to analyze these data statistically.ResultPEDF protected ARPE-19 cells from H₂O₂-induced cell death with low toxicity.The results of cell viability showed the viable cells decreased with the increasing concentrations of H₂O₂,while the number of these cells was increased after PEDF treatment.We also discovered the shrinkaged cells and the numbers of cell death were reduced in PEDF groups.Cell activity decreased gradually with the increased concentrations of H₂O₂（p<0.05）.The expression of apoptosis genes caspase 3 and Bax were inhibited,while anti-apoptotic gene Bcl2 was improved by PEDF treatment in different passages of ARPE-19 cells（p<0.05）.RT-PCR and Real-time PCR results showed that PEDF increased the level of UCP2 gene expression.The mRNA level of UCP2 gene were found significant differences between PEDF treated group and H₂O₂alone（p<0.05）.Labeling of the UCP2 detector in the confocal images confirmed a decreased UCP2 protein staining both in RPE cells and RPE layers under H₂O₂ injury,while we found an inhibition of this effect by PEDF.According to HE staining,we measured the thickness of the RPE layer,which were significantly increased in the section with PEDF treatment under oxidative stress,in accordance with the increased number of RPE cells.ConclusionPEDF increased the UCP2 gene expression in both ARPE-19 cells and animal RPE layers under oxidative stress,prompting that PEDF may act a role of protection RPE cells and tissues under oxidative injury.