Quercetin Phospholipid Complex Significantly Protects Against Oxidative Injury In ARPE-19 Cells Associated With Activation Of Nrf2 Pathway

Objective:we prepared the phospholipid complex of quercetin (quercetin-PC),then established tert-butyl hydroperoxide (t-BHP)-induced oxidative injury model in ARPE-19 cells to examine the effects of quercetin-PC. Furthermore, we examined the protective effects of quercetin-PC on oxidative stress-induced damages in ARPE-19 cells, and explored the roles of Nrf2 pathway and the downstream phase II metabolizing enzymes and antioxidases in quercetin-PC effects.Methods:We used the solvent method to prepare quercetin-PC and characterized its structure by differential scanning calorimetry, infrared spectrum and x-ray diffraction. The equilibrium solubility of quercetin and quercetin-PC in water and chloroform was measured respectively. We used t-BHP to treat ARPE-19 cells leading to oxidative damages within cells, then detect protective effects quercetin-PC against cell death in ARPE-19 cells treated with t-BHP by MTT and AnnexinV-FITC/PI on cell proliferation and apoptosis. ARPE-19 cell is divided into five groups:vehicle group (DMSO), oxidative stress group, Nrf2 siRNA group, drug treated group and Nrf2 siRNA+drug treated group. Quantitative PCR was used to detect the Nrf2、HO-1、 NQO-1、GCL mRNA transcription, and western blot to detect nuclear Nrf2, cytoplasmic Nrf2、 HO-1、NQO-1 and GCL protein expression in each group cells.The levels of ROS、T-AOC、SOD and MDA in cells were measured by Dichloride fluorescent yellow chemical colorimetry, xanthine oxidase andSulfide barbituric acid method respectively.Results:Results of differential scanning calorimetry, infrared spectrum and x-ray diffraction ifferential scanning analysis show that quercetin phospholipid complex is a new compound; Quercetin-PC had equilibrium solubility of 38.36 and 1351.27 μg/ml in water and chloroform, respectively, which was remarkably higher than those of quercetin alone. Quercetin-PC, stronger than quercetin, promoted cell proliferation, and the proliferation rate was increased to be 78.89% when treated with Quercetin-PC at 400μM. Moreover, quercetin-PC effectively prevented ARPE-19 cells from apoptosis, and the apoptotic rate was reduced to be 3.1% when treated with Quercetin-PC at 200 μM. In addition, quercetin-PC at 200μM significantly increased the activities of SOD and level of T-AOC, reduced the levels of reactive oxygen species and MDA in t-BHP -treated ARPE-19 cells, but diminished in NRF2 siRNA cells. Molecular examinations revealed that quercetin-PC at 200 μM significantly activated Nrf2 nuclear translocation and significantly enhanced the expression of target genes HO-1, NQO-1 and GCL by different folds at both mRNA and protein levels, but diminished in NRF2 siRNA cells.Conclusion:Our current data collectively indicated that complex with phospholipid significantly enhanced the water solubility of quercetin in water and chloroform; Compared with quercetin, quercetin-PC had stronger protective effects against oxidative-induced damages in ARPE-19 cells, which was associated with activation of Nrf2 pathway and its target genes implicated in antioxidant defense.