槲皮素磷脂复合物激活Nrf2信号通道增强对氧化损伤ARPE-19细胞的保护作用

Protective effects of quercetin phospholipid complex on oxidative injury in ARPE-19 cells associated with activation of Nrf2 pathway

目的研究槲皮素磷脂复合物对叔丁基氢过氧化物(tert-butyl hydroperoxide,tBHP)诱导ARPE-19细胞氧化损伤的保护作用及相关机制。方法采用溶剂法制备槲皮素磷脂复合物,并测定磷脂复合物在水和氯仿中的溶解度。实验细胞分组:溶媒对照组、模型对照组、槲皮素及其磷脂复合物组(含400μmol·L-1、200μmol·L-1和100μmol·L-1三个浓度)。MTT法检测细胞活力,Annexin V-FITC/PI法检测细胞凋亡,酶标仪检测细胞内超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶活性及丙二醛、总抗氧化能力水平,荧光素DCFH-DA探针法测定细胞内活性氧含量,Real time PCR、Western Blot检测细胞核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、血红素加氧酶-1、醌氧化还原酶-1和谷氨酸半胱氨酸连接酶的表达水平。结果形成磷脂复合物后槲皮素在水和氯仿中的溶解度分别为(38.36±2.91)μg·m L-1、(1351.27±28.12)μg·m L-1,较单体显著提高;浓度为400μmol·L-1、200μmol·L-1和100μmol·L-1的槲皮素及其磷脂复合物对氧化损伤ARPE-19细胞有保护作用,而且高、中浓度的槲皮素磷脂复合物的作用优于单体;浓度为200μmol·L-1的槲皮素及其磷脂复合物均能降低氧化损伤ARPE-19细胞的凋亡率,槲皮素磷脂复合物作用明显优于单体;槲皮素磷脂复合物能显著提高细胞内超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶活性,降低细胞内活性氧、丙二醛水平,槲皮素单体则不能,但槲皮素及其磷脂复合物能提高细胞内总抗氧化能力;槲皮素及其磷脂复合物能诱导Nrf2核转位,激活Nrf2通道,上调血红素加氧酶-1、醌氧化还原酶-1和谷氨酸半胱氨酸连接酶的表达。结论槲皮素形成磷脂复合物后,水溶性、脂溶性提高,对氧化损伤ARPE-19细胞的保护作用比单体强,其作用机制与激活Nrf2信号通路,上调下游Ⅱ相代谢酶和抗氧化酶的表达有关。

Objective To investigate the protective effects of quercetin phospho- lipid complex on oxidative injury in ARPE-19 cells induced by tert-butyl hydroperoxide （t-BHP） and elucidate the underlying mechanism. Methods The phospholipid com- plex of quercetin （quercetin-PC） was prepared by solvent method,the equilibrium solu- bility of quercetin and quercetin-PC in water and chloroform was measured respective- ly. Experimental cells are divided into four groups： control group, oxidative damage, quercetin and quercetin-PC group. The cell viability was detected with MTT assay, the apoptosis was measured by annexinV/PI double staining flow cytometry, the activities of superoxide dismutase （ SOD）, catalase （ CAT）, glutathione peroxidase （GSH-PX） and intracellular level of malondialdehyde （MDA） ,total antioxidant capacity （T-AOC） were detected by enzyme linked immunosorbent assay, intracellular level of reactive oxygen species （ROS） was detected by DCFH-DA staining method. The mRNA and protein ex- pressions of Nrf2, HO-1, NQO-1 and GCL were detected by real-time PCR and Western blot, respectively. Results Quercetin-PC had equilibrium solubility of （ 38.35 ± 2.91 ） μg. mL-1 and （1351.27 ± 28. 12） μg mL-1 in water and chloroform,respectively, which was remarkably higher than those of quercetin alone. Pretreatment with both quercetin and quercetin-PC significantly increased cell proliferation in a dose-dependent manner, especially, quercetin-PC at 200 μmol L-1 and 400 μmol L-1 showed more significantly protective effects on cell proliferation than quercetin alone. Both quercetin and quercetin-PC at 200 μmol L-1 significantly reduced t-BHP-induced apoptosis in APRE-19 cells and quercetin-PC was more effective. In addition, quercetin-PC at 200 μmol L-1 significantly increased the activities of SOD, CAT and GSH-PX, and reduced the levels of ROS and MDA in t-BHP-treated ARPE-19 cells ,but quercetin at 200 μmol L-1 failed to do so,both quercetin and quercetin-PC at 200 μmol L-1 significantly in- creased intracellular level of T-AOC. Molecular examinations revealed that quercetin-PC at 200 μmol L - 1 significantly activated Nrf2 nuclear translocation and enhanced the ex- pression of target genes HO-1, NQO-1 and GCL by different folds at both mRNA and protein levels. Conelusion Quercetin complex with phospholipid significantly en- hance the water and fat solubility of quercetin, quercetin-PC has stronger protective effects on oxidative-induced damages in ARPE-19 cells, which is associated with activa- tion of Nrf2 pathway and its target genes implicate in antioxidant defense.