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Lutein reverses hyperglycemia-mediated blockage of Nrf2 translocation by modulating the activation of intracellular protein kinases in retinal pigment epithelial (ARPE-19) cells.
Journal of Cell Communication and Signaling ( IF 4.1 ) Pub Date : 2019-12-09 , DOI: 10.1007/s12079-019-00539-1
Arpitha Haranahalli Shivarudrappa 1, 2 , Ganesan Ponesakki 1, 2, 3
Affiliation  

Diabetic retinopathy (DR) is a major cause of acquired blindness among working adults. The retinal pigment epithelium (RPE), constitutes an outer blood-retinal barrier, is vastly affected in diabetic humans and animals. Lower levels of lutein in the serum and retina of diabetic population, and beneficial effects of carotenoids supplementation in diabetic retinopathy patients created an interest to examine the protective effect of lutein on hyperglycemia-mediated changes in oxidative stress and antioxidant defense system in ARPE-19 cells. The WST-1 assay was performed to analyze the impact of glucose, and lutein on the viability of ARPE-19. The intracellular oxidative stress was measured by a DCF (dichlorofluorescein) assay, mitochondrial membrane potential (MMP) was monitored using a JC-10 MMP assay kit and GSH level was examined using GSH/GSSG ratio detection kit. The oxidative stress markers, protein carbonyl and malondialdehyde were spectrophotometrically measured using 2,4-dinitrophenylhydrazine and 2-thiobarbituric acid, respectively. The expression of endogenous antioxidant enzymes and regulatory proteins in ARPE-19 was quantified by western blotting. The localization of Nrf2 protein was examined by immunofluorescent staining. The results show that lutein (up to 1.0 μM) did not affect the viability of ARPE-19 grown in both normal and high-glucose conditions. Lutein treatment blocked high glucose-mediated elevation of intracellular ROS, protein carbonyl and malondialdehyde content in ARPE-19 cells. The decreased MMP and GSH levels observed in ARPE-19 grown under high-glucose condition were rescued by lutein treatment. Further, lutein protected high glucose-mediated down-regulation of a redox-sensitive transcription factor, Nrf2, and antioxidant enzymes, SOD2, HO-1, and catalase. This protective effect of lutein was linked with activated nuclear translocation of Nrf2, which was associated with increased activation of regulatory proteins such as Erk and AKT. Our study indicates that improving the concentration of lutein in the retina could protect RPE from diabetes-associated damage.

中文翻译:

叶黄素通过调节视网膜色素上皮 (ARPE-19) 细胞中细胞内蛋白激酶的激活来逆转高血糖介导的 Nrf2 易位阻断。

糖尿病性视网膜病变 (DR) 是工作成年人中获得性失明的主要原因。视网膜色素上皮细胞 (RPE) 构成外层血-视网膜屏障,在糖尿病人和动物中受到极大影响。糖尿病人群血清和视网膜中较低水平的叶黄素,以及补充类胡萝卜素对糖尿病视网膜病变患者的有益作用,引起了人们对检查叶黄素对高血糖介导的 ARPE-19 细胞氧化应激和抗氧化防御系统变化的保护作用的兴趣. 进行 WST-1 测定以分析葡萄糖和叶黄素对 ARPE-19 活力的影响。通过 DCF(二氯荧光素)测定法测量细胞内氧化应激,使用 JC-10 MMP 测定试剂盒监测线粒体膜电位 (MMP),并使用 GSH/GSSG 比率检测试剂盒检查 GSH 水平。分别使用 2,4-二硝基苯肼和 2-硫代巴比妥酸通过分光光度法测量氧化应激标记物、蛋白质羰基和丙二醛。通过蛋白质印迹法定量 ARPE-19 中内源性抗氧化酶和调节蛋白的表达。通过免疫荧光染色检查 Nrf2 蛋白的定位。结果表明,叶黄素(高达 1.0 μM)不影响在正常和高葡萄糖条件下生长的 ARPE-19 的活力。叶黄素治疗可阻断高葡萄糖介导的 ARPE-19 细胞内 ROS、蛋白质羰基和丙二醛含量升高。在高葡萄糖条件下生长的 ARPE-19 中观察到的 MMP 和 GSH 水平降低通过叶黄素处理得以挽救。此外,叶黄素保护高葡萄糖介导的氧化还原敏感转录因子 Nrf2 和抗氧化酶 SOD2、HO-1 和过氧化氢酶的下调。叶黄素的这种保护作用与 Nrf2 的激活核转位有关,而 Nrf2 与 Erk 和 AKT 等调节蛋白的激活增加有关。我们的研究表明,提高视网膜中叶黄素的浓度可以保护 RPE 免受糖尿病相关损伤。叶黄素的这种保护作用与 Nrf2 的激活核转位有关,而 Nrf2 与 Erk 和 AKT 等调节蛋白的激活增加有关。我们的研究表明,提高视网膜中叶黄素的浓度可以保护 RPE 免受糖尿病相关损伤。叶黄素的这种保护作用与 Nrf2 的激活核转位有关,而 Nrf2 与 Erk 和 AKT 等调节蛋白的激活增加有关。我们的研究表明,提高视网膜中叶黄素的浓度可以保护 RPE 免受糖尿病相关损伤。
更新日期:2019-12-09
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