17β-雌二醇通过PI3K/AKT和雌激素受体信号通路上调NRF2抑制光损伤诱导的SD大鼠视网膜神经变性

朱春晖; 李红波; 王少兰; 王宝英; 胡成虎; 杜芳英; 赵盼盼; 李昂; 俞小瑞

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17β-Estradiol Up-Regulates NRF2 via PI3k/AKT and Estrogen Receptor Signaling Pathways to Suppress Light-Induced Retinal Degeneration in Rat

首发时间：2015-06-24

ZHU Chunhui ¹
ZHU Chunhui (Birth 1985 May),female,PhD candidate,focus on the mechanism study of degeration diseases
LI Hongbo ² WANG Shaolan ¹ WANG Baoying ¹ HU Chenghu ¹ DU Fangying ¹ ZHAO Panpan ¹ LI Ang ¹ YU Xiaorui ²
Yu Xiaorui (Birth 1958 July),female,biochemistry and molecular biology, focus on the mechanism study of retina degeneration diseases and estrogen neuroprotection

1、Department of Genetics and Molecular Biology, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, China
2、Department of Periodontology for Stomatology, Stomatological Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an 710004, China

Abstract：Human age-related retinal diseases, such as age-related macular degeneration (AMD), are intimately associated with decreased tissue oxygenation and hypoxia. Different antioxidants have been investigated to reverse AMD. In the present study, we describe the antioxidant 17β-estradiol (βE2) and investigate its protective effects on retinal neurons. Fourteen days after ovariectomy, adult Sprague-Dawley rats were exposed to 8000-lux light for 12 h to induce retinal degeneration. Reactive oxygen species (ROS) levels were assessed by confocal fluorescence microscopy using 2, 7-dichlorofluorescein diacetate. Nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant enzyme mRNA expression were detected by real-time PCR. Western blotting was used to evaluate NRF2 activation. NRF2 translocation was determined by immunohistochemistry, with morphological changes monitored by hematoxylin and eosin staining. Following light exposure, βE2 significantly reduced ROS production. βE2 also up-regulated NRF2 mRNA and protein levels, with maximal expression at 4 and 12h post-exposure, respectively. Interestingly, following βE2 administration, NRF2 was translocated from the cytoplasm to the nucleus, primarily in the outer nuclear layer. βE2 also up-regulated NRF2, which triggered phase-2 antioxidant enzyme expression (superoxide dismutases 1 and 2, catalase, glutaredoxins 1 and 2, and thioredoxins 1 and 2), reduced ROS production, and ameliorated retinal damage. However, the beneficial effects of βE2 were markedly suppressed by pretreatment with LY294002 or ICI182780, specific inhibitors of the phosphotidylinositol 3-kinase-Akt (PI3K/AKT), and estrogen receptor (ER) signaling pathways, respectively. Taken together, these observations suggest that βE2 exerts antioxidative effects following light-induced retinal degeneration potentially via NRF2 activation. This protective mechanism may depend on two pathways: a rapid, non-genomic-type PI3K/AKT response, and a genomic-type ER-dependent response. Our data provides evidence that βE2 is potentially effective for treatment of retinal degeneration diseases.

keywords： 17β-estradiol neuroprotection oxidative stress nuclear factor-E2-related factor 2 PI3K/Akt estrogen receptor

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17β-雌二醇通过PI3K/AKT和雌激素受体信号通路上调NRF2抑制光损伤诱导的SD大鼠视网膜神经变性

朱春晖 ^{1
2}
ZHU Chunhui (Birth 1985 May),female,PhD candidate,focus on the mechanism study of degeration diseases
李红波 ¹ 王少兰 ¹ 王宝英 ¹ 胡成虎 ¹ 杜芳英 ¹ 赵盼盼 ¹ 李昂 ^{2
3} 俞小瑞 ^{1
4}
Yu Xiaorui (Birth 1958 July),female,biochemistry and molecular biology, focus on the mechanism study of retina degeneration diseases and estrogen neuroprotection

1、西安交通大学医学院遗传与分子生物学系，西安，710061
2、西安交通大学口腔医院牙周粘膜科，陕西西安，710004
3、西安交通大学口腔医院研究中心，陕西西安，710004
4、西安交通大学医学院环境与基因重点实验室，西安,710061

摘要：人类年龄相关性视网膜疾病，如年龄相关性黄斑变性（AMD），与组织的氧合水平降低及缺氧密切相关。当前，不同的抗氧化剂被用以缓解AMD的发生发展。本研究通过玻璃体腔注射17-β雌二醇（βE2），观察其对视网膜神经细胞的抗氧化保护作用。雌性SD大鼠去势14天后经8000-lux白光照射12小时构建视网膜光损伤模型。2,7-二氯二乙酸酯染色法测量视网膜组织活性氧（ROS）的水平并用共聚焦荧光显微镜采集图像；RT-PCR检测Nrf2和抗氧化酶基因的表达；Western blot评估NRF2的激活；NRF2核转位通过免疫组织化学法进行检测；HE染色观察视网膜组织形态学变化。结果显示，光损后，βE2可显著减少ROS的产生。玻璃体腔注射βE2可以显著上调NRF2的表达，其中mRNA水平在4小时最高，蛋白水平在12小时最高。有趣的是，βE2给药后，NRF2从胞浆向胞核易位，这一过程主要发生在视网膜的外核层细胞。随后，我们发现光损后，βE2也能显著上调NRF2表达并激活NRF2依赖的2相抗氧化酶的表达（Sod1和2，Cat，Glx1和2，和Txn1和2），从而降低ROS的产生并缓解视网膜损伤。然而，大鼠玻璃体腔注射βE2前给药PI3K / AKT或雌激素受体（ER）的特异性抑制剂LY294002或ICI182780，可显着地抑制βE2的保护作用。总之，这些观察结果表明，βE2通过激活NRF2发挥抗氧化效应从而保护视网膜神经细胞免受光损伤，该保护机制可能通过两种途径发挥效应：一种依赖非基因组型PI3K / AKT反应，另一种依赖基因组型ER反应。本研究为βE2有效治疗视网膜变性疾病提供理论依据。

关键词： 17β-雌二醇;神经保护;氧化应激;核因子E2相关因子2; PI3K / Akt信号;雌激素受体

基金：

1. The National Natural Science Foundation of China （no.30672286 and no. 81271013) and the National Research Foundation for the Doctoral Program of Higher Education of china）

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ZHU Chunhui,LI Hongbo,WANG Shaolan,et al. 17β-Estradiol Up-Regulates NRF2 via PI3k/AKT and Estrogen Receptor Signaling Pathways to Suppress Light-Induced Retinal Degeneration in Rat[EB/OL]. Beijing:Sciencepaper Online[2015-06-24]. https://www.paper.edu.cn/releasepaper/content/201506-302.

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论文编号	201506-302
论文题目	17β-雌二醇通过PI3K/AKT和雌激素受体信号通路上调NRF2抑制光损伤诱导的SD大鼠视网膜神经变性
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