Nrf2/ARE通路在团头鲂鱼油氧化应激损伤中的作用及其大黄素的表观遗传调控研究

Animal growth, fat oxidative stress and nutritional epigenetic regulation become the research focus in this field. Traditional Chinese herbs are potent medicines for treating numerous diseases, yet their specific mechanisms of action are not clear. Our objective in this study is to investigate the effects of emodin on epigenetic regulation and signal transduction mechanisms in the intestine of Wuchang bream, an important aquaculture species and valuable model for exploring the effects of medicinal herbs. Both in vivo and in vitro protocols will be used..Using cell culture,flow cytometry, immunochemistry, western blotting, bisulfite DNA pyrosequencing,confocal microscopy and electron microscopy techniques, we will investigate the effects of emondin on the signal transduction proteins, Keapl, Nrf2 and Bach1 (associated with intestinal oxidaive stress in the cytoplasm and nucleus), quantify the changes of key enzymes (ROS, HO-1, GST, NQO1,et al), evaluate expression of key components in the signal transduction pathway KNrf2/ARE and quantify key proteins invovled in cell apotosis (P53, apsase-3, HSPs). Oxidated fish oil, a stimulator of DNA methylation, will be used to further explore the change of DNA methylation by quantifying DNMT1 and DNMT3a expression induced by emodin. We expect to clarify signaling mechanisms of intestinal oxidative stress induced by oxidated fish oil and regulated by emodin in Wuchang bream mediated by Nrf2/ARE, to reveal the reason dietary emodin modifies the stress response in Wuchang bream, and to develop additional some research ideas on nonspecific immunity and epigenetic regulation. The results will provide a theroetical basis for the development efficient feed and health for Wuchang bream, but also provide important information on neutraceuticals for human food.

动物生长、脂肪氧化应激及其营养表观遗传调控成为大家关注的热点。针对脂肪氧化应激损伤可降低鱼体饲料利用和生长的关键问题，我们前期研究表明日粮大黄素可提高团头鲂抗应激的作用，然而大黄素对鱼油诱导氧化应激损伤保护作用机制还待急需解析。鉴于此，以团头鲂为研究对象，大黄素为调节剂，采用整体动物和离体细胞手段，运用流式细胞仪、甲基化测序及共聚焦显微镜等方法，建立氧化鱼油应激模型，采用抑制剂阻断Nrf2代谢途径，重点解析Nrf2/ARE的信号通路ROS、Keapl、Nrf2、HO-1、GST、NQO1等关键因子与大黄素抗鱼油氧化应激损伤的关系，并深入解析细胞调亡相关蛋白P53、Caspase-3、HSPs等变化。在此基础上，探索大黄素对团头鲂肠道甲基转移酶、DNA甲基化、非编码RNA等影响，进一步阐明大黄素对脂肪氧化应激保护的表观遗传调控作用，为大黄素缓解团头鲂饲料脂质过氧化提供依据。

针对针对肠道氧化应激损伤可降低鱼体饲料利用和生长的关键问题，以团头鲂为研究对象，大黄素为调节剂，采用整体动物和离体细胞等手段，运用细胞培养、流式细胞仪、DNA甲基化测序及共聚焦显微镜等方法，建立氧化鱼油应激模型，采用抑制剂阻断Nrf2代谢途径，重点解析Nrf2/ARE的信号通路ROS、Keapl、Nrf2、HO-1、GST、NQO1等关键因子与大黄素抗鱼油氧化应激损伤的关系，并深入解析细胞调亡相关蛋白P53、Caspase-3、HSPs等变化，进一步阐明大黄素对脂肪氧化应激保护的表观遗传调控作用，为大黄素缓解团头鲂饲料脂质过氧化提供依据。结果表明：.（1）获得0.32mg/ml环磷酰胺可建立团头鲂白细胞的免疫抑制氧化应激模型，为进一步探索大黄素抗氧化应激保护作用打基础。.（2）0.20μg/ml大黄素对环磷酰胺致团头鲂外周白细胞的损伤有一定的缓解保护作用，其机理可能是通过调节细胞内抗氧化应激相关因子以降低对细胞的损伤，刺激胞内Nrf2-Keap1通路，缓解环磷酰胺致团头鲂外周白细胞的氧化损伤。.（3）氧化鱼油导致团头鲂生长受阻，代谢紊乱，免疫力降低，肠道组织损伤病变，抗氧化防御机制启动。氧化鱼油提高了膜受体TLR2表达，激活PI3K-Akt信号，进而激活NF-κB信号，提高下游免疫炎症因子TNF-α、IL-1β、IL-6和IL-12，抗氧化酶GPx1和GSTm表达，提高了机体的抗氧化防御机能。同时，氧化应激激活MHCII-β，经TCR激活NF-κB信号，通过TGF-β和TNF-α抑制肠道炎症。.（4）日粮补充大黄素能够缓解氧化鱼油诱导的生长受阻、提高免疫及抗氧化能力，改善肠道组织形态。miR-144、miR-153a、miR-200a、miR-155和miR-196a与Nrf2-Keap1信号的关键因子Nrf2、Keap1和Bach1存在负调控，其表达趋势表明，miRNA/Nrf2-Keap1信号共同参与了机体氧化应激损伤及大黄素修复调控。