中药活性多糖对病毒感染免疫细胞组蛋白乙酰化修饰及相关基因表达调控的研究

The objective of this project is to investigate the regulatory effect and its molecular mechanism of active polysaccharides isolated from the roots of Sophora subprosrate or Sargassum on the histone acetylation induced by virus infection in immunocytes in vivo and in vitro. The study involves a intersection field of new technology of molecular biology and basic theory of immunopharmacology as well as free radicalbiology. In this project, the immunocytes of mice and pigs will be chosen as the target cells for the two kinds of polysaccharides. Regulatory effect of the polysaccharides on oxidative stress will be observed by investigating free radical related enzymes, reactive oxygen species level and redox status in immunocytes infected by virus. The actions of virus infection and reactive oxygen species on histone acetylation will be investigated by measuring levels of histone acetylation and activities of related enzymes. The regulatory effect of Sophora subprosrate or Sargassum on histone acetylation induced by virus infection in immunocytes in vivo and in vitro will be observed by investigating levels of inflammatory factors and activities of both histonacetylase and histonedeaoetylase. The modulatory mechanism of the polysaccharides on histone acetylation induced with virus infection will be studied by observing gene expression in immunocytes. The new idea will be of benefit to develop immunomodulator and control virus infections in animals.

本项目拟选择小鼠、猪的免疫细胞为研究对象，以免疫药理学、自由基生物学的理论为基础，结合分子生物学新技术，通过体内外实验，探讨免疫细胞组蛋白乙酰化修饰在病毒感染中的作用，研究山豆根多糖、马尾藻多糖对病毒感染免疫细胞组蛋白乙酰化修饰的调节作用。从免疫细胞活性氧产生酶活性、活性氧水平和氧化还原状态方面，观察病毒对免疫细胞氧化胁迫的影响；从组蛋白乙酰化水平及相关酶活性方面，观察病毒对体外、体内免疫细胞组蛋白乙酰化的影响及活性氧的作用；从免疫细胞组蛋白乙酰化酶和组蛋白去乙酰化酶活性、炎性因子等方面，观察多糖对病毒感染免疫细胞组蛋白乙酰化修饰的调节作用；从免疫细胞组蛋白乙酰化相关基因表达方面，观察病毒介导的组蛋白乙酰化的分子机制及多糖的调控作用。从而明确病毒感染在免疫细胞乙酰化中的作用及多糖调控机制，可为免疫药物研究和畜禽病毒性疾病防制提供新思路，具有重要学术意义和潜在应用价值。

优化并获得了山豆根多糖、马尾藻多糖分离提取最佳工艺。成功建立了PCV2、PRRSV感染诱发的猪脾淋巴细胞、猪肺泡巨噬细胞（3D4/2）、小鼠单核巨噬细胞系RAW264.7细胞氧化胁迫体外模型和PCV-2诱导小鼠体内免疫细胞氧化胁迫模型、PRRSV诱导小鼠体内免疫细胞氧化胁迫模型。PCV2、PRRSV病毒感染能增加免疫细胞组蛋白乙酰化水平；病毒感染免疫细胞导致细胞处于氧化胁迫状态，活性氧水平增加，上调乙酰化水平。山豆根多糖、马尾藻多糖能升高病毒感染免疫细胞组蛋白乙酰化水平；山豆根多糖、马尾藻多糖对病毒感染免疫细胞炎性因子分泌具有一定的调节作用，可缓解炎症反应，与细胞内组蛋白乙酰化水平的变化相关。山豆根多糖通过抑制组蛋白乙酰化相关基因（HAT1、HDAC1）、转录相关基因表达水平，进而抑制炎性基因表达而干预免疫细胞炎症反应。马尾藻多糖能降低炎症基因表达水平，调控炎性因子分泌，缓解炎症。研究发现，山豆根多糖、马尾藻多糖对病毒感染诱发的免疫细胞组蛋白乙酰化具有调节作用。