辣蓼黄酮对病毒感染猪免疫细胞活性氧介导的炎性反应干预作用及其信号机制研究

The objective of this project is to investigate the regulatory effect and its molecular mechanism of flavonoids from Polygonum flaccidum Meissn on reactive oxygen species (ROS)-mediated inflammatory reaction in swine immunocytes infected by PRRSV. 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 pigs will be chosen as the target cells for the flavonoid and PRRSV to observe regulatory effect of the flavonoid on levels of ROS and inflammatory factors in vivo and in vitro. The effect of the flavonoid on the inflammatory cascade reaction mediated with ROS in swine immunocytes infected by PRRSV will be studied by observing gene expression associated with ROS and inflammatory factors. The molecular mechanism of flavonoids from Polygonum flaccidum Meissn on ROS-mediated inflammatory reaction in swine immunocytes will be will be observed by investigating signaling pathways involved in NF-kB, MAPKs, AMPK, protein phosphorylation and prostaglandins. The new idea will be of benefit to develop anti-inflammatory drugs and control virus infections in animals.

本项目拟选择猪的免疫细胞为研究对象，以免疫药理学、自由基生物学理论为基础，结合现代分子生物学新技术，探讨辣蓼黄酮对病毒感染免疫细胞内活性氧介导的炎性反应干预作用及其分子机制。分离提取及纯化辣蓼总黄酮；采用猪繁殖与呼吸综合征（经典猪蓝耳病）病毒体内外感染猪免疫细胞，通过体外实验和整体动物实验，观察病毒感染对免疫细胞内活性氧、炎性因子水平影响及辣蓼黄酮的调节作用；从细胞内活性氧、炎性因子相关基因表达方面，探讨辣蓼黄酮对活性氧介导的炎性级联反应的影响；从细胞内NF-kB信号通路、MAPKs信号通路、AMPK信号通路、蛋白磷酸化信号通路和前列腺素信号体系等方面探讨辣蓼黄酮对活性氧介导的免疫细胞炎性反应干预的分子机制。从而明确天然活性黄酮的抗炎作用与机体自由基反应的相关性，阐明生物活性黄酮调控炎性反应的分子机理，为动物病毒性疾病的预防和治疗提供新的思路，具有重要的学术意义和潜在应用价值。

本研究优化并获得了辣蓼黄酮提取和分离纯化的最佳工艺，分离提取出辣蓼黄酮及活性部位。通过体内、体外实验，探讨了活性氧在病毒感染免疫细胞中的作用，发现PRRSV体外感染能导致猪肺泡巨噬细胞活性氧水平明显升高，活性氧参与PRRSV感染的炎性反应过程。在LPS诱导的体外炎症模型和小鼠内毒素血症体内炎症模型的基础上，建立了PRRSV、PCV2、PRV分别感染诱发的猪肺泡巨噬细胞、猪肺泡巨噬细胞系（3D4/2）、小鼠单核巨噬细胞系（RAW264.7）体外炎症模型、PRRSV感染猪的整体动物炎症模型和PCV-2、PRV感染小鼠整体动物炎症模型；辣蓼黄酮通过调节病毒感染免疫细胞内活性氧、炎性因子水平和氧化还原状态干预氧化应激和炎症反应。辣蓼黄酮体外通过下调PRRSV感染的猪肺泡巨噬细胞AKT、c-fos、c-jun、c-myc的mRNA表达水平，体内通过下调c-fos、c-jun、c-myc、NF-κB、p-AKT和MAPK的mRNA表达水平及MAPK、PI3K/Akt和NF-κB磷酸化通路中炎症反应相关蛋白的表达水平，干预PRRSV感染诱导的猪肺泡巨噬细胞炎症反应。辣蓼黄酮通过下调IL-1β、TNF-α、IL-6、IL-8、IL-10、COX-2、iNOS、c-fos、c-jun、c-myc、P65、AKT、P38 MAPK和ERK的mRNA表达水平，并下调PI3K/AKT、p44/42、p38和NF-κB及其相应蛋白磷酸化的表达，抑制MAPK和PI3K/Akt信号通路而干预PCV2、PRV感染的3D4/2、RAW264.7细胞的炎性反应。本研究明确了病毒感染诱发免疫细胞炎性反应中活性氧的作用及辣蓼黄酮调控的分子机制，具有重要的学术价值和潜在应用前景。