动物疫病的防控急需高效、安全、作用机制明确的新型免疫增强剂。本项目在前期成功研制出藿蜂酮免疫增强剂（EPI），并证明脂质体能进一步提高其免疫增强活性的基础上，探讨EPI脂质体调控Kupffer细胞（KCs）活化作用及机制。首先制备EPI脂质体，并用Box-Behnken响应面分析法对制备条件进行优化；然后运用流式细胞术、实时荧光定量PCR、荧光素酶报告基因、凝胶电泳迁移率、RNA干扰、microRNA芯片、染色体免疫共沉淀等技术，检测EPI脂质体对KCs的活化作用，从体外、体内测定EPI脂质体对KCs中NF-κB信号通路的调控，并考察microRNA在其调控中的作用，阐明EPI脂质体发挥免疫增强作用的分子机制；最后进行临床验证。本项目研究结果不仅对EPI的推广应用和提高动物疫病的防控效果具有重大的实用价值，而且对促进中兽药现代化和研发新型免疫增强剂也具有重要的理论意义。

The prevention and control of animal epidemic diseases are in urgent need of new immunopotentiators with high efficiency, safety and clear mechanism of action. Based on our previous experimental results that epimedium polysacharide-propolis flavone immunopotentiator (EPI) had been developed successfully and liposome could further improve its immune-enhancing activity, this project plans to investigate the activation and mechanism of EPI liposome on regulating Kupffer cells (KCs). First, EPI will be made into EPI liposome, and response surface methodology with Box-Behnken design will be used to optimize the preparation condition of EPI liposome; Next, flow cytometry, real-time fluorescent quantitative PCR, luciferase reporter gene, electrophoretic mobility shift assay, RNA interference, microRNA microarrays, chromatin immunoprecipitation and other methods will be used to detect the activation of EPI liposome on KCs, determine its regulating effect on NF-κB signaling pathway in vivo and in vitro, and investigate the effect of microRNA in regulating action of EPI liposome on NF-κB signaling pathway, the aim is to illuminate the molecular mechanism of EPI liposome on enhancing immune effect. Finally, the clinical verification will be carried out. This study will not only have significant practical value for the popularization and application of EPI and improving the prevention and control effect of animal epidemic diseases, but also have great theoretical significance for promoting the modernization of traditional Chinese veterinary medicines and the development of new immunopotentiators.

动物疫病的防控急需高效、安全、作用机制明确的新型免疫增强剂。本项目在前期成功研制出藿蜂酮免疫增强剂（EPI），并证明脂质体能进一步提高其免疫增强活性的基础上，探讨EPI脂质体调控Kupffer细胞（KCs）活化作用及机制。首先制备EPI脂质体，并用Box-Behnken响应面分析法对制备条件进行优化；然后运用流式细胞术、实时荧光定量PCR、microRNA芯片等技术，检测EPI脂质体对KCs的活化作用，测定EPI脂质体对KCs中NF-κB信号通路的调控，并考察microRNA在其调控中的作用，阐明EPI脂质体发挥免疫增强作用的分子机制；最后进行临床验证。结果显示：EPI脂质体的最优制备工艺为: 脂药比为14:1，膜材比为6:1，超声时间为19 min；作用于KCs 后，EPI脂质体能够显著活化KCs；EPI脂质体能够调控KCs中的NF-κB通路，能够显著促进TLR4、MyD88和NF-κB的mRNA的表达；通过进一步的研究，筛选并确定调控NF-κB通路的6个关键miRNAs。此外，体内试验结果显示，EPL能够显著提高免疫应答效果，且效果显著高于EPI。本项目研究结果不仅对EPI的推广应用和提高动物疫病的防控效果具有重大的实用价值，而且对促进中兽药现代化和研发新型免疫增强剂也具有重要的理论意义。