杨树花保护仔猪肠黏膜屏障物质基础及干预TLR4/NF-κB通路机制的研究

Flos Populi was used for treatment of intestinal disease of young animals in veterinary medicine, which came from Veterinary Pharmacopoeia of People’s Republic of China. It was of obvious curative effect in protecting intestinal mucosal barrier function of weaned piglets. However, little information has been reported about effective substance and mechanism of Flos Populi so far.. Therefore, the spectrum-effect relationships between HPLC fingerprints and activities were investigated using canonical correlation analysis (CCA). The active components were screened from Flos Populi using bioactivity and fingerprint as index. Then, the main effective components were seperated and identified. Furthermore, the effect of main effective components was verified by the model of LPS infecting piglets.. Base on the model of LPS infecting IPEC-J2, the expression of TNF-α, IL-1β, IL-6, IL-8, TLR4, NF-κB, IκB、IKK were detected by Real time-PCR and Western blot analysis. Moreover, the target point of TLR4/ NF-κB will be determined by RNA interference test in vitro. The study will clarity the mainly effective components and the mechanism of the protective effects of Flos Populi on intestinal mucosal barrier. It can also provide important theoretical basis for the application of Flos Populi as green feed additive.

杨树花是《中华人民共和国兽药典》收载的用于治疗肠道疾病的中兽药，本课题组在前期研究中，发现杨树花总黄酮对肠黏膜有明显的保护作用，但其药效物质基础及作用机制尚未明确。本项目拟在建立杨树花指纹图谱基础上，结合杨树花对大肠杆菌脂多糖(LPS)致猪小肠上皮细胞（IPEC-J2）损伤保护作用的活性参数，采用谱效分析法辨识出与活性相关的成分，进一步分离制备有效组分，利用LPS致仔猪肠黏膜损伤的模型进行验证；利用LPS损伤IPEC-J2的模型，采用Real-time PCR、Western blot技术研究有效组分对TLR4/NF-κB通路及下游炎症因子的表达水平的影响，进一步通过体外培养IPEC-J2的RNA干扰试验，确定有效组分作用于该通路的靶点。本研究不仅可阐明杨树花保护仔猪肠黏膜的物质基础，揭示其干预TLR4/NF-κB信号通路的机制，也能为杨树花作为绿色添加剂的应用提供重要的理论基础。

肠黏膜屏障可防止致病性病原体及毒素的侵入，是宿主与病原微生物双向联系的第一道防御，用天然药物进行肠黏膜保护也成为当前研究的热点。杨树花为《中国兽药典》各版所收载，对仔猪腹泻及肠黏膜保护具有明显的疗效，但其药效物质基础及作用机制尚未明确。本项目在建立杨树花指纹图谱基础上，结合杨树花保护肠上皮细胞的药效学试验，经谱效关系分析辨识出有6个色谱峰与肠黏膜保护密切相关。经有机溶剂萃取、柱层析分离、对照品比对鉴定出其中的5个成分分别为槲皮素、木樨草素、芹黄素、松属素和白杨素。. 本项目选取健康雄性BALB/c小鼠建立LPS诱发小鼠肠黏膜损伤模型，采用RNA-seq转录组学方法对其进行测序，结果显示空白组与LPS模型组之间存在345个差异表达基因，包括238个表达量上调的基因和107个表达量下调的基因，其差异表达基因在KEGG中富集的通路主要包括：PPAR信号通路、ECM受体相互作用、补体和凝血级反应以及细胞黏附分子等。将此转录组测试结果结合文献分析，我们推断PPAR/TLR4/NF-κB可能是其关键通路。进一步利用LPS诱导肠黏膜损伤的体外模型，检测有效组分对TLR4/NF-κB通路及下游炎症因子表达水平的影响，从干预TLR4/NF-κB信号通路的角度揭示了杨树花保护肠黏膜屏障的机制。. 此外，本项目还应用响应面法和大孔吸附树脂法对杨树花中木犀草素等有效成分的超声提取工艺及富集工艺进行优化，为杨树花有效成分的开发利用提供重要参考，为杨树花药用资源的开发提供了扎实的理论基础。