纳米猴头菇多糖对猪小肠上皮细胞免疫应激的调控作用及其机制

Our previous researches have shown that nanopartide Hericium erinaceus polysaccharides (nano-HEP) can significantly decreased the concentration of IL-6, TNF-α than Hericium erinaceus polysaccharides (HEP) on the immunological stress of intestinal epithelial cells. The project, on the basis of our previous researches, will take nanopartide Hericium erinaceus polysaccharide as the research object. Firstly, the immunological stress models of the intestinal porcine epithelial cells will be established, to study the immunological stress effect of nano-HEP. Secondly, the effects on its signal transduction pathway and related the transcription factor will be studied by using RNA-seq and CRISPR/Cas9 technology, which is study of nano-HEP for intestinal immunological stress effect on the molecular mechanisms. Finally, the effect and mechanism of nano-HEP for intestinal immunological stress activity will be studied by immunological stress swine models. The aim of this project is not to accumulate good experiment data for the effect and mechanism of the nano-HEP on the intestinal immunological stress, but also to provide theoretical basis for further clinical application of polysaccharides for the prevention and treatment of intestinal disease in swine.

前期研究发现，纳米猴头菇多糖能显著抑制肠道免疫应激模型小鼠肠道IL-6、TNF-α的产生，有效降低炎症反应，效果显著优于天然猴头菇多糖。本项目拟在此基础上，建立猪小肠上皮细胞免疫应激模型，研究纳米猴头菇多糖对肠道上皮细胞免疫应激的调控作用；然后利用RNA-seq技术筛选调节小肠上皮细胞免疫应激的主要信号通路和转录因子（即下游靶基因），并利用CRISPR/Cas9技术进行功能缺失性实验，验证靶基因在小肠上皮细胞免疫应激中的功能，揭示纳米猴头菇多糖调节小肠上皮细胞免疫应激的分子机制；最后，通过仔猪肠道免疫应激模型，验证纳米猴头菇多糖对猪小肠上皮细胞免疫应激的调控作用及其机制。研究结果不但对阐明纳米猴头菇多糖调控肠道免疫应激的作用和机制具有重要意义，而且对开发猪肠道保健新兽药、提高猪肠道疾病的防控效果具有重要价值。

本项目成功建立了猪肠上皮细胞系（IPEC-J2 细胞）免疫应激模型，通过运用多种细胞生物学手段研究纳米猴头菇多糖对免疫应激的调控作用，筛选出具有显著免疫保护作用的纳米猴头菇多糖（7.81 μg/mL）；然后利用RNA-seq和CRISPR/Cas9技术筛选出调节小肠上皮细胞免疫应激的主要信号通路MAPK/NF-κB，揭示纳米猴头菇多糖调节小肠上皮细胞免疫应激的分子机制；最后，通过仔猪肠道免疫应激模型，验证纳米猴头菇多糖对猪小肠上皮细胞免疫应激的调控作用及其机制。研究结果首次明确纳米猴头菇多糖对肠道免疫应激的保护作用与MAPK/NF-κB信号通路密切相关。在后续的研究中进一步研究其确切作用机制。