白术多糖对断奶应激诱导肠粘膜屏障损伤的调控作用及机制研究

Maintenance the integrity of intestinal mechanical barrier, the balance of intestinal microbiota and the normal intestinal immune function is crucial during early weaning of piglets. Thus, studies on variation and nutritional regulation of intestinal mucosal barrier functions under weaning stress are needed, which have become the key issues to be solved in pig production. We previously found that Atractylodes macrocephala polysaccharides (AMP) could increase the height of duodenum and jejunum villus, and improve the balance of the intestinal microbiota of weaning piglets. Those results indicate that AMP might regulate the intestinal mucosal barrier functions of piglets under weaning stress..In the present research, AMP will be isolated and prepared, the structural features of which will be measured. And then, weaning piglets and intestinal epithelial cells will be infected using pathogenic E. coli to establish the early weaning stress models. The regulatory effects and mechanism of AMP on intestinal mucosal barrier dysfunction induced by weaning stress based on microbiota and TLR/NF-κB signaling pathway will be investigated using nutritional and molecular biological methods from the overall-cells-molecules levels. This study will provide a theoretical basis for the structure-activity relationship study of polysaccharide and the regulation of intestinal mucosal barrier functions based on microbiota and TLR/NF-κB signaling pathway. Meanwhile, it will create new ideas for the health research of weaning piglets.

早期断奶过程中，保护仔猪肠道机械屏障完整性、微生物区系平衡及肠道免疫功能正常发挥对仔猪健康至关重要。因此，深入研究断奶应激条件下早期断奶仔猪肠道粘膜屏障功能的变化规律及其机制，寻求安全有效的营养调控措施具有重要的科学意义。申请者前期研究发现，白术多糖可以提高断奶仔猪十二指肠和空肠的绒毛高度，调节肠道微生物区系的平衡。结果提示，白术多糖可能对断奶应激诱导的肠道粘膜屏障功能损伤具有一定的调控作用。.基于此，本研究拟在白术多糖提取制备及结构表征的基础上，利用细感染构建仔猪断奶应激模型及肠道上皮细胞应激模型，从整体-细胞-分子层面揭示肠道微生态及TLR/NF-κB信号通路介导白术多糖调节断奶应激诱导肠道粘膜机械屏障、生物屏障及免疫屏障功能损伤的作用机制，为多糖的构效关系研究及白术多糖作为肠道功能调节制剂的应用提供理论依据，并为断奶应激条件下仔猪肠道粘膜屏障功能调控和健康研究奠定基础。

维持肠道机械屏障完整性、微生物区系平衡及肠道免疫功能对仔猪健康至关重要。因此，深入探究肠道屏障功能损伤的潜在机制并研发安全高效的营养调控策略具有重要的科学意义。本研究在白术多糖提取制备及结构表征的基础上，采用葡聚糖硫酸钠（DSS）成功构建了小鼠结肠炎及猪肠道上皮细胞IPEC-J2损伤模型，利用微生物组测序技术、基因沉默技术、RNA-Seq及lncRNA-Seq高通量测序技术等手段，深入探究了白术多糖对肠黏膜屏障功能损伤的缓解作用。高效液相色谱、红外光谱及核磁共振等分析表明，白术多糖是由甘露糖、氨基葡萄糖、核糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖和阿拉伯糖组成（摩尔百分比为1.57%：0.23%：0.22%：2.05%：0.47%：4.68%：58.38%：9.21%：0.29%：22.94%）的杂多糖，平均分子量为3714Da。动物实验结果表明，白术多糖能显著降低DSS诱导的结肠炎小鼠血清及肠道促炎因子水平，缓解肠道嗜中性粒细胞浸润，促进紧密连接蛋白和黏蛋白表达，并调控肠道微生态区系平衡。细胞实验结果显示，白术多糖能显著促进DSS处理的IPEC-J2细胞增殖，降低炎症反应，促进紧密连接蛋白表达，缓解DSS诱导IPEC-J2细胞屏障功能损伤。此外，本研究筛选获得了白术多糖调控肠道屏障功能的靶点lncRNA ITSN1-OT1，阐明了白术多糖通过上调lncRNA ITSN1-OT1阻断磷酸化STAT2的入核转运，防止紧密连接蛋白表达降低和结构破坏，进而维持肠道屏障功能。本研究从动物整体-细胞-分子层面揭示了白术多糖缓解肠道屏障功能损伤的分子机制，为多糖构效关系的研究及其作为肠道功能调节剂的应用提供了理论依据，并为断奶应激条件下仔猪肠道黏膜屏障功能调控和健康研究奠定了基础。