基于Th17/Treg细胞平衡研究壳寡糖调控仔猪肠黏膜免疫对ETEC感染的防御机制

The intestinal problem is the main cause of diarrhea in piglets, and the imbalance of Th17/Treg cells is an important cause of the intestinal mucosal immunity. Our previous study found that chitosan oligosaccharide could improve the mucosal immunity of piglets, what’s more, chitosan oligosaccharide could affect the key effect factors IL-17 and Foxp3 and TLR4, STAT3, MAPK signaling pathway, which regulate the balance of Th17/Treg cells, but the mechanism is still not clear. Therefore, the effect of chitosan oligosaccharide on the balance of mucosal Th17/Treg cells in ETEC diarrhea piglets will be clarified in this study, and its pathway will be verified in mice by FCM, PCR Array and Western Blot technology and so on, then the regulation mechanism of chitosan oligosaccharides on the major cytokines, transcription factors and TLR4, Tim-3 and STAT3 signal pathway that impact the balance of Th17/Treg cells will be interpreted from the cellular immunity, gene, protein and molecular signaling pathway angle, furthermore, the molecular mechanism of chitosan oligosaccharide on regulating the balance of Th17/Treg cells will be verified by ETEC stimulated IL-17A gene knockout mice. Hence, the research results will provide new ideas for the nutrition intervention and drug treatment to piglets diarrhea, and provide a scientific experimental basis for the theoretical basis of chitosan oligosaccharide application as feed additives.

仔猪肠道问题是引起腹泻的主要原因，而Th17/Treg细胞失衡是影响肠黏膜免疫的重要原因。我们前期研究发现壳寡糖能提高仔猪肠黏膜免疫，对影响Th17/Treg细胞平衡的关键因子IL-17、Foxp3和TLR4、STAT3、MAPK信号通路有调节作用，但其作用机制尚不明确。本项目通过FCM、PCR Array、Western Blot等技术阐明壳寡糖对ETEC腹泻仔猪肠黏膜Th17/Treg细胞平衡的作用，并用小鼠验证其作用途径，从细胞免疫、基因、蛋白与分子信号通路角度阐释壳寡糖对影响Th17/Treg细胞平衡的主要细胞因子、转录因子和TLR4、Tim-3、STAT3信号通路的调控机制，再用ETEC刺激的IL-17A基因敲除小鼠验证壳寡糖调节Th17/Treg细胞平衡的分子机制。预期研究结果将为仔猪腹泻病的营养干预及药物治疗提供新思路，为壳寡糖作为饲料添加剂应用的理论基础提供科学实验依据。

本项目以小鼠为研究对象，开展了壳寡糖靶向调控小鼠肠黏膜Th17细胞和Treg细胞分化与Th17/Treg 细胞平衡的研究。研究结果得出：COS对ETEC刺激小鼠上皮损伤有缓解作用，对炎性浸润程度、组织损伤评分无明显作用，对小鼠回肠Th17/Treg平衡相关细胞因子IL-6、TGF-β、IL-17、IL-21、IL-23、IL-2、IL-10、IFN-γ水平均表现出明显作用，但对Th17/Treg平衡相关基因的mRNA表达的IL-6、Jak2、STAT3、RORγt、IL-17、IL-2、Jak3、STAT5、Foxp3、IL-10 mRNA表达量无明显作用。然后，本项目以仔猪为研究对象，开展了基于Th17/Treg细胞平衡研究壳寡糖调控ETEC诱导的腹泻仔猪肠黏膜免疫的分子机制研究。研究结果得出：ETEC攻毒使仔猪产生了腹泻和炎症反应，COS可以提高仔猪生长性能，缓解仔猪外周血炎性细胞浸润，促进肠道发育，优化仔猪肠道菌群结构；COS能抑制ETEC刺激的仔猪肠道黏膜IL-6/JAK/STAT3和TGF-β1/STAT5/FOXP3信号通路的激活。.本项目研究表明COS对ETEC攻毒诱导的肠道炎症仔猪在生长性能、血常规指标、肠道微生物稳态方面起作用，COS主要通过IL-6/JAK/STAT3信号通路维护Th17/Treg细胞失衡方面，从而缓解ETEC刺激对肠道炎症的损伤作用。本研究成果为壳寡糖增强动物免疫功能、缓解炎症反应损伤和促进机体健康提供营养调控手段，为降低动物炎症反应的营养调控及药物干预策略研究提供新思路，为COS作为饲料添加剂应用提供了重要科学试验依据，对畜牧业可持续性发展具有重要的理论和实践意义。