基于ROS-HMGB1信号通路研究断奶仔猪肠上皮细胞自噬机制及胞外多糖调控作用

Weaning stress in piglets is a bottleneck problem that restricts the development of the pig industry. Weaning up-regulates intestinal cell autophagy in piglets. Excessive autophagy can damage intestinal mucosal barrier and induce weaning stress in piglets. ROS can induce cytoplasmic migration of HMGB1 to cytoplasm, where HMGB1 binding to Beclin1 can up-regulate autophagy. Our previous study showed that weaning leaded to intestinal ROS accumulation in piglets. It's still unclear whether ROS-HMGB1 pathway can mediate intestinal autophagy in weaned piglets. This project is going to determine intestinal ROS, autophagy, HMGB1 migration from nucleus to cytoplasm and HMGB1-Beclin1 complex of piglets at different times after weaning, to clarify ROS-HMGB1 pathway-mediated effects on intestinal cell autophagy. With IPEC-J2 oxidative stress model and silencing HMGB1 technique, we are going to explore the mechanism of autophagy in porcine intestinal epithelial cells mediated by ROS-HMGB1 pathway. By feeding weaned piglets with ROS scavenger exopolysaccharide, we are going to determine intestinal ROS, HMGB1 migration and HMGB1-Beclin1 complex, to verify the role of ROS-HMGB1 pathway in mediating autophagy of weaned piglets. This study could propose new strategies to prevent weaning stress in piglets.

仔猪断奶应激是制约养猪业发展的瓶颈问题。断奶上调仔猪肠道细胞自噬，过度自噬损伤肠黏膜屏障功能，诱发仔猪断奶应激。ROS可诱导细胞核HMGB1向胞质迁移与Beclin1 结合上调自噬，前期研究发现断奶导致仔猪肠道ROS积累，ROS-HMGB1通路是否介导断奶仔猪肠道细胞自噬尚不清楚。本项目采用体内-细胞-体内研究策略，测定仔猪断奶后不同时间肠道ROS、自噬、HMGB1由细胞核向胞质迁移及其与Beclin1结合水平，明确ROS-HMGB1通路介导作用；利用猪肠上皮细胞氧化应激模型和沉默HMGB1技术，探究ROS-HMGB1通路介导猪肠上皮细胞自噬机制；以ROS清除剂胞外多糖饲喂断奶仔猪，测定肠道ROS、HMGB1迁移及其与Beclin1结合水平，探究胞外多糖调控断奶仔猪肠道细胞自噬作用，验证ROS-HMGB1通路介导断奶仔猪肠道细胞自噬的靶点作用；为防治仔猪断奶应激提供新思路新策略。

仔猪断奶应激是制约养猪业发展的瓶颈问题。申请者所在团队前期研究发现，断奶可导致仔猪肠道ROS积累。已知ROS可诱导细胞核HMGB1向胞质迁移与Beclin1结合上调自噬，然而ROS-HMGB1通路能否介导断奶仔猪肠道细胞自噬尚不清楚。本项目首先测定了仔猪断奶过程中肠道ROS、自噬和HMGB1变化规律，发现断奶后仔猪肠道组织ROS水平存在先升高再恢复的变化趋势，而肠道组织自噬水平却呈降低趋势。为了进一步明确仔猪断奶后肠道组织ROS水平与自噬的关系，采用具有清除ROS能力的微生物源性抗氧剂饲喂断奶仔猪，基于转录组学分析微生物源性抗氧剂对断奶仔猪肠道组织自噬的影响。研究结果表明，微生物源性抗氧剂能够显著降低断奶仔猪肠道组织ROS水平，且具有激活肠道细胞自噬相关通路的潜在作用。此外，肠道菌群结构紊乱普遍发生于断奶仔猪腹泻过程中，肠道炎症和屏障功能损伤是断奶仔猪易发生腹泻主要内因，调节ROS的产生可改善炎症反应和屏障功能。因此，本项目以肠道菌群为切入点，研究微生物源性ROS清除剂对断奶仔猪和仔鼠肠道菌群组成的调控作用。研究结果表明，微生物源性氧化剂可降低断奶仔猪肠道潜在致病菌丰度、调控肠道菌群组成，研究成果为防治仔猪断奶应激提供了新思路。