基于ROS-NLRP3信号通路研究断奶仔猪肠道炎症反应机制及雪莲果酚酸的调控作用

Diarrhea of weaned piglets is a key problem that hinder the development of pig industry. The main causes of diarrhea in weaned piglets are the intestinal inflammation and injury of intestinal barrier. In intestinal inflammation, Interleukin-1β and Interleukin-18 are most important pro-inflammatory factors. It has been verified that ROS mediates NLRP3 inflammasome activation. The activated NLRs connect with ASC and Caspase-1 to form inflammasomes, produce IL-1β and IL-18 and induce inflammatory response. In our previous studies, over-production of ROS and inflammatory response were both occurred in intestines of weaning piglets. Thus, we speculate that ROS can mediate the activation of intestinal NLRP3 inflammasome, and lead to intestinal inflammation and barrier dysfunction. In this study, by in vivo-cell-in vivo study ，we are going investigate the variations of ROS, NLRP3, IL-1β and IL-18 inside intestinal issues of weaning piglets, and the connection of these variations with the intestinal inflammation and barrier function. With the LPS-induced inflammation model in porcine macrophages and H2O2-induced oxidative stress model in porcine intestinal epithelial cells, we are going to explore the mechanism of intestinal inflammation and barrier function regulated by ROS-intermediated NLRP3, based on ROS inhibition technology and NLRP3-silencing technology. Intestinal inflammatory response and barrier function are going be observed in weaned pigs fed with phenolic acid from yacon as well. This study will reveal the role of ROS-NLRP3 signaling pathway in diarrhea of weanling piglets, and provide a new target for prevention and treatment of diarrhea in weaned piglets.

断奶仔猪腹泻是阻碍养猪业发展的关键问题，肠道炎症和屏障功能损伤是腹泻易发的主要内因。IL-1β和IL-18是肠道炎症中最重要的促炎因子。已证实ROS介导了NLRP3激活并与ASC及Caspase-1形成炎症小体，产生IL-1β和IL-18，诱发炎症反应。先期研究发现断奶仔猪肠道ROS过量产生和肠道炎症反应，推测ROS介导了肠道NLRP3炎症小体激活导致肠道炎症和屏障功能障碍。本课题采用体内-细胞-体内研究策略，观察断奶仔猪肠道ROS、NLRP3及IL-1β和IL-18变化及其与炎症反应和屏障功能的关系；利用猪巨噬细胞炎症模型和猪肠上皮细胞氧化应激模型，采用ROS抑制和NLRP3沉默技术探究ROS介导的NLRP3在肠道炎症反应和屏障功能作用机制；以雪莲果酚酸饲喂断奶仔猪，观察肠道炎症反应和屏障功能。综合分析，明确ROS-NLRP3信号通路在仔猪断奶腹泻中的作用，为断奶仔猪腹泻防治发现新靶点。

断奶仔猪腹泻是阻碍养猪业发展的关键问题，肠道炎症和屏障功能损伤是腹泻易发的主要内因。IL-1β和IL-18是肠道炎症中最重要的促炎因子。本课题系统研究仔猪断奶过程肠道氧化还原状态、NLRP3 炎症小体、肠道形态结构、紧密连接蛋白的时空变化规律；建立了LPS诱导的巨噬细胞炎症模型和H2O2诱导的小肠上皮细胞氧化应激模型，采用siRNA沉默技术，结合体内体外试验，研究发现ROS通过激活NLRP3/Caspase-1/IL-1β信号通路诱发断奶仔猪肠道炎性反应；IL-1β可下调紧密连接蛋白ZO-1和Occludin的表达，诱发屏障功能损伤；揭示了ROS-NLRP3-IL-1β信号通路介导仔猪断奶过程中炎性反应和肠道屏障功能的机制；阐明了微生物源性抗氧化剂（MA）通过促进Nrf2进入细胞核，活化Nrf2-抗氧化信号，抑制ROS/NLRP3/IL-1β信号通路减缓肠道氧化应激、炎性反应，促进肠道屏障功能的作用。研究成果进一步揭示了仔猪断奶应激综合征的发生机制，丰富了仔猪肠道发育理论，为预防仔猪早期断奶应激综合征提供新的靶点和方法。