基于MAPKs通路研究淫羊藿苷及磷酸化修饰物对ETEC诱导的断奶腹泻仔猪肠道屏障功能的保护作用

Enterotoxigenic Escherichia coli (ETEC) can damage the intestinal barrier function of weaned pigs and induce diarrhea. The use of traditional Chinese medicine chemical ingredients can avoid the disadvantages of antibiotics in treating diarrhea in weaned piglets to some extent. The previous study by the applicant found that Icariin (ICA) has a good protective effect on Duck embryo hepatocytes, and phosphorylated modified icariin (pICA) can significantly enhance this protective effect. This project intends to study the protective effect and mechanism of ICA and pICA on the intestinal barrier of ETEC-induced weaned diarrhea piglets in vitro and in vivo. Based on the establishment of etec-induced diarrhea in piglets and Ipec-j2 cell injury model, the effect of ICA and pICA on intestinal epithelial structure and cell permeability, tight junction protein, mitochondrial function and apoptotic protein was studied by transmission electron microscopy, Ussing Chamber, qRT-PCR, Western blot, immunofluorescence, flow cytometry and the protective effect of ICA and pICA on intestinal barrier was analyzed. Furthermore, specific blockers were used to inhibit the MAPKs signaling pathway to verify the protective mechanism of ICA and pICA on intestinal barrier. In this study, the protective effects of ICA and pICA on intestinal barrier were analyzed at the cellular, animal and molecular levels, which provided a scientific basis for the subsequent commercialization of veterinary preparations for the protection of intestinal health of piglets.

ETEC可以使断奶仔猪肠道屏障功能受损而诱发腹泻，利用中药化学成分可以从一定程度上避免抗生素在治疗时存在的种种弊端。申请人前期研究发现，淫羊藿(ICA)对雏鸭肝细胞具有良好的保护作用，磷酸化修饰后(pICA)则可以显著提高这一保护作用。本项目拟通过体外和体内实验，研究ICA与pICA对ETEC诱导的断奶腹泻仔猪肠道屏障的保护作用及机制，在构建ETEC诱导的仔猪腹泻模型和IPEC-J2细胞损伤模型的基础上，应用透射电镜、Ussing Chamber、qRT-PCR、WB、免疫荧光、流式细胞等技术研究二者对肠道上皮结构与细胞通透性、紧密连接蛋白、线粒体功能、细胞凋亡的影响，分析二者对肠道屏障的保护作用；进一步通过特异性阻断剂抑制MAPKs信号通路，验证二者对肠道屏障的保护机制。本研究从细胞、动物和分子水平解析二者对肠道屏障的保护效果，为后续保障仔猪肠道健康的天然产物兽用制剂产品化提供科学依据。

产肠毒素型大肠杆菌K88（ETEC K88）导致的断奶仔猪腹泻严重危害我国养猪业，其呈现的多重耐药性趋势对防治工作带来巨大挑战。因此，寻找替代传统抗菌药物的新兽药具有重要意义。本项目主要开展淫羊藿苷（ICA）和磷酸化淫羊藿苷（pICA）缓解ETEC K88诱导的肠道屏障损伤的机制研究。结果表明，首先，ICA和pICA可以显著降低ETEC K88导致的断奶仔猪腹泻率；其次，体内外实验发现ETEC K88可以通过增加促炎细胞因子表达、激活氧化应激、抑制抗氧化酶活性的方式来破坏肠道通透性，进而导致肠道屏障损伤，而ICA和pICA可以通过逆转这一过程来缓解ETEC K88造成的肠道屏障损伤；最后，通过p38 MAPK抑制剂(SB203580) 和激活剂（anisomycin）干预实验证明了ICA和pICA通过调节p38 MAPK信号通路对ETEC K88诱导的肠屏障功能损伤起到保护作用。本项目旨在为后续保障仔猪肠道健康的天然产物兽用制剂产品化提供理论基础，并为运用中药防控其他动物疾病提供参考。本项目作为第一标注已发表SCI论文1篇，作为第二标注发表已SCI论文1篇；参加学术会议3次；培养研究生2名。