解淀粉芽孢杆菌对巨噬细胞极化的影响及其信号通路解析

Bacillus amyloliquefaciens SC06 (BaSC06), a strain of probiotic screened by our research group, has been proved to have remarkable effects on growth and immunity function of animals. M1 macrophage polarization mediated by BaSC06 may be one of. key mechnisms related to its beneficial effects, such as improving phagocytic capacity of animals. In order to examine whether macrophage polarization induced by BaSC06 is directly related to immunologic function and its resistance to pathogens, we will systematically study the influences of BaSC06 on macrophage polarization and the proteins phosphorylation levels of key transcriptional factors(STAT1/2、NF-κB、AP1、IRF3、IRF5 and IRF9 for M1， STAT3/6 for M2) by culturing BMDM in vitro. According to the results obtained, the signaling pathway involved in BaSC06-induced macrophage polarization is deduced , which is verified by antibody blocking experiment. After that, we will analyze the effect of BaSC06 on macrophage polarization and intestinal mucosal immune function of normal mice and infectious mice in vivo. Eventually, we will further test the importance of BaSC06 in porcine macrophage polarization, signaling pathway and intestinal mucosal immune function by porcine-originated macrophage in vitro experiments and piglets feeding trails. Results will provide theoretical basis for elucidating mechanism of macrophage polarization induced by BaSC06，and also supply the technique and theoretical guidance for BaSC06 applied in pig and feed industry.

解淀粉芽孢杆菌SC06（Ba SC06）是一株由本课题组筛选的具提高动物免疫功能的益生菌，它对巨噬细胞M1极化的调节是其提高动物对病原菌的清除、发挥益生作用的重要原因之一。本项目拟通过体外培养小鼠原代骨髓源Mφ，研究Ba SC06对Mφ极化及其信号通路中关键转录因子（M1型为STAT1/2、NF-κB、AP1、IRF3、IRF5和IRF9，M2型为STAT3/6）磷酸化水平的影响，并通过抗体阻断技术验证Ba SC06介导Mφ极化的信号通路。随后通过小鼠体内试验，研究Ba SC06对正常小鼠和病原菌感染模型小鼠体内Mφ极化和肠粘膜免疫功能的影响，探明Ba SC06对体内Mφ极化的调控是否与机体免疫功能及对病原菌的清除力的提高直接相关；最后通过猪源Mφ细胞体外培养和仔猪饲养试验，进一步研究Ba SC06对猪Mφ极化、信号通路和肠粘膜免疫功能的影响。研究结果将为芽孢杆菌调节Mφ极机理的阐明提供理论基础。

调节巨噬细胞极化是益生菌降低动物病原菌感染的重要作用机理之一。本项目研究了解淀粉芽孢杆菌 SC06（BaSC06）对小鼠体内外Mφ极化及信号通路的影响，对正常和沙门氏菌感染小鼠生长、肠道结构、菌群和巨噬细胞极化的影响，对仔肠生长、肠粘膜结构、免疫功能及巨噬细胞极化的影响。结果发现， SC06单独处理可显著上调骨髓源巨噬细胞M1型标志基因表达、增加iNOS含量、抑制Arg-1表达，但对P- STAT1和P- STAT6表达无显著影响。ST感染巨噬细胞后可降低CD16/32的表达、增加CD206表达， SC06与ST共处理可增加CD16/32并抑制CD206的表达。另外，SC06 促进小鼠腹腔MφM1极化，而ST可促进M2极化并抑制M1极化，同时向小鼠腹腔注射SC06和ST可显著降低脾脏中ST含量。无菌小鼠进一步研究发现，SC06促进肠道MφM1极化，抑制ST诱导的M2极化。最后细胞回输实验发现，回输SC06和M2型处理的Mφ可分别显著降低或增加脾脏中ST含量，回输M2型Mφ显著增加ST含量。以上结果表明BaSC06可通过非经典信号通路诱导MφM1极化，抑制ST的M2极化，增强Mφ和小鼠的杀菌功能。小鼠养殖实验结果表明，口服SC06可显著降低由ST引起的小鼠体重下降及ST移位，显著降低由ST感染所致的IL-1β和IL-12含量的增加，提高IL-10含量，并增加小鼠盲肠中M1和M2型Mφ数量及NO含量。SC06还可改善ST引起的菌群失衡，移植BaSC06组小鼠粪便菌群可够缓解ST引起的盲肠中性粒细胞浸润和炎症，促进Mφ向M2极化。以上结果提示，BaSC06可同时提高小鼠肠道MφM1/M2极化和肠粘膜功能、缓解ST感染引起的炎症。猪体内外实验表明，BaSC06可直接诱导猪巨噬细胞M1极化、增加其吞噬杀菌功能。BaSC06可通过改善肠道菌群结构平衡、抑制MAPKs信号通路和调节炎症相关细胞因子表达，同时通过上调STAT3激活巨噬细胞M2极化，提高肠道M1和M2巨噬细胞数量，维持肠道稳态。