德氏乳杆菌通过调控细胞自噬缓解仔猪肠道炎症的机制研究

Intestinal inflammation is the leading cause of diarrhea in piglets and has been a difficult problem in pig industry. Lactobacillus delbrueckii has the characteristics of immunomodulation, anti-bacteria, and anti-inflammation, and plays an important role in improving intestinal health and promoting animal growth. Our previous experimental study found that Lactobacillus delbrueckii alleviated intestinal mucosal barrier damage, coupled with the alteration of autophagy-related genes (LC3 and Beclin-1) expression and the decrease of pro-inflammatory cytokines mediated by NF-κB, indicating that autophagy-mediated intracellular signaling pathway is involved in the protective mechanism of Lactobacillus delbrueckii on inflammatory injury of intestine. Therefore, the present project will be conducted using a piglet model with inflammation induced by lipopolysaccharide to evaluate the effects of Lactobacillus delbrueckii on intestinal inflammation and autophagy from the molecular, cellular, and organizational levels. In vitro, through intervention of autophagy in IPEC-J2 cells, we would like to further clarify the regulation effects of Lactobacillus delbrueckii on mTOR\ULK1\Beclin-1 signaling pathway (confirmed to be one of the major pathways to regulate autophagy), and elucidate the molecular protective mechanism of Lactobacillus delbrueckii on intestinal inflammation. In this respect, this project provides an intervention to prevention and treatment of diseases related to intestinal mucosal barrier injury, and provides a theoretical basis for development of Lactobacillus delbrueckii and its utilization in livestock production.

肠道炎症是仔猪易发生腹泻的主要内在原因，是养猪生产中亟待解决的一个难题。德氏乳酸杆菌具有免疫调节和抗菌消炎的特性，在改善肠道健康和促进动物生长方面发挥重要作用。本课题组前期的研究结果发现，德氏乳杆菌缓解肠黏膜屏障损伤并伴有自噬基因LC3和Beclin-1表达变化以及NF-κB介导的促炎细胞因子的分泌减少，提示细胞自噬介导的胞内信号通路参与了德氏乳杆菌对肠道炎性损伤的保护机制。为此，本项目拟利用脂多糖刺激仔猪肠道建立炎症模型，从分子、细胞、组织等水平研究德氏乳杆菌对肠道炎症反应和自噬活性的影响。再结合IPEC-J2细胞试验，通过体外干预自噬，进一步探究德氏乳杆菌对mTOR\ULK1\Beclin-1信号通路（该通路是介导细胞自噬的重要途径之一）的调控，揭示其缓解肠道炎症的分子机制，为推进肠黏膜屏障损伤相关疾病的防治提供干预手段，并为德氏乳杆菌产品的开发和在畜牧生产中的应用提供理论依据。

肠道炎症是仔猪易发生腹泻的主要内在原因，是养猪生产中亟待解决的一个难题。德氏乳酸杆菌具有免疫调节和抗菌消炎的特性，在改善肠道健康和促进动物生长方面发挥重要作用。自噬能通过调控天然免疫反应控制炎症的过程，而德氏乳杆菌在抗炎过程中的作用是否与细胞自噬有关，尚未见报道。因此，本项目首先利用脂多糖（LPS）刺激仔猪肠道建立炎症模型，以研究德氏乳杆菌对肠道炎症反应和自噬活性的影响。结果表明，德氏乳杆菌能够显著降低断奶仔猪腹泻指数，改善LPS诱导的仔猪肠黏膜形态结构，缓解其肠道炎性细胞浸润，降低肠道IL-1β、IL-6和IL-8等促炎细胞因子的浓度，提高肠道IL-10等抗炎细胞因子的浓度，减少肠道NF-κB（P65）入核，从而缓解肠道炎症损伤状况。通过透射电镜观察发现，LPS可导致仔猪肠道自噬小体数量增多，线粒体形态异常，伴随着细胞自噬相关基因Atg13、Beclin-1、Atg5表达上调，同时自噬形成的标志LC3-II/ LC3-I蛋白表达水平比值明显提高；而德氏乳杆菌能够在一定程度上下调细胞自噬相关基因的表达水平，缓解LPS诱导所致的肠上皮细胞自噬小体的异常增多。最后，再结合IPEC-J2细胞试验，通过体外促进或抑制自噬，检测了细胞炎症水平和自噬活性，进一步发现德氏乳杆菌能通过调控细胞自噬缓解LPS诱导的肠上皮细胞炎症反应，且mTOR\ULK1\Beclin-1信号通路参与其自噬调控作用。本项目从细胞自噬角度揭示了德氏乳杆菌缓解仔猪肠道炎症的分子机制，为减缓仔猪免疫应激、推进肠黏膜屏障损伤相关疾病的防治提供了干预手段，并为德氏乳杆菌（饲用益生菌）的产品开发和在畜牧生产中的应用提供理论依据。