益生芽孢杆菌对断奶仔猪自噬的影响及其缓解氧化应激的分子机理

Probiotics with anti-oxidative capacities are able to alleviate oxidative stress in weaned piglets. However, there are few reports about the effects of probiotics on autophagy and its molecular mechanisms involved in antioxidant defense. To this end, we will utilize western blot, immunofluorescence confocal and transmission electron microscopy, as well as RNAi technology to evaluate the effects of probiotics with antioxidant capacities (selected in our lab) on the expression of four main transcription factors (Nrf2, FoxO1, NF-κB and AP1) and key proteins in signaling pathways related to autophagy/anti-oxidation (p62, keap1, Nrf2, MAPK and p53) in rat intestinal epithelial cell 6 (IEC6). Then, we will study the potential autophagy/anti-oxidation-related signaling pathways mediated by probiotic Bacillus in rats under normal and stress conditions. Eventually, we will utilize intestinal porcine epithelial cell 1 (IPEC-1) and weaned piglets exposed to oxidative stress to further clarify the impacts of probiotic Bacillus on autophagy and its regulation in oxidative stress in weaned piglets. Results will illustrate the effects of probiotic Bacillus on autophagy/anti-oxidation and the underlying molecular mechanisms in weaned piglets.

本项目拟利用western blot、免疫荧光和透射电镜等方法，研究具抗氧化功能的益生芽孢杆菌（本实验室筛选保存）对体外培养大鼠空肠上皮细胞（IEC6）自噬/抗氧化及其信号通路（p62/ keap1/ Nrf2、MAPK、p53）中关键蛋白和主要转录因子（Nrf2、FoxO1、NF-κB、AP1）的影响，并通过阻断技术验证益生芽孢杆菌介导的自噬信号通路在抗氧化中的作用。随后研究益生芽孢杆菌对正常和氧化应激条件下大鼠自噬/抗氧化功能及其相关信号通路的影响，探明益生芽孢杆菌对大鼠自噬的影响及其所介导的信号通路在缓解氧化应激中的作用。最后通过猪肠上皮细胞体外培养和氧化应激条件下断奶仔猪试验，进一步研究益生芽孢杆菌对断奶仔猪自噬/抗氧化的影响及其自噬信号通路在抗氧化中的作用。研究结果将为解析益生芽孢杆菌对断奶仔猪自噬的影响及其缓解仔猪氧化应激的分子机制提供理论基础。

自噬是细胞内的一种保守的降解机制，能够将细胞中受损的器官、蛋白等进行降解并循环利用，从而抵抗外界应激。本项目旨在探究芽孢杆菌对动物肠道自噬的影响及其缓解氧化应激的作用机理。结果发现益生菌BaSC06能缓解由氧化应激诱导的大鼠肠道和肝脏损伤、提高抗氧化酶GSH-Px、SOD活性和HO-1蛋白表达及自噬水平，降低细胞凋亡和氧化应激。此外，SC06芽孢杆菌还可通过调节肠道菌群结构及其代谢通路(碳水化合物、氨基酸等)和细菌结构组装合成(鞭毛、生物膜、分泌系统等)，从而缓解氧化应激、降低肝脏损伤。大鼠肠上皮细胞实验发现，BaSC06能通过p38 MAPK信号通路提高自噬缓解氧化应激、降低细胞凋亡。仔猪实验发现，BaSC06可提高抗氧化酶表达、自噬水平和nrf2信号通路并缓解断奶仔猪氧化应激。最后通过猪肠上皮细胞实验验证了BaSC06能够通过降低IPEC-J2细胞ROS的产量、提高抗氧化酶活性及促进自噬显著减轻DQ诱导的IPEC-J2细胞凋亡和氧化应激损伤。RNA-seq测序结果表明，BaSC06影响自噬的信号通路有：MAPK信号通路、AMPK信号通路、PI3K-Akt信号通路、P53信号通路、FOXO信号通路、JAK-STAT信号通路、NF -κB信号通路、TNF信号通路、TGF-beta信号通路和mTOR信号通路等。Western-blotting和实时荧光定量PCR实验结果表明BaSC06可以通过抑制AKT-FOXO信号通路调节其下游自噬相关靶基因表达、促进Atg12-Atg5复合物的形成，从而提高自噬水平缓解IPEC-J2细胞氧化应激。以上结果阐明了BaSC06通过自噬缓解氧化应激降低细胞凋亡的分子机理，为益生菌应用于畜禽健康养殖提供了理论依据。