TLRs信号通路在热应激肉鸡组织损伤中的作用及营养调控

Heat stress-induced oxidative stress is an important mediator of tissue injury caused by heat stress in broiler chickens. However, it is unclear how oxidative stress mediates tissue damage under heat stress. Recently, Toll-like receptors (TLRs) have been shown to mediate the inflammatory response induced by oxidative stress, thus leading to tissue and organ damage. Our previous study demonstrated that the TLRs expression pattern was changed under the condition of heat stress in broiler chickens. So the question arises that whether the TLRs signaling pathways play an important role in mediating heat stress induced tissue injuries? Based on our previous research work, this project, therefore, aims to investigate the redox state of tissues, the expressions of heat shock proteins, high mobility group box-1 protein (HMGB1), TLRs and its downstream signaling molecular, negative regulators of TLRs signaling using quantitative RT-PCR, ELISA, western blotting, immunohistochemistry and histopathology methodologies by in vitro and in vivo experiments, and thus to demonstrate the underlying mechanism by which the heat stress induces tissue injury from the heat stress-oxidative stress-DAMPs/TLRs signaling point of view, and to clarify the molecular mechanism underlying the anti-heat stress effects of specific nutrients, which would contribute to enriching the heat stress theory and finding new potential biomarker for evaluating heat stress-induced injury in broilers.

热应激诱导的氧化应激是热应激损伤的重要介导者，然而，热应激诱导的氧化应激是通过何种方式介导引起肉鸡组织损伤？Toll样受体（TLRs）在介导氧化应激损伤中发挥关键作用。我们前期研究发现，热应激引起肉鸡组织TLRs表达模式发生改变，那么，TLRs信号通路是否在介导热应激肉鸡组织损伤中发挥重要作用？为此，本研究拟运用荧光定量RT-PCR、ELISA、免疫印迹、免疫组化、组织病理学等技术手段，通过体内试验和体外实验研究热应激条件下肉鸡组织氧化应激状态、热休克蛋白、HMGB1、TLRs及其下游信号分子、信号通路负调控因子表达变化规律，从热应激-氧化应激-DAMPS/TLRs信号通路及其负调控的角度探讨肉鸡组织热应激损伤机理，进一步明确抗热应激损伤的分子营养调控机制。项目研究有助于更深入揭示肉鸡热应激损伤的作用机制，丰富和完善热应激理论，且可能发现判定肉鸡热应激损伤的新生物标志物。

热应激诱导的氧化应激是热应激损伤的重要介导者，然而，热应激诱导的氧化应激是通过何种方式介导引起肉鸡组织损伤？Toll样受体（TLRs）在介导氧化应激损伤中发挥关键作用。我们前期研究发现，热应激引起肉鸡组织TLRs表达模式发生改变，那么，TLRs信号通路是否在介导热应激肉鸡组织损伤中发挥重要作用？针对这些科学问题，本项目从组织器官和分子水平探讨了热应激/氧化应激条件下肉鸡组织氧化应激状态、组织结构的完整性以及损伤相关分子模式（DAMPs）、TLRs及其下游信号分子和负调控因子的表达变化及丁酸钠、益生菌、葛根素和色氨酸的调控作用，从热应激-氧化应激-DAMPs/TLRs信号通路及其负调控的新角度进一步探讨了热应激对肉鸡组织器官的损伤反应及营养调控的分子机理。研究发现，慢性热应激/氧化应激降低肉鸡生产性能，破坏肉鸡肠道黏膜绒毛结构和黏膜完整性，降低血液、组织器官（小肠、肝脏、脾脏、胸腺和法氏囊）的抗氧化能力，破坏机体组织器官炎性细胞因子/抗炎细胞因子平衡引发炎症反应和损伤。饲粮添加丁酸钠、益生菌、葛根素和色氨酸可逆转以上变化而缓解热应激/氧化应激诱导的机体组织器官损伤。进一步发现，热应激/氧化应激提高肉鸡小肠和免疫器官热休克蛋白70和90、高迁移率族蛋白B1 (HMGB1）、TLR4、TLR5、TLR15以及TLRs下游基因的表达，同时降低组织器官TLRs信号通路负调控因子A20、Tollip和SIGIRR的表达。总之，慢性热应激/氧化应激激活肉鸡组织器官DAMPs-TLRs-MAPKs/NF-κB介导的信号通路，促进促炎细胞因子表达，降低抗炎细胞因子和TLRs信号通路负调控因子表达而引起组织器官炎症性损伤，饲粮添加丁酸纳和葛根素可通过抑制DAMPs-TLRs（尤其是HMGB1-TLR4信号通路）-MAPKs/NF-κB信号通路缓解热应激脂引起的组织器官损伤。本项目研究结果有助于深入阐明热应激损伤肉鸡组织器官功能的分子机理，丰富肉鸡热应激损伤理论，为有效预防和缓解热应激损伤提供新的措施，并为预防和缓解肉鸡热应激损伤的营养调控策略提供丰富的理论依据。