猪下丘脑TLR4炎症通路在食欲调节中的作用及其分子机制

Increased the feed intake has been the crucial factor for pig production. Generally, the high-grade inflammation induces anorexia and weight loss. However, recent evidences demonstrated the low-grade inflammation in hypothalamus induced hyperphagia of animals. Toll like receptors, a key receptor family of innate immunine response, play a key role in the process of inflammation. Our pilot studies suggested that the TLR4 might be the potential receptor to mediate the orexigenic effect of local low-grade inflammation at hypothalamus. To identify the role of TLR4 involved in the regulation of pig appetite, we will firstly detect the distribution and the colocalization of TLR4 and other neuropeptide by IHC. Then, the effects of TLR4 overexpression and knock-down on the neuropeptide expression, receptor signalling pathway and transcriptional mechanism will be investigated on N38 cell lines and brain slices of pig. Moreover, the hypothalamic nucleus micro-injection and Cre-LoxP strategy will also be applied to find out the exact nucleus and neuron target of TLR4 to modulate animal appetite. Finally, the effect of natual ligand of TLR4, Reishi polysaccharides and Astragalus polysaccharides, on pig feed intake and growth performance were also investigated by the in vivo experiments of pig. The main purpose of this project is to reveal the potential mechanisms underlines the effect of TLR4 on pig appetite, and the results will also provide the fundamental data for the development of novel nutritional and efficient feed additives to promote pig appetite.

提高食欲一直是养猪生产中亟需解决的重要问题。过度的炎症反应通常导致动物厌食和消瘦，但最新研究表明：下丘脑局部低水平炎症反而能够促进动物采食。Toll样受体（TLR）作为一类天然免疫受体直接参与了炎症反应。本实验室前期研究发现：TLR4很可能是介导炎症与食欲调节的关键受体。为深入研究TLR4调控猪采食的机制，项目拟以猪下丘脑为靶点，首先明确TLR4在各核团的分布与共定位；进而分别以N38细胞系和猪下丘脑脑片为模型，研究TLR4过表达和干扰对食欲调节因子表达和分泌的影响、受体信号通路及转录调控机制；同时采用核团注射和Cre-LoxP转基因小鼠模型，进一步明确TLR4调控食欲的中枢靶点。最后通过仔猪饲养试验，在日粮中添加TLR4天然多糖类配体，研究其对猪采食行为和生产性能的影响。研究结果不仅能够阐明TLR4参与动物食欲调节的机制，也为研制新型的诱食剂提供试验依据，具有重要的学术价值和实践意义。

采食量不足是限制猪生产性能的重要障碍，影响采食量的核心因素是食欲，提高食欲一直是养猪生产中亟需解决的重要问题。过度的炎症反应通常导致动物厌食和消瘦，但最新研究表明下丘脑局部低水平炎症反而能够促进动物采食。本课题为探究猪下丘脑炎症信号通路在采食调控中的详细作用机制，我们首先通过不同能量状态下下丘脑TLRs家族表达变化明确TLR4可能在动物食欲调节方面发挥重要作用。但是进一步利用Cre/LoxP技术对AgRP或POMC神经元特异性过表达或敲除TLR4均对动物采食和体重无影响。随后通过外源添加不同TLR4配体激活或抑制TLR4通路发现，LPS在显著提高动物采食的同时，还可以导致动物焦虑行为的发生。CRH神经元作为动物应激调控中枢，存在高表达的TLR4，暗示CRH神经元可能在调控动物采食和体重方面发挥重要作用。为此，我们首先检测肥胖状态下CRH神经元敏感性发现肥胖可显著降低CRH神经元的敏感性。随后对CRH神经元过表达NaChBac或Kir2.1持续激活或抑制其活性，发现均可引起动物采食量增加和肥胖的出现。本课题研究结果不仅阐述了下丘脑TLR4炎症通路在动物食欲调节中的作用，更揭示了CRH神经元作为应激调控中枢，其敏感性的高低直接影响动物的食欲。研究结果不仅能够阐明TLR4参与动物食欲调节的机制，也为研制新型的诱食剂提供试验依据，具有重要的学术价值和实践意义。