PiT-1介导ERK/NHERF1调控肉鸡肠道磷吸收的分子机制研究

Improving intestinal phosphorus absorption efficiency is of great significance for reducing phosphorus pollution from livestock and poultry excreta. The intestinal sodium-phosphorus transport system (NaPi) is an important target for the development of phosphorus-reducing strategies. Intestinal NaPi-IIb is mainly responsible for phosphorus absorption, while NaPi-III functions as "phosphorus sensor". We found that the intestinal phosphorus absorption efficiency was significantly increased in broilers when the NaPi-III subtype PiT-1 was inhibited by using an own-developed PiT-1 antibody. Further experiments showed that PiT-1 could sense extracellular phosphorus concentration signal and down-regulate the protein expression of NaPi-IIb, but the mechanism remains unknown. In this project, we will first intervene with PiT-1 in the primary intestinal epithelial cells from broilers, the PiT-1-mediated intermediate signaling molecules that regulate NaPi-IIb would be identified using techniques such as co-immunoprecipitation. Then, the selected intermediate signal molecules will be interfered by specific inhibitors or siRNAs, and the signal network will be clarified using techniques such as phosphorylation proteomics. Finally, PiT-1 will be inhibited in vivo to explore the intrinsic mechanism by which PiT-1 regulates NaPi-IIb and affects intestinal phosphorus absorption in broilers. The results of this project will clarify the role of PiT-1 in limiting broiler intestinal phosphorus absorption and provide a new target for the development of phosphorus-reducing strategies.

改善肠道磷吸收效率对于减轻畜禽排泄物磷污染有重要意义。肠道钠磷转运系统（NaPi）是节磷减排研究的重要靶位；此系统中，NaPi-IIb主要负责磷吸收，而NaPi-III则作为“磷感受器”发挥作用。申请人用自主研制的抗体抑制NaPi-III的亚型PiT-1后，发现肉鸡肠道磷吸收效率显著提高。进一步试验显示，PiT-1可感知细胞外磷浓度信号并下调NaPi-IIb蛋白表达，但机制不明。本项目拟首先在肉鸡原代肠上皮细胞中靶向干预PiT-1，通过免疫共沉淀等技术探明PiT-1调控NaPi-IIb可能涉及的中间信号分子；然后使用抑制剂或RNAi对筛选出的中间信号分子进行干预，利用磷酸化蛋白质组学等技术明晰信号网络；最后，对PiT-1进行在体干预，探究PiT-1通过调控NaPi-IIb进而影响肉鸡肠道磷吸收的内在机制。本项目研究结果将阐明PiT-1对肠道磷吸收的限制作用，可为节磷减排技术研发提供新靶点。

本项目初步明确了鸡肠道中PiT-1对NaPi-IIb的调控作用，当肠腔中磷浓度升高时，肠上皮细胞膜PiT-1可感知该信号，并通过MAPK等下游信号网络，调控细胞功能（调控NaPi-IIb的表达），以适应磷环境变化，即：PiT-1介导了机体对高磷摄入的生理适应性反应。. 按照项目规划，项目组重点开展了以下6个方面的研究工作：（1）明确肠上皮细胞对胞外磷浓度的响应（增加胞外磷浓度，PiT-1的蛋白表达水平升高，NaPi-IIb的蛋白表达水平降低）；（2）探索肠上皮细胞中PiT-1调控NaPi-IIb的可能机制（增加胞外磷浓度，PiT-1的蛋白表达水平升高，伴随着MAPK信号通路的活化）；（3）制备anti-PiT-1卵黄抗体（以期用于缓解高磷刺激下PiT-1对NaPi-IIb蛋白表达的抑制作用，即用于优化肠道的磷吸收效率）；（4）探讨anti-PiT-1卵黄抗体在商品肉鸡饲料中的应用效果（一种有利于肉鸡生产“节磷减排”的技术方案）；（5）探讨anti-PiT-1卵黄抗体在商品肉鸡饲料中的应用效果（十二指肠原位结扎灌注实验）；（6）项目延申（在明确PiT-1对肉鸡肠道磷吸收调控作用的基础上，进一步探究产蛋鸡肠道PiT-1的表达及调控规律，促使项目成果的未来应用延申）。. 上述试验结果提示，家禽肠腔中磷浓度升高时，PiT-1可适应性抑制NaPi-IIb的表达，以避免机体磷元素的过量摄入。但是，在实际生产中，家禽配合饲粮中磷元素的浓度受到科学控制，磷元素过量摄入的情况鲜见。因此，PiT-1对NaPi-IIb的抑制作用，并不利于现代规模化养殖家禽对磷元素的高效利用。. 项目组认为，以PiT-1为靶点，开发家禽节磷减排调控技术，具备潜在的可行性。