AMP依赖的蛋白激酶调控慢性热应激肉鸡小肠营养物质吸收功能的研究

The expression of AMPK in the small intestinal cells was significantly changed during chronic heat stress. It is important to further reveal that AMPK regulates the absorption function of small intestine nutrients of the environmental adaptability mechanism of heat stress broilers. In this study, the artificial climate chamber was used to simulate the chronic heat stress environment of broilers, and then the content, activity and distribution of AMPK and three important nutrient carriers in the small intestine were measured. The dose-effect relation of AMPK and nutrient uptake was revealed in chronic heat stress. The in vitro maintenance and perfusion system of small intestine of broiler chickens was established by using the Ussing Chamber. Through the specific activation and blockage of AMPK signal, the three nutrient uptake capacities of the intestinal tract were analyzed dynamically, which revealed that AMPK regulate nutrient uptake dynamically in the process. To establish a small intestinal cell culture model, through specifically enhancing and blocking the AMPK signal, small intestinal cell activity, proliferation and differentiation characteristics, cell stability and nutrient absorption related protein changes were detected to reveal the AMPK regulation of small intestinal nutrient absorption cytology basis. The effect of AMPK signal on the intestinal absorption of heat-stressed broilers was analyzed from the whole, tissue, cell and molecular level, and the theoretical basis was established to control the heat stress from the angle of regulation of AMPK signal.

肉鸡慢性热应激时小肠细胞应激适应因子AMPK发生显著改变。进一步揭示AMPK对小肠营养物质吸收功能的调控是阐明热应激肉鸡环境适应性机制的重要环节之一。本研究利用人工气候室模拟肉鸡慢性热应激环境，测定各小肠段中AMPK、三大营养物质吸收载体含量、活性及分布，揭示肉鸡慢性热应激时AMPK与营养物质吸收功能的量效关系。利用尤斯室建立肉鸡小肠的体外维持与灌注系统，通过特异性激活和阻断AMPK信号，动态分析慢性热应激时肠道三大营养物质吸收能力，揭示AMPK在营养物质吸收过程中的动态调控作用。建立小肠细胞培养模型，通过特异激活和阻断AMPK信号，检测小肠细胞活性、增殖及分化特性、细胞稳态以及营养物质吸收功能相关蛋白的变化，分析AMPK对小肠细胞稳态及营养物质吸收蛋白的调控。从整体、组织、细胞和分子等层次分析AMPK信号对热应激肉鸡肠道吸收功能的影响，为从调节AMPK信号的角度防控热应激奠定理论基础。

肉鸡体表几乎无汗腺、基础代谢率高的遗传特性和近年来夏季高热天气频现等因素的影响，导致肉鸡热应激时常发生。而强度低、发生时间长的慢性热应激在生产过程中极易忽视。本项目以热应激肠道损伤为主要研究对象，研究了小肠在慢性热应激时多种变化以及AMPK在这一过程中的变化，为通过调节肠道功能的方式防控热应激提供理论支持和思路。研究发现，慢性热应激环境下肉鸡十二指肠、空肠、回肠等肠段肠道绒毛长度、隐窝深度、杯状细胞数量等与肠道相关的形态结构发生了显著变化。而血清中二胺氧化酶、D-乳酸、LPS含量均有升高，说明慢性热应激对肉鸡的肠道黏膜细胞有显著的损伤作用，肠壁黏膜的通透性升高。肠道营养物质代谢与消化结果分析表明，与常温处理组相比，热应激使肉鸡对干物质、有机物以及粗蛋白的表观消化率均显著降低。此外，通过测定肠道、肝脏、胸肌等组织中糖转运载体的表达量的研究表明，慢性热应激时影响了肠道对糖类物质的吸收，进而影响机体内能量的供给方式。通过对胸肌、腿肌、肠道等组织AMP/AMPK含量的测定发现，AMPK在慢性热应激发生过程中产生了重要的调节作用，且不同部位AMP/AMPK含量变化规律并不相同。慢性热应激肉鸡肠道细胞有多个特异性差异产物。代谢路径分析发现，肠道细胞糖代谢路径、谷胱甘肽代谢路径、类固醇合成等10个细胞内代谢路径发生显著改变。慢性热应激时糖、脂、蛋白质类各种营养物质变化的规律表明慢性热应激对肉鸡的糖、脂肪和蛋白质代谢均有显著的影响。体外细胞学的研究表明，热应激时肠道细胞增殖显著降低20%以上。流氏细胞仪检测发现，热应激显著增加肠道细胞的凋亡和坏死率。Western-blotting结果表明，热应激时可导致肠道细胞Bcl-2表达增加，Bax变化不显著，且Caspase-3蛋白表达增加，促进了肠道细胞凋亡。此外，慢性热应激时延胡索酸、硫辛酸、甜菜碱、维生素C、益生菌等调控措施进行缓解热应激的危害，取得了明显的效果。