肠道水转运通道在仔猪营养性腹泻形成中的作用及营养调控

The major problem in pig production is how to effectively control diarrhea of piglets. Intestinal water transport channels, including ion/nutrient co-transporters, aquaporins, and tight junction proteins, are reported to be closely associated with the water absorption and water balance. However, the role of water transport channels in weaning-associated diarrhea and their nutritional regulation remain largely unknown. This study is conducted to investigate the effect of intestinal water transport channels on piglet diarrhea using an experimental model of glycinin-induced diarrhea and small intestinal epithelial cell incubation. The real-time PCR, western blot, immunohistochemistry, and Ussing chamber methods are used to determine the distribution and function of water channels. Meanwhile, the confocal microscopic technology, RNA interference, and Stopped-flow spectrometry are utilized to determine the distribution and function of AQP8 in enterocytes, while the PKA activator (Bt2cAMP) and inhibitor (H89) are employed to elucidate the role of cAMP-PKA signaling pathway in AQP8 expression. Additionally, the effects of Gln and NAC on expression of water channels are explored. The results of this study will aid in understanding the role of intestinal water transporters in the formation and regulation of piglet nutritional diarrhea with related molecular mechanisms, and will provided important theoretical basis and new technical clues for nutritional regulation of piglet diarrhea.

控制仔猪腹泻是养猪生产中的一个难题。肠道水转运通道与水分的吸收和平衡密切相关，但其在仔猪腹泻形成中的作用及调控机制尚不清楚。本项目以大豆球蛋白诱发仔猪营养性腹泻为实验模型，结合猪小肠上皮细胞体外培养实验，通过采用real-time PCR、免疫印迹、免疫组化和尤斯灌流室技术检测水转运通道（离子/营养素共转运载体、水通道蛋白和紧密连接蛋白）在肠道组织中的定位和作用；采用激光共聚焦、RNA干扰技术和停流色谱法检测水通道蛋白8（AQP8）在猪小肠上皮细胞中定位及作用，并结合PKA激活剂和抑制剂实验研究cAMP-PKA信号通路在调控AQP8表达中的作用；同时以谷氨酰胺（Gln）和N-乙酰半胱氨酸（NAC）为营养调节剂，探讨它们调控肠道水转运通道的作用效果及靶点，从而全面认识肠道水转运通道在仔猪营养性腹泻形成与调控中的作用及相关分子机理，为仔猪腹泻的有效控制提供新的理论依据和技术思路。

控制仔猪腹泻是养猪生产中的一个难题。肠道水转运通道与水分的吸收和平衡密切相关，但其在仔猪腹泻形成中的作用及调控机制尚不清楚。本项目以大豆抗原蛋白诱发仔猪营养性腹泻为实验模型，结合猪小肠上皮细胞体外培养实验，通过用real-time PCR和免疫印迹技术检测水转运通道（离子/营养素共转运载体、水通道蛋白和紧密连接蛋白）在肠道组织中的表达；采用免疫组化技术检测水通道蛋白3（AQP3）和AQP4在猪肠道细胞中的定位与表达，并结合激光共聚焦、自噬抑制剂和诱导剂试验研究自噬在肠道AQP3和AQP4表达中的作用。同时以谷氨酰胺（Gln）和N-乙酰半胱氨酸（NAC）为营养调节剂，探讨了它们调控肠道水转运通道的作用效果及靶点。结果表明，β-伴大豆球蛋白（β-CG）仔猪生长性能降低，腹泻率增加。β-CG仔猪的水转运通道（包括CFTR、SGLT-1、AQP3、AQP4、AQP10等）表达量显著降低，但小肠AQP8的基因表达量却上调；水通道蛋白AQP3在小肠绒毛顶端和结肠均有分布，而AQP4在小肠绒毛顶端、固有层、及结肠均有分布，可见AQP3和AQP4在肠道水分吸收中具有重要作用。而cAMP-PKA信号通路并不参与调控肠道AQP3和AQP4的表达，但是自噬可能参与了水转运通道的降解；NAC和Gln均可改善β-CG仔猪肠道水转运通道的表达，其作用机制可能与改善肠道屏障功能和抑制肠道自噬活性相关。本项目的研究结果为仔猪肠道水转运通道的营养调控提供了崭新的学术观点，亦为NAC和Gln作为饲料添加剂在防治仔猪腹泻上的应用提供了重要的理论依据。