AQP3和NHE3基因甲基化在仔猪肠道水分转运中的作用及营养调控

肠道水分转运不良引起的腹泻严重影响仔猪生长，现已证实水转运通道表达异常是肠道水分转运不良的主要诱因，但水转运通道表达的调控机制仍不清楚。项目利用致病性大肠杆菌感染诱导的仔猪腹泻模型，结合体外肠上皮细胞培养试验，通过亚硫酸氢钠测序法检测AQP3和NHE3基因甲基化水平，通过体外人工补丁甲基化技术和RNA干扰技术检测启动子甲基化对AQP3和NHE3表达的影响，通过抑制和激活DNA甲基化酶（DNMT）的转录因子NF-κB及DNMT对接位点分析，探明AQP3和NHE3基因甲基化调控机制，并利用标记物H218O和22NaCl检测养分转运，阐明AQP3和NHE3基因甲基化调控肠道水分转运的作用机制。进一步以葛根素为调控剂，探明葛根素干预AQP3和NHE3基因甲基化调控肠道水分转运的作用效果及机制，以期在肠道水分转运的营养调控方面形成新的学术观点，为揭示仔猪腹泻的形成机理、建立营养调控技术奠定理论基础。

Diarrhea caused by abnormal water transport can severely retard the growth of piglets. It has been shown that abnormal expression of water transport channels, such as aquaporins (AQPs) and Na+/H+ exchanger 3 (NHE3), is a major cause of intestinal water transport dysfunction. However, the regulatory mechanism(s) of water transport channels expression in the intestine remain(s) largely unknown. Our preliminary study found that DNA methylation may be involved in the regulation of the expression of water transporters, which needs further confirmation. In the present study, both in vitro cellular model and in vivo animal model infected with enterotoxigenic Escherichia coli K88 (ETEC) will be employed to determine the role of promoter methylation of AQP3 and NHE3 on intestinal water transport. Within the experiment, the levels of methylated AQP3 and NHE3 promoters are determined by bisulfite sequencing (BS) method. In vitro methylation and si-RNA technologies are used to measure the promoter activity in the transcription of AQP3 and NHE3. The regulatory mechanism of AQP3 and NHE3 methylation is also investigated through inhibiton and activation of NF-κB in enterocytes, as well as in silico target fishing technology. The nutrient transport by small intestine and enterocytes is determined by the administration of H218O and 22NaCl, which levels in the portal vein blood and enterocytes will be analyzed by mass spectrometry technology and Ussing chamber method, thereby to reveal the underlying mechanisms where DNA methylation regulates water transport. Moreover, we will investigate the intervention of puerarin on DNA methylation regulating intestinal water transport in enterocytes and piglets infected with ETEC. The results of this study will provide novel academic viewpoints on the nutritional regulation of water transport by piglets’ small intestine, and will also provide important theoretical basis for understanding the formation mechanism of diarrhea and establishment of new nutritional regulation technology.