AMPK/SIRT1-PGC1α信号通路在母体纤维营养调控猪胎儿肠道发育中的作用研究

The intestinal development in late gestation is crucial for piglet growth and health. Unfortunately, the previous studies focus on the role of nutrition on modulating intestinal development of weaning piglets, it is quite less about the modulating effect of nutrition on fetal intestinal development. Our previous study has shown that the higher intake of fiber enhanced the fetal growth, intestinal index and nutrient utilization in pigs or rodent animals. Short chain fatty acids, as metabolites by fermenting dietary fiber, have various biological effects on regulating cell metabolism, cell proliferation and inflammation et al. However, it is unknown if maternal high fiber intake could enhance fetal intestinal development through SCFAs, and underlying mechanism needs to be explored. The previous study has shown that PPAR-γ coactivator 1α (PGC1α) is the upstream gene for governing the fetal intestinal maturation. As the metabolic sensors, AMP-Activated Protein Kinase（AMPK） and NAD-dependent protein deacetylases（SIRT1） modulate the activity of PGC1α, and it is known that short chain fatty acids (SCFAs) could regulate the biological process such as cell proliferation and energy metabolism through AMPK or AMPK-SIRT1 signal pathway. Therefore, this project firstly aims to investigate the effects of maternal high fiber intake on the fetal intestinal development and the production of SCFAs. Moreover, the modulating role of SCFAs on the intestinal development is validated by jugular vein infusion of SCFAs. Finally, the role of AMPK/SIRT1-PGC1α signal pathway in the regulation of SCFAs on the epithelial cells function will be clarified using in vitro cell model. Results of the project would reveal the molecular mechanism linking maternal high fiber intake with the intestinal development of piglets, and providing a new idea for the nutritional modulation on the intestinal health.

胎儿肠道发育显著影响生后生长性能和肠道健康。遗憾的是，过去研究集中在断奶仔猪肠道发育及营养调控，有关猪胎儿肠道发育营养调控机制研究，十分稀少。前期研究表明，母体高纤维摄入促进了猪或鼠胎儿生长发育、肠道指数和养分利用；纤维微生物代谢产物-短链脂肪酸（SCFAs）具有广泛的营养生理效应，调控了细胞代谢、增殖分化和炎症等多种生物学过程。母体高纤维摄入是否通过SCFAs促进胎儿肠道发育，尚不清楚；SCFAs调控猪胎儿肠道发育的分子机制，有待研究。前期研究发现，PPARγ辅助激活因子1α(PGC1α)是调控猪胎儿肠道发育成熟关键基因；细胞“代谢感受”蛋白激酶AMPK和去乙酰化酶SIRT1信号调控PGC1α活性；纤维代谢产物-短链脂肪酸（SCFAs）可能通过激活AMPK或AMPK-SIRT1信号调控细胞增殖分化和代谢等多种生物过程。本项目首先研究母体高纤维摄入调控猪胎儿肠道发育的效应及SCFAs动态代谢规律，进一步通过静脉恒流灌注技术和细胞培养，验证SCFAs是纤维调控胎儿肠道发育的重要代谢物，并证实AMPK/SIRT1-PGC1α信号通路是SCFAs调控肠细胞功能的关键分子机制。母体营养代谢程序化地影响后代生长发育和疾病已成为现代生命科学研究热点。研究母体纤维营养调控猪胎儿肠道发育的营养代谢关系及分子机制，对于深入认识仔猪肠道发育规律具有重要意义，并为营养调控仔猪肠道发育提供新思路。

妊娠后期是猪胎儿肠道发育快速成熟期及关键准备期，以启动生后肠道吸收和免疫屏障等功能；过去研究集中在断奶仔猪肠道发育及营养调控，有关猪胎儿肠道发育营养调控机制研究，十分稀少。前期研究表明，母体高纤维摄入促进了猪或鼠胎儿生长发育，母体高纤维摄入是否通过SCFAs促进胎儿肠道发育，尚不清楚；SCFAs调控猪胎儿肠道发育的分子机制，有待研究。项目研究首先明确了母体高纤维摄入对胎儿肠道发育的调控效应，以及对母猪-血液循环和羊水中SCFAs浓度的影响，建立母体纤维营养调控胎儿肠道发育的营养代谢关系；进一步通过静脉恒流灌注及体外肠细胞培养，研究纤维营养代谢产物SCFAs调控肠上皮细胞功能的关键信号通路。研究发现：（1）妊娠后期高纤维摄入增加新生仔猪体长和回肠隐窝深度、提高结肠乙酸及异丁酸水平，并改变仔猪结肠食糜微生物群和回肠蛋白质组；妊娠后期高纤维摄入还增加了与氧化状态、能量代谢、免疫和炎症反应相关的蛋白表达，降低了与细胞凋亡、细胞结构和运动相关的蛋白表达；相关性分析表明，肠道微生物在母体高纤维摄入对后代肠道发育的影响中具有潜在作用；（2）研究阐明了长期高纤维摄入对后代大鼠DSS诱导结肠炎的影响及可能机制，发现DSS攻毒处理降低大鼠体重和结肠长度，增加肝脏指数和脾脏指数，造成结肠结构和屏障功能损伤，且增加肠道炎症；而惊奇地发现，母体长期高纤维摄入加剧后代大鼠DSS诱导下肠道损伤和炎性，这可能与肠道菌群结构有关；关联分析表明，肠道微生物在肠道炎症调节中具有重要作用；（3）进一步研究了颈静脉灌注短链脂肪酸SCFAs对猪胎儿肠道发育及关键信号通路调控，发现母猪血液代谢物中丁酸含量显著升高，但羊水中SCFAs含量未显著变化，且新生仔猪回肠隐窝深度有增加趋势，通过考察肠道发育关键调控因子，重点检测SCFAs调控仔猪肠道发育差异表达基因及蛋白，尤其是AMPK/SIRT1-PGC1α信号通路分子；结果表明，SCFAs对新生仔猪肠道组织pAMPK2α/总AMPK2α蛋白表达，PGC1α磷酸化水平及表达和PGC1α下游调控因子表达产生显著影响；尽管如此，体外采用丁酸与IPEC-J2细胞共培养超过一定剂量时却抑制了细胞活力。通过上述研究阐明母体纤维营养调控猪胎儿肠道发育的营养代谢关系及分子机制，对于深入认识仔猪肠道发育规律具有重要意义，并为营养调控仔猪肠道发育提供新思路。