猪乳腺V-ATPase/mTORC1信号通路的基因表达及营养调控

The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway plays a key role in regulating cellular metabolism and growth. Vacuolar H+-ATPase (V-ATPase) is the first downstream target discovered thus far in activation of mTORC1 by amino acids. Oxidizing agents result in reversible inactivation of V-ATPase activity through disulfide bond formation between cysteine residues of V-ATPase, which suggests the potential role of V-ATPase/mTORC1 in dealing with oxidative stress. It has been demonstrated that sows are under severe catabolic status during late gestation and lactation causing increased oxidative stress and impaired milk production, however, little is known about the underlying mechanism. In this study, sows in different physiological stages are used to determine the mutual relations of mTORC1 pathway signaling to mammary growth, organic metabolism and nutrition status, through modern genomics, metabolomics and bioinformatics techniques. Swine mammary epithelial cells are further used to investigate the inhibition effect of oxidative stress on V-ATPase activity and mTORC1 activation, and to test the potential modulation effect of methionine sources on V-ATPase activity and mTORC1 activation. Through this study it may reveal from the whole animal, cellular and molecular basis the mechanism of expression and manipulation of mTORC1 pathway signaling in the mammary glands, which may provide a new ideal and theory basis for the manipulation of mammary growth, anabolism and anti-stress capacity.

mTORC1是哺乳动物细胞调节代谢和生长的枢纽。V-ATPase是迄今发现位于氨基酸下游并通过其活性调节mTORC1的第一个靶标。在氧化环境下，V-ATPase的半胱氨酸残基形成二硫键导致V-ATPase可逆失活，预示了V-ATPase/mTORC1应对氧化应激的潜在作用。母猪妊娠后期和哺乳期机体大量分解代谢引发氧化应激，损害乳产量，但其机制并不清楚。本项目以不同生理阶段的母猪为对象，采用现代基因组学、代谢组学和生物信息学等技术，研究mTORC1信号通路与乳腺发育、机体代谢和营养供给的相互关系；进一步以乳腺细胞为模型，研究氧化应激对V-ATPase/mTORC1的抑制效应，探索不同蛋氨酸源对V-ATPase活性及mTORC1通路的调节作用。本研究意义在于从动物整体、细胞和分子三个层面揭示乳腺mTORC1信号通路的表达调控机制，为乳腺生长发育、合成代谢和抗应激力的调控提供新的思路和理论依据。

母猪泌乳不足不仅是引起仔猪断奶前死亡的一个主要因素，也是最根本的因素。研究表明，乳产量在很大程度上取决于乳腺上皮细胞数量，妊娠后期和哺乳期是乳腺快速发育时期，同时也是母猪机体代谢引发氧化应激的时期，合理的营养对促进乳腺发育和健康至关重要。mTOR 信号通路可以对能量状态、氨基酸、生长因子和应激等多种多样的环境因素进行精确感应和整合，以控制蛋白质合成、脂类合成和自噬反应等许多生物过程，从而在调节细胞生长和增殖上发挥巨大作用。本项目分为三个试验分别研究乳腺mTORC1 信号通路的表达与母猪生理期代谢的关系，营养供给对母猪机体代谢及乳腺mTORC1 信号通路的影响，氧化应激抑制乳腺细胞mTORC1信号通路的机制研究，和蛋氨酸促进乳腺上皮细胞蛋白质合成的研究。妊娠期试验结果表明，妊娠90天比妊娠60天乳腺发育更加完善的同时也遭受着更加严重的氧化应激，同时，乳腺利用蛋氨酸进行抗氧化。泌乳期营养限制的母猪同样也产生了更加严重的氧化应激，且乳腺也遭受了氧化应激，并阻碍了溶酶体v-ATPase/mTORC1信号通路的表达，最终下调了酪蛋白的合成。进一步的细胞试验同样证明，氧化应激下调了mTORC1信号通路，并阻碍细胞增殖和引起细胞自噬。乳腺上皮细胞中添加抗氧化物质的前体物质蛋氨酸及其羟基类似物能激活mTORC1信号通路促进乳蛋白的合成。并综上所述，氧化应激会抑制乳腺v-ATPase/mTORC1信号通路，并阻碍乳腺功能的实现。但是蛋氨酸的添加可以激活mTORC1信号通路和促进乳蛋白合成。本研究结果对进一步优化营养方案，促进乳腺发育和健康，进而提高母猪泌乳力具有极其重要的理论意义。