蛋氨酸调控羔羊肝脏组织抗氧化应激的分子机制研究

Methionine is one of essential amino acids in animal nutrition that would have limiting effect on animal growth and development, and proper concentration of methionine in feedings plays a vital role in promoting potential growth and maintaining wellness of animals. Methionine is closely related to antioxidative stress effect while present reports are unable to explain the mechanism of methionine regulating antioxidant. The previous studies by our group suggested that proper concentration of methionine significantly changed the expression abundance of genes related to mitochondrial respiratory chain and antioxidation in liver. The model of lamb primary liver cells treated by different levels of methionine is planned to establish in the present study, and the expression abundance of mRNA and protein related to mitochondrial respiratory chain and antioxidation would be determined by using QPCR and Western Blot method. Gene silencing and over-expression and co-immunoprecipitation assay technology would be used to specify the key genes on ERK-Nrf2 pathway and its action mechanism. And the molecular mechanism of methionine regulating antioxidant stress in lamb liver tissue would be elaborated in two aspects, mitochondrial respiratory chain function and ERK-Nrf2 signaling pathway regulation, which would reveal mechanism of the methionine regulating antioxidant stress in lamb liver tissue in theory and provide theoretical basis for using methionine to improve animals’ wellness in practice.

蛋氨酸是动物生长的限制性氨基酸，适宜的蛋氨酸水平对于发挥动物的生长潜能、维护机体健康具有重要作用。蛋氨酸水平与机体氧化应激关系密切，国内外现有理论不能完全解释蛋氨酸水平调控机体氧化应激的作用机制。本课题组此前研究表明，日粮中蛋氨酸水平能够显著改变羔羊肝脏组织线粒体呼吸链和抗氧化酶基因的表达水平。本研究拟通过建立不同蛋氨酸水平培养的羔羊肝脏原代细胞模型，采用QPCR及Western Blot方法检测线粒体呼吸链以及抗氧化基因在mRNA和蛋白水平的表达情况，结合基因沉默、过表达和免疫共沉淀技术，明确ERK-Nrf2通路上关键基因及作用途径，从线粒体呼吸链功能以及ERK-Nrf2信号通路的调控作用两个角度揭示蛋氨酸调控羔羊肝脏组织抗氧化应激的分子机制，明确蛋氨酸调控羔羊肝脏组织抗氧化应激的作用途径，在生产上能够为蛋氨酸的合理应用和改善动物的健康水平提供理论依据。

蛋氨酸是畜禽必需的氨基酸，在机体中具有广泛而重要的生物学功能。近年来研究表明，限制饮食中的蛋氨酸（MetR）可产生许多生理效应，如延长寿命、预防代谢性疾病和参与调节炎症性疾病。作为评价机体健康水平的重要指标，氧化应激得到越来越多的关注。大量研究证实了MetR 对机体氧化应激的正面效应，MetR 可通过减少线粒体活性氧产生、调控关键信号通路以及改变miRNAs 表达等方式参与氧化应激的调控，从而对畜禽健康产生广泛的影响。本课题组此前研究表明，日粮中蛋氨酸水平能够显著改变羔羊血清抗氧化酶的活力。本项目通过建立不同蛋氨酸水平培养的羔羊肝脏原代细胞模型，采用QPCR及Western Blot方法检测线粒体呼吸链以及抗氧化基因在mRNA和蛋白水平的表达情况，通过RNA-seq技术明确蛋氨酸调控羔羊肝脏组织抗氧化应激的作用途径。本研究表明，蛋氨酸限制影响了羔羊肝脏组织的氧化还原状态。GSTA基因表达的上调似乎与GSH前体的缺乏有关，而GSH前体又激活了Nrf2信号通路。在蛋氨酸限制期间，细胞通过上调抗氧化酶（如GSH-Px和SOD）来适应缺乏的蛋氨酸条件，以维持氧化还原状态。细胞稳态没有恢复到正常水平，这可能是由于蛋氨酸补充期间肝脏蛋氨酸代谢滞后引起的。本研究为低蛋白日粮在反刍动物上的合理利用提供理论依据。