日粮亮氨酸水平对早期断奶仔猪骨骼肌蛋白质组影响的研究

发布时间：2018-06-14 08:17

本文选题：亮氨酸 + 骨骼肌　；参考：《华中农业大学》2017年硕士论文

【摘要】：仔猪早期断奶措施会导致仔猪出现早期断奶应激反应,诱导仔猪骨骼肌、肝脏和脾脏发生自噬。自噬是一种在进化上高度保守的分解代谢过程,参与到细胞的多个生理和病理过程,如生长发育、分化、代谢、免疫、炎症和凋亡。亮氨酸不仅是蛋白质合成的底物,还可以通过mTORC1、Sestrin2和谷氨酸脱氢酶等信号通路发挥调节细胞自噬的功能。因此,亮氨酸对细胞自噬的调控机制有待进一步研究,以期为断奶仔猪的精准饲养提供新的研究资料。本研究通过对早期断奶仔猪分别饲喂1.66%和2.10%亮氨酸水平的日粮,在21日龄、22日龄和26日龄各选取3头仔猪进行屠宰,取其背最长肌样品,运用蛋白质组学iTRAQ技术鉴定仔猪骨骼肌中的差异表达蛋白,并筛选受亮氨酸调节的关键自噬相关蛋白。在仔猪骨骼肌中共鉴定到蛋白质1919个,差异表达蛋白402个,且特有肽段数、肽段序列长度和肽段序列覆盖率都符合质谱测序标准。差异表达蛋白的GO分析结果显示,差异表达蛋白主要分布在细胞内组分和细胞器部分,参与营养物质的代谢过程,主要发挥结合与催化功能。差异表达蛋白代谢通路分析显示,蛋白质合成相关的信号通路显著增强,包括eIF2、eIF3E、eIF5、eEF1信号通路和40S核糖体相关蛋白等。通过Western blot验证发现,无论是mTOR的活性,还是其下游底物S6K1的活性都显著增强。同时,与能量合成相关的氧化磷酸化通路中的F0F1-ATP合成酶、NADH脱氢酶、铁硫蛋白,三羧酸循环通路中的顺乌头酸酶、异柠檬酸脱氢酶、琥珀酸脱氢酶、酮戊二酸脱氢酶、琥珀酰辅酶A,糖酵解通路中的丙酮酸激酶,磷酸戊糖途径中的磷酸葡萄糖异构酶的含量都显著升高。通过高效液相色谱法也发现在试验组中,AMP含量极显著降低,ADP含量显著降低,ATP含量显著升高,AMP/ATP比值极显著降低。最后,我们筛选到CAPNS1、RACK1和PRKAR1A三个自噬相关蛋白,且在断奶应激诱导自噬反应的过程中CAPNS1、RACK1和PRKAR1A的表达量都显著升高。另外,在26日龄试验组中,p-AMPK的含量显著下降,p-mTOR的含量显著升高。因此,亮氨酸可以激活仔猪骨骼肌中的多条能量合成途径,很可能通过抑制AMPK的活性,增强mTOR的活性,并抑制RACK1的表达,最终起到抑制自噬的作用。综上所述,得出以下结论:(1)2.10%的亮氨酸水平日粮促进断奶仔猪骨骼肌中蛋白质合成相关的信号通路,包括mTOR信号通路和eIF2信号通路等;(2)2.10%的亮氨酸水平日粮促进断奶仔猪骨骼肌中的能量合成,极显著降低AMP含量,显著提高ATP含量;(3)断奶应激可以诱导自噬发生,显著提高CAPNS1、RACK1和PRKAR1A的表达量;(4)亮氨酸很可能通过AMPK-mTOR信号通路抑制RACK1的表达,发挥抑制细胞自噬的功能。
[Abstract]:Early weaning can lead to early weaning stress and induce autophagy in skeletal muscle liver and spleen of piglets. Autophagy is an evolutionarily conserved catabolism process, which is involved in many physiological and pathological processes, such as growth, differentiation, metabolism, immunity, inflammation and apoptosis. Leucine is not only a substrate for protein synthesis, but also regulates autophagy through signal pathways such as mTORC1Sestrin2 and glutamate dehydrogenase. Therefore, the regulation mechanism of leucine on autophagy needs to be further studied in order to provide new data for precision feeding of weaned piglets. In this study, three piglets were slaughtered at the age of 21 days, 22 days old and 26 days old respectively, and the longissimus dorsi muscle samples were collected by feeding 1. 66% and 2. 10% leucine level of early weaning piglets respectively. Differential expression proteins in skeletal muscle of piglets were identified by proteomics iTRAQ technique and the key autophagy related proteins regulated by leucine were screened. A total of 1919 proteins and 402 differentially expressed proteins were identified in skeletal muscle of piglets. Go analysis of the differential expression protein showed that the differential expression protein mainly distributed in the intracellular components and organelles, participated in the metabolism of nutrients and played a major role in the binding and catalytic functions. The metabolic pathway analysis of differentially expressed proteins showed that the signal pathways associated with protein synthesis were significantly enhanced, including eIF2eIF3EIF3EIF5EF1 signaling pathway and 40s ribosomal associated protein. Western blot analysis showed that both the activity of mTOR and the activity of substrates S6K1 were significantly increased. At the same time, the F0F1-ATP synthase nadh dehydrogenase, ferrithionein, cisaconitase, isocitrate dehydrogenase, succinate dehydrogenase, ketoglutarate dehydrogenase, isocitrate dehydrogenase, ketoglutarate dehydrogenase were found in the oxidative phosphorylation pathway associated with energy synthesis. The contents of succinyl coenzyme A, pyruvate kinase in glycolysis pathway and phosphoglucose isomerase in pentose phosphate pathway were significantly increased. It was also found by HPLC that the content of AMP in the experimental group was very significantly decreased, the content of ADP was significantly decreased, the content of ATP was significantly increased and the ratio of AMP / ATP was significantly decreased. Finally, three autophagy related proteins, CAPNS1, RACK1 and PRKAR1A, were screened, and the expression levels of CAPNS1, RACK1 and PRKAR1A were significantly increased during the induction of autophagy response under weaning stress. In addition, the content of p-AMPK in 26 days old group decreased significantly, and the content of p-mTOR increased significantly. Therefore, leucine can activate multiple energy synthesis pathways in skeletal muscle of piglets, which may inhibit autophagy by inhibiting the activity of AMPK, enhancing the activity of mTOR and inhibiting the expression of RACK1. To sum up, the following conclusions can be drawn: 1% of leucine level diet promotes the signal pathway related to protein synthesis in skeletal muscle of weaned piglets. 2.10% leucine level diet including mTOR signaling pathway and eIF2 signaling pathway promoted energy synthesis in skeletal muscle of weaned piglets, significantly reduced AMP content, significantly increased ATP content and induced autophagy. Leucine could inhibit the expression of RACK1 and PRKAR1A through AMPK-mTOR signaling pathway and inhibit the function of autophagy.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828.5

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