缬氨酸通过AMPK/mTOR信号通路调控牛成肌细胞增殖

doi:10.11843/j.issn.0366-6964.2025.09.029

摘要/Abstract

摘要： 旨在探究缬氨酸(Val)对牛成肌细胞增殖的影响及潜在的分子调控机制。采用单因素试验设计，通过CCK-8和EdU检测不同浓度Val对细胞增殖的影响并确定其最佳作用浓度；使用分子对接方法预测Val分别与AMPK和mTOR蛋白之间的相互作用；将细胞分为6组：对照组(0 mmol·L^-1 Val)、0.5 mmol·L^-1 Val组(0.5 mmol·L^-1 Val)、Val+AMPK激活剂AICAR组(0.5 mmol·L^-1 Val+1 mmol·L^-1 AICAR)、Val+AMPK抑制剂Compound C组(0.5 mmol·L^-1 Val+5 μmol·L^-1 Compound C)、AMPK激活剂AICAR组(1 mmol·L^-1 AICAR)和AMPK抑制剂Compound C组(5 μmol·L^-1 Compound C)。试验采用qRT-PCR和Western blot技术检测Val对牛成肌细胞中增殖相关因子PAX7、PCNA和CDK1以及AMPK/mTOR信号通路关键因子AMPK和mTOR的mRNA和蛋白表达影响。结果表明：与对照组相比，0.5 mmol·L^-1 Val作用牛成肌细胞24 h后细胞活力极显著提高(P<0.001)，EdU阳性细胞率极显著上升(P<0.01)，PAX7和PCNA的mRNA表达水平(P<0.05)、PAX7的蛋白表达水平均显著升高(P<0.05)。分子对接预测结果显示，Val与AMPK之间由2个氢键连接，Val与mTOR之间可形成3个氢键，氢键的作用使Val与AMPK和mTOR之间的结合更加稳定。与对照组相比，0.5 mmol·L^-1 Val单独作用于牛成肌细胞后mTOR的蛋白表达水平显著升高(P<0.05)，AMPK的蛋白表达水平显著降低(P<0.05)。与0.5 mmol·L^-1 Val组相比，0.5 mmol·L^-1 Val+1 mmol·L^-1 AICAR组mTOR的mRNA表达水平、mTOR和PAX7的蛋白表达水平均极显著降低(P<0.01)，AMPK的mRNA和蛋白表达水平均显著升高(P<0.05)；0.5 mmol·L^-1 Val+5 μmol·L^-1 Compound C组mTOR的mRNA表达水平显著升高(P<0.05)。本研究表明，Val可通过负调控AMPK/mTOR信号通路促进牛成肌细胞增殖，揭示了Val通过AMPK/mTOR信号通路影响牛成肌细胞增殖的分子机理。

关键词: 缬氨酸, 牛, 成肌细胞, AMPK/mTOR信号通路, 细胞增殖

Abstract: The purpose of this study was to explore the effect of valine (Val) on the proliferation of bovine myoblasts and its underlying molecular regulatory mechanisms. A single variate experimental design was used to detect the effects of different concentrations of Val on cell proliferation by CCK-8 and EdU in order to determine its optimal concentration. Molecular docking methods were used to predict the interactions between Val and AMPK and mTOR proteins, respectively. Cells are divided into 6 groups: Control group (0 mmol·L^-1 Val), 0.5 mmol·L^-1 Val group (0.5 mmol·L^-1 Val), Val+AMPK activator AICAR group (0.5 mmol·L^-1 Val+1 mmol·L^-1 AICAR), Val+AMPK inhibitor Compound C group (0.5 mmol·L^-1 Val+5 μmol·L^-1 Compound C), AMPK activator AICAR group (1 mmol·L^-1 AICAR) and AMPK inhibitor Compound C group (5 μmol·L^-1 Compound C). In this study, qRT-PCR and Western blot were used to detect the effects of Val on the mRNA and protein expressions of proliferation-related factors PAX7, PCNA and CDK1, as well as AMPK/mTOR signaling pathway key factors AMPK and mTOR in bovine myoblasts. The results showed that compared with the control group, after 24 h of 0.5 mmol·L^-1 effected on bovine myoblasts, cell viability was extremely significantly increased (P<0.001), and the rate of EdU-positive cells was extremely significantly increased (P<0.01), the mRNA expression levels of PAX7 and PCNA (P<0.05)and the protein expression levels of PAX7 were significantly increased (P<0.05).The molecular docking prediction results showed that Val and AMPK were connected by two hydrogen bonds, and three hydrogen bonds could be formed between Val and mTOR, and the combination between Val and AMPK and mTOR was more stable. Compared with the control group, the protein expression level of mTOR was significantly increased after 0.5 mmol·L^-1 Val was applied to bovine myoblasts alone (P<0.05), and the protein expression level of AMPK was significantly decreased (P<0.05). Compared with the 0.5 mmol·L^-1 Val group, the mRNA expression level of mTOR and the protein expression levels of mTOR and PAX7 in the 0.5 mmol·L^-1 Val+1 mmol·L^-1 AICAR group were extremely significantly decreased (P<0.01), and the mRNA and protein expression levels of AMPK were significantly increased (P<0.05); The mRNA expression level of mTOR in 0.5 mmol·L^-1 Val+5 μmol·L^-1 compound C group was significantly increased (P<0.05).This study showed that Val could promote the proliferation of bovine myoblasts by negatively regulating the AMPK/mTOR signaling pathway, revealing the molecular mechanism by which Val affect the proliferation of bovine myoblasts through the AMPK/mTOR signaling pathway.

Key words: valine, cattle, myoblasts, AMPK/mTOR signaling pathway, cell proliferation

中图分类号:

S823.5

夏春秋, 苗舒, 李志青, 刘磊, 万发春, 沈维军. 缬氨酸通过AMPK/mTOR信号通路调控牛成肌细胞增殖[J]. 畜牧兽医学报, 2025, 56(9): 4491-4506.

XIA Chunqiu, MIAO Shu, LI Zhiqing, LIU Lei, WAN Fachun, SHEN Weijun. Valine Regulates Bovine Myoblast Proliferation through the AMPK/mTOR Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4491-4506.

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