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