亮氨酸通过PI3K-AKT信号通路促进牛成肌细胞的增殖

doi:10.11843/j.issn.0366-6964.2024.01.015

摘要/Abstract

摘要： 旨在探究外源补充亮氨酸（Leu）对牛成肌细胞增殖的影响及其作用机理。本试验以牛成肌细胞为研究对象，利用CCK-8法和EdU染色法检测不同浓度的Leu对牛成肌细胞增殖的影响，确定Leu的最佳添加浓度；以最佳浓度Leu为处理组、0 mmol·L^-1 Leu为对照组进行转录组测序（RNA-Seq），筛选Leu调控牛成肌细胞增殖的关键通路。利用qRT-PCR验证关键通路中的关键调控基因，并通过添加抑制剂进行进一步验证。结果显示，添加Leu可提高牛成肌细胞活力，其中0.5 mmol·L^-1 Leu显著提高成肌细胞活力和EdU阳性率（P<0.05）。Leu组和对照组差异表达基因有1 290个，其中688个上调，602个下调。KEGG富集分析，所注释的309个上调基因涉及47条显著富集通路，其中与骨骼肌生长发育相关通路中最为显著的是PI3K-AKT信号通路，包含34个上调基因。随机挑选PIK3R1、FOXO3、IRS1、RPTOR、MTOR和MET等6个差异表达基因进行qRT-PCR验证，结果与RNA-Seq一致。添加PI3K-AKT信号通路中PI3K的抑制剂LY294002后，阻断了Leu对牛成肌细胞增殖的促进作用。综上所述，Leu可通过PI3K-AKT信号通路促进牛成肌细胞的增殖。

关键词: 亮氨酸, 牛, 骨骼肌, 成肌细胞增殖, RNA-Seq, PI3K-AKT

Abstract: This study was conducted to investigate the effect of exogenous leucine supplementation on the proliferation of bovine myogenic cells and its mechanism. The effects of different concentrations of Leu on the proliferation of bovine myoblasts were examined by CCK-8 and EdU staining, and the optimal concentration of Leu was determined; transcriptome sequencing (RNA-Seq) was performed with the optimal concentration of Leu as the treatment group and 0 mmol·L^-1 Leu as the control group to screen the key pathways of Leu regulating the proliferation of bovine myoblasts. Key regulatory genes in the key pathway were screened using qRT-PCR and validated by adding inhibitors or activators. Addition of Leu increased bovine myogenic cell viability, with 0.5 mmol·L^-1 Leu significantly increasing myogenic cell viability and EdU positive rate (P<0.05). There was 1 290 differentially expressed genes between Leu group and control group, of which 688 were upregulated and 602 were downregulated. The 309 up-regulated genes annotated by KEGG enrichment analysis were involved in 47 significantly enriched pathways, among which the most enriched pathway related to skeletal muscle growth and development was the PI3K-AKT signaling pathway, containing 34 up-regulated genes. Six differentially expressed genes, including PIK3R1, FOXO3, IRS1, RPTOR, MTOR and MET, were randomly selected for qRT-PCR validation, and the results were consistent with the result of RNA-Seq. The addition of LY294002, an inhibitor of PI3K in the PI3K-AKT signaling pathway, blocked the proliferation-promoting effect of Leu on bovine myogenic cells. In conclusion, Leu can promote the proliferation of bovine myogenic cells through PI3K-AKT signaling pathway.

Key words: Leu, bovine, skeletal muscle, myoblast proliferation, RNA-Seq, PI3K-AKT

中图分类号:

S823.2

苗舒, 安济山, 王祚, 肖定福, 兰欣怡, 刘磊, 沈维军, 万发春. 亮氨酸通过PI3K-AKT信号通路促进牛成肌细胞的增殖[J]. 畜牧兽医学报, 2024, 55(1): 142-152.

MIAO Shu, AN Jishan, WANG Zuo, XIAO Dingfu, LAN Xinyi, LIU Lei, SHEN Weijun, WAN Fachun. Leucine Promotes the Proliferation of Bovine Myoblasts through PI3K-AKT Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 142-152.

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