| Skeletal muscle is widely distributed in the animal body and maintains many important functions in the body.Its structure consists of a variety of muscle fibres with different metabolic functions.Different types of muscle fibres have different effects on the growth and development of skeletal muscle and on the quality of meat produced.The typing and transformation of muscle fibre types is influenced by a complex regulatory network,in which the role of small molecules in the organism cannot be ignored.Butyric acid is a short-chain fatty acid secreted by the gastrointestinal tract,which is essential in the regulation of livestock and poultry production performance and also plays a key role in muscle growth and development and myofiber type transformation.In this study,an in vitro model of bovine muscle cell differentiation was developed.Firstly,the optimal treatment concentration of sodium butyrate was screened to investigate its effect on myoblast differentiation and muscle fibre type transformation;then the CDS region of bovine AMPKα1 gene was cloned in vitro and its molecular characteristics and tissue expression pattern were analysed;finally,an inhibitor of AMPK pathway was used to investigate whether sodium butyrate acts through this pathway.The main results of this paper are as follows:1.The optimal treatment concentration of sodium butyrate was screened by CCK-8 at 1 mM,and the effect of sodium butyrate on the expression levels of bovine myoblast differentiation marker genes and the four myosin heavy chain isoforms was examined by RT-qPCR and WB.The results revealed that sodium butyrate significantly up-regulated the mRNA and protein expression levels of the differentiation marker genes MYOG and MYF6 during cell differentiation(P<0.05),significantly up-regulated the mRNA of MyHc Ⅰ and MyHC Ⅱa and the protein expression levels of slow MyHC(P<0.05),and significantly down-regulated the mRNA of MyHC Ⅱb and the protein expression of fast MyHC-related(P<0.05).The above results suggest that sodium butyrate promotes myoblast differentiation and slow myofibrillogenesis and inhibits fast myofibrillogenesis.2.AMPKα1 gene is one of the important upstream genes in the AMPK pathway.The AMPKα1 gene was selected as a candidate gene for coding region cloning,molecular characterization and tissue expression characterization.The results showed that the coding region of AMPKα1 gene in Qinchuan cattle is 1377 bp long and encodes 458 amino acids.Protein interaction analysis showed that AMPKα1 protein interacts mainly with PRKAA2,TSC2,ACACA and other proteins related to muscle development.Tissue expression profiling showed that the AMPKα1 gene had the highest expression in the longest muscle tissue of the bovine dorsum,followed by the cardiac muscle;the expression of AMPKα1 in muscle tissues of calves and adults at different developmental stages showed significant differences,and the expression of this gene in adult cattle tissues was significantly higher than that in calves(P<0.05).The AMPKα1 gene and its AMPK pathway play an important regulatory role in the development of bovine muscle.To investigate whether sodium butyrate mediates the role of AMPK signaling pathway,by exogenously adding sodium butyrate,it was found that sodium butyrate significantly up-regulated the mRNA expression levels of AMPKαl,AMPKα2,PGC-1α and SIRT1 during cell differentiation(P<0.05),all of the above genes belong to AMPK pathway;bovine myoblasts were treated with AMPK pathway inhibitor and it was found that The mRNA expression levels of AMPKα1,AMPKα2,PGC-1α and SIRT1 were significantly(P<0.05)suppressed,and the mRNA and protein expression levels of the differentiation marker genes MYOG and MYF6 were significantly(P<0.05)down-regulated,and the mRNA of MyHC Ⅰ and MyHC Ⅱa and the protein expression of slow MyHC The mRNA and protein expression levels of MyHC Ⅰ,MyHC Ⅱa and slow MyHC were all significantly down-regulated(P<0.05),while the mRNA and protein expression levels of MyHC Ⅱb and fast MyHC were all significantly upregulated(P<0.05);and the AMPK pathway inhibitor could significantly alleviate the effect of sodium butyrate(P<0.05).In conclusion,the present study initially investigated the effects of sodium butyrate on the differentiation of bovine muscle cells and the transformation of skeletal muscle fibre types.It was hypothesised that the AMPKα1 gene and its AMPK pathway play an important regulatory role in the growth and development of bovine muscle,and further studies indicated that sodium butyrate may promote cell differentiation and slow-type muscle fibre production through the AMPK/Sirt1/PGC-1α signalling pathway.The results of this study provide some reference and new ideas for improving beef quality through exogenous addition of small molecules. |
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