| With the improvement of living standard,people’s demand for beef and meat quality are increasing,and the meat production and meat quality of livestock and poultry directly affect the economic benefits of livestock industry.Skeletal muscle as the main component of livestock body weight,the exploration of its development mechanism is of great significance to livestock and poultry production.In addition to genetic factors,factors such as feed intake and nutrient addition are also involved in the regulation of muscle development.Butyric acid is one of the short-chain fatty acids,mainly produced by fermentation of fibers by intestinal microorganisms,and acts as a signaling molecule to regulate body metabolism and muscle fiber formation through different pathways.In order to clarify the effect of butyric acid on the proliferation and differentiation of bovine muscle fibers and their formation,this experiment isolated bovine skeletal satellite cells(SSCs)and added sodium butyrate to investigate the biological regulation of bovine SSCs by sodium butyrate treatment.The main contents are as follows:1、Bovine primary SSCs were isolated by enzyme digestion,and the isolated cells were confirmed to be bovine SSCs by Desmin immunofluorescence identification and induced differentiation identification.The expression of skeletal muscle differentiation marker genes MyoD1,MYF6 and Myog after induced differentiation of bovine SSCs was verified by qRT-PCR.The results showed that the bovine primary SSCs were successfully isolated;2、The expression of genes related to proliferation,differentiation and apoptosis of primary bovine SSCs treated with sodium butyrate was detected by qRT-PCR,CCK-8 and flow cytometry.Sodium butyrate significantly down-regulated the expression of cell proliferation-related genes CDK1、CDK2、CCNB2、MCM3 and PCNA(P<0.01);Sodium butyrate significantly down-regulated the expression of skeletal muscle cell differentiation marker genes MyoD1、Myf6 and Myog(P<0.01);At the same time,sodium butyrate significantly down-regulated the expression of apoptosis-inhibiting gene Bcl-2(P<0.01),and significantly up-regulated the expression of proapoptosis-related genes p53 and Bax(P<0.01).The results showed that sodium butyrate inhibited the proliferation and differentiation of bovine SSCs and promoted their apoptosis;3、The effects of sodium butyrate treatment on mitophagy and biosynthesis in bovine SSCs were detected by Werstern blot,qRT-PCR,electron microscopy and immunofluorescence.Sodium butyrate treatment could promote the production of mitophagosomes in bovine SSCs,while sodium butyrate treatment increased cellular ROS accumulation,increased cellular ATP and mitochondrial copy number,and decreased mitochondrial membrane potential.In addition,sodium butyrate treatment could significantly up-regulate the mRNA and protein expressions of key mitophagy enzymes PINK1 and LC3B,and significantly down-regulate the mRNA and protein expressions of p62.It was shown that sodium butyrate treatment promoted mitochondrial autophagy and regulated mitochondrial biosynthesis in bovine SSCs.4、The effect of sodium butyrate treatment on the mTOR pathway of bovine SSCs was detected by qRT-PCR method.The results showed that sodium butyrate significantly down-regulated the expression of mTOR,FOXO1,AKT1 and EIF4EBP1 genes in the mTOR signaling pathway of bovine SSCs(P<0.01).By treating bovine SSCs with mTOR signaling pathway inhibitor,it was found that the regulation of mTOR pathway inhibitor on the proliferation,differentiation,apoptosis and mitophagy of bovine SSCs was consistent with the results of sodium butyrate treatment.This study preliminarily confirmed that sodium butyrate treatment of bovine SSCs could inhibit the proliferation and differentiation of SSCs,while promoting apoptosis and mitophagy.The effect of sodium butyrate on SSCs cemay realize the proliferation and differentiation of bovine SSCs by mediating the mTOR signaling pathway. |
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