密骨胶囊通过NF-κB和AKT-mTOR信号通路对肌少症的作用机制

Study on the mechanism of the Migu capsule in the treatment of sarcopenia via NF-κB and AKT-mTOR signaling pathway

目的 利用网络药理学和小鼠C2C12肌细胞体外细胞实验,研究密骨胶囊治疗肌少症的主要活性成分和潜在作用机制。方法 从TCMSP等网络数据库对密骨胶囊的中药成分进行活性成分和主要靶点的收集,并从GeneCards等数据库中对肌少症的疾病基因进行搜索,交集预测密骨胶囊作用于肌少症的靶点和通路。体外实验对小鼠C2C12肌细胞进行培养及诱导分化。用密骨胶囊含药血清预处理细胞,再用TNF-α炎症因子进行刺激,造成肌细胞萎缩。免疫印迹法和聚合酶链式反应法证明其对肌细胞内NF-κB通路和AKT-mTOR通路中相关蛋白表达情况。结果 在密骨胶囊对肌肉的作用中,核心靶点为MTOR、ESR1、EGFR、SIRT1、CTNNB1、VEGFA、ALB、IL6、TP53、TNF、AKT1,可能干预HIF-1、AGE-RAGE、TNF、PI3K-AKT-mTOR等信号通路。密骨胶囊含药血清干预后C2C12肌细胞增粗,降低MuRF1 mRNA表达。免疫印迹实验结果中,密骨胶囊含药血清降低TNF-α刺激的Atrogin-1蛋白量,抑制iκbα磷酸化降解,减少蛋白降解。密骨胶囊含药血清减少TNF-α对p-AKT、p-4EBP1的抑制,增加蛋白合成。结论 通过网络药理学和体外实验验证,密骨胶囊可能对肌少症有防治作用。密骨胶囊抑制NF-κB通路,减缓肌蛋白降解。密骨胶囊激活AKT-mTOR通路,促进肌细胞内蛋白合成。

Objective To study the main active components and potential mechanism of the Migu capsule in the treatment of sarcopenia by network pharmacology and the experiment of mouse C2C12 muscle cells in vitro.Methods The active components and main targets of traditional Chinese medicine components of Migu capsule were collected from TCMSP databases.The disease genes were searched from GeneCards and other databases.The intersection between the two sets of the targets and pathways of sarcopenia that the Migu capsule may be related was predicted.After culturing and inducing differentiation of mouse C2C12 muscle cells in vitro,the cells were pretreated with the Migu capsule and then stimulated with TNF-inflammatory factors.Western blotting and polymerase chain reaction showed its effect on the expression of related proteins in NF-κB pathway and AKT-mTOR pathway in muscle cells.Results In the action of the Migu capsule on muscle,the core targets were MTOR,ESR1,EGFR,SIRT1,CTNNB1,VEGFA,ALB,IL6,TP53,TNF,and AKT1,which interfered with HIF-1,AGE-RAGE,TNF,and PI3K-AKT-mTOR signal pathways.The Migu capsule decreased the expression of MuRF1 mRNA.The result of Western blotting showed that the drug-containing serum decreased the amount of Atrogin-1 protein stimulated by TNF-αand inhibited the phosphorylation and degradation of iκbαin NF-κB pathway.It also increased the amount of p-AKT and p-4EBP1 protein by the inhibition of TNF-αin AKT-mTOR pathway.Conclusion Through network pharmacology and in vitro experiments,the Migu capsule may have a preventive and therapeutic effect on sarcopenia.The Migu capsule inhibits NF-κB pathway and slows down the degradation of muscle proteins.The Migu capsule activates AKT-mTOR pathway and promotes protein synthesis in muscle cells.