氯化两面针碱抑制小鼠C2C12细胞成肌分化

Nitidine Chloride inhibits myogenesis of murine C2C12 cells

关键词:  氯化两面针碱  C2C12  分化  骨骼肌  分子机制  P38 MAPK  AKT

Key words:  nitidine chloride  C2C12  myogenesis  skeletal muscle  molecular mechanism  p38 MAPK  AKT

摘要:

[目的] 探讨氯化两面针碱（NC）对小鼠C2C12细胞成肌分化的影响及其可能的分子机制。[方法] 将C2C12细胞分为空白组和NC（100 μmol/L）组，干预24 h，采用免疫荧光染色观察肌管形态。为了分析NC对肌管分化的影响，通过定量逆转录聚合酶链式反应（qRT-PCR）检测关键标志基因水平，同时检测了细胞增殖标记基因Ki67的表达并使用细胞计数试剂盒-8（CCK-8）试剂盒以分析NC对细胞增殖的影响。通过DIA定量蛋白质组学分析差异表达蛋白（DEPs），基于基因本体（GO）富集、京都基因与基因组百科全书（KEGG）富集和蛋白-蛋白相互作用（PPI）网络分析，进一步通过蛋白印迹（Western Blot，WB）验证高富集通路蛋白的磷酸化水平。[结果] 免疫荧光染色显示，与空白组相比，NC组肌管数量减少。qRT-PCR结果显示，与空白组相比，NC组肌源性分化基因和不同肌纤维类型标记基因表达均下调，NC组Ki67表达也显著降低。CCK-8细胞活力检测结果发现，NC（100 μmol/L）组细胞活力显著降低；蛋白质组学分析显示，共鉴定出1 297种DEPs；GO分析结果显示，差异蛋白主要参与细胞外结构组织、细胞外基质组织、外部封装结构组织、细胞黏附等过程，KEGG分析结果显示，DEPs主要富集的肌分化相关通路有转化生长因子β（TGF-β）信号通路、磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（AKT）信号通路、丝裂原活化蛋白激酶（MAPK）信号通路；PPI分析结果显示，丝裂原活化蛋白激酶11（MAPK11/P38-β MAPK）占据主要的节点位置。WB结果证明NC处理后，C2C12细胞中P38丝裂原活化蛋白激酶（P38 MAPK）和AKT的磷酸化水平被显著下调。[结论] 通过蛋白质组学分析和WB结果表明NC可能通过下调P38 MAPK和AKT的磷酸化水平进而抑制C2C12细胞成肌分化。

Abstract:

[Objective] To investigate the effects of nitidine chloride（NC）on the differentiation of C2C12 cells and its possible molecular mechanisms. [Methods] C2C12 cells were divided into control and NC（100 μmol/L）groups and treated for 24 h. Myotube morphology was observed by immunofluorescence staining. To analyze the effect of NC on myotube differentiation，the expression of skeletal muscle-related genes was detected by quantitative reverse transcription polymerase chain reaction（qRT-PCR）. The expression of the cell proliferation marker gene Ki67 was also detected，and CCK-8 kits were used to analyze the effect of NC on cell proliferation. Differentially expressed proteins（DEPs）were analyzed by DIA quantitative proteomics based on Gene Ontology（GO）enrichment，Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment，and protein-protein interaction（PPI）analyses，and the phosphorylation levels of the highly enriched pathway proteins were further verified by Western blot（WB）. [Results] Immunofluorescence staining showed that the number of myotubes was reduced in the NC group compared with the control group. qRT-PCR showed that the expression of myogenic differentiation genes and different myofiber type marker genes was down-regulated in the NC group compared with the control group，and the expression of Ki67 was significantly reduced in the NC group. CCK-8 cell viability assay showed that the viability of the cells was significantly reduced in the NC（100 μmol/L）group. Proteomics analysis showed that a total of 1 297 DEPs were identified. GO analysis revealed that the differentially expressed proteins were primarily associated with extracellular structural organization，extracellular matrix organization，external encapsulating structure organization，and cell adhesion. KEGG analysis indicated that the pathways predominantly enriched with the differentially expressed proteins related to myogenic differentiation were the transforming growth factor- β（TGF-β） signaling pathway，phosphoinositide 3-kinase（PI3K）/protein kinase B（AKT）signaling pathway，and mitogen-activated protein kinase（MAPK）signaling pathway. PPI network analysis showed that mitogen- activated protein kinase 11（MAPK11/p38-β MAPK）occupied the main node position. WB results proved that the phosphorylation levels of p38 mitogen-activated protein kinase（p38 MAPK）and AKT were significantly down-regulated in C2C12 cells after NC treatment. [Conclusion] Proteomics analysis and WB results indicate that NC may inhibit myogenic differentiation of C2C12 cells by down-regulating the phosphorylation levels of p38 MAPK and AKT.