三棱提取物抑制糖酵解限速酶抗黑色素瘤机制研究

Study on Anti-Melanoma Mechanism of Sparganii Rhizoma Extract Inhibiting Glycolysis Rate-limiting Enzyme

    摘要
  • 摘要:
      目的  探究三棱抗黑色素瘤的分子作用机制及其有效物质基础。
      方法  通过中药系统药理学数据库与分析平台(TCMSP)检索三棱的活性成分及对应的靶点, 利用GeneCards数据库检索黑色素瘤疾病靶点, 借助Cytoscape3.7.2软件绘制药物-疾病靶点蛋白互作(PPI)网络, 并筛选出核心靶点, 再根据DAVID数据库平台进行KEGG通路富集分析; 通过细胞增殖与活性检测(CCK-8)实验、EdU增殖检测实验、海马能量代谢分析实验、代谢产物及酶活性检测试剂盒实验、蛋白质免疫印迹法(Western blot)实验进一步验证上述分析结果。
      结果  从TCMSP数据库中获得5个三棱中活性成分, 对应的靶点共70个, 与疾病靶点构建PPI网络并进一步筛选得到由86个核心靶点组成的关键网络, KEGG信号通路富集分析结果表明, 三棱治疗黑色素瘤主要涉及代谢相关通路、p53信号通路、AMPK信号通路、MAPK信号通路、PI3K/Akt信号通路、凋亡信号通路等; 体外实验结果表明, 三棱提取物可剂量依赖性抑制黑色素瘤A375细胞的增殖, 并降低细胞的胞外酸化率及糖酵解代谢产物丙酮酸、乳酸、ATP的生成, 进一步结果表明, 三棱提取物可抑制丙酮酸激酶的活性, 并降低丙酮酸脱氢酶(PKM2)、乳酸脱氢酶(LDHA)、单羧酸转运蛋白(MCT4)的蛋白表达水平。
      结论  三棱提取物具有明显的抗黑色素瘤的作用, 其作用机制可能与抑制糖酵解相关蛋白PKM2、LDHA、MCT4表达、干扰肿瘤细胞糖酵解代谢相关。

     

    Abstract:
      OBJECTIVE  To explore the molecular mechanism of Sparganii Rhizome anti-melanoma and its effective material basis.
      METHODS  The active ingredients and corresponding targets of Sparganii Rhizome were retrieved through the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), and the melanoma disease targets were retrieved by GeneCards database. The drug-disease target protein-protein interaction (PPI) was constructed by the cytoscape 3.7.2 software. The core targets were screened out and uploaded to the DAVID platform for KEGG pathway enrichment analysis. Further verification was carried out through cell proliferation and activity detection (CCK-8) experiment, EdU proliferation testing experiment, Seahorse XF cell mito stress analysis experiment, metabolites and enzyme activity detection kit experiment and Western blot.
      RESULTS  Obtained from the TCMSP database, the five active ingredients in Sparganii Rhizome, corresponding to a total of 70 targets, constructed a PPI network with disease targets and further screened to obtain a key network composed of 86 core targets. The results of enrichment analysis of the KEGG signaling pathway showed that Sparganii Rhizome treatment of melanoma mainly involved metabolic-related pathways, p53 signal pathways, AMPK signal pathways, MAPK signal pathways, PI3K/Akt signal pathways, apoptosis signal pathways, etc. The results of in vitro experiments showed that the Sparganii Rhizome extract (SLTQW) inhibits the proliferation of melanoma A375 cells in a dose-depentent manner and reduces the rate of extracellular acidification of cells and the production of glycolytic metabolites pyruvate, lactic acid, ATP. The results of the kit and Western blot showed that the SLTQW inhibited the activity of PK and reduced the protein expression levels of PKM2, LDHA, MCT4.
      CONCLUSION  SLTQW has obvious anti-melanoma effect, and its mechanism may be related to inhibiting the glycolysis-related proteins PKM2, LDHA, MCT4 expression and interfering with tumor cell glycolysis metabolism.

     

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