三棱提取物抑制糖酵解限速酶抗黑色素瘤机制研究

刘洋; 郑秀芹; 杨葛俊; 赵涵; 陆茵; 王爱云; 陈文星

doi:10.14148/j.issn.1672-0482.2022.0331

三棱提取物抑制糖酵解限速酶抗黑色素瘤机制研究

doi: 10.14148/j.issn.1672-0482.2022.0331

刘洋^{1, 2,},
郑秀芹^{1, 2},
杨葛俊^{1, 2},
赵涵^{1, 2},
陆茵^{1, 2},
王爱云^{1, 2},
陈文星^{1, 2, ,}

1.
南京中医药大学药学院, 江苏南京 210023
2.
江苏省中药药效与安全性评价重点实验室, 江苏南京 210023

基金项目:

国际(地区)合作与交流项目 81961128020

国家自然科学基金面上项目 81673648

国家自然科学基金面上项目 81673725

国家自然科学基金面上项目 81973734

国家自然科学基金面上项目 81973587

江苏省中医药领军人才项目 SLJ0229

江苏省研究生科研创新计划项目 KYCX21_1781

详细信息

作者简介:
刘洋, 男, 硕士研究生, E-mail: 20190372@njucm.edu.cn

通讯作者:
陈文星, 男, 教授, 主要从事活血化瘀中药对肿瘤转移的影响研究, E-mail: chenwx@njucm.edu.cn

中图分类号: R285.5
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- 文章访问数: 228
- HTML全文浏览量: 38
- PDF下载量: 21
- 被引次数: 0
出版历程
- 收稿日期: 2021-12-15
- 网络出版日期: 2022-04-13
- 发布日期: 2022-04-10

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

LIU Yang^{1, 2
,},
ZHENG Xiu-qin^{1, 2},
YANG Ge-jun^{1, 2},
ZHAO Han^{1, 2},
LU Yin^{1, 2},
WANG Ai-yun^{1, 2},
CHEN Wen-xing^{1, 2
, ,}

1.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing 210023, China

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。
- 三棱 /
- 网络药理学 /
- 黑色素瘤 /
- 能量代谢 /
- 糖酵解
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.
- Sparganii Rhizome /
- network pharmacology /
- melanoma /
- energy metabolism /
- glycolysis

HTML全文

图 1 三棱活性成分潜在靶点图

Figure 1. Potential targets map of active ingredients in Sparganii Rhizome

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图 2 PPI网络核心靶点筛选图

Figure 2. Core targets of PPI network

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图 3 KEGG通路富集分析气泡图

注：与对照组相比, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001。

Figure 3. KEGG pathway enrichment analysis

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图 4 不同浓度三棱提取物分别在24、48 h对A375细胞活力的影响

注：与对照组相比, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001。x±s，n=6。

Figure 4. Effects of different concentrations of Sparganii Rhizome extract on A375 cell viability at 24 h and 48 h

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图 5 三棱提取物对A375细胞增殖的影响

注：与对照组相比, ^*P < 0.05, ^***P < 0.001。x±s, n=3。

Figure 5. Effect of Sparganii Rhizome extract on proliferation of A375 cells

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图 6 三棱提取物对A375细胞ECAR、OCR的影响(x±s，n=5)

Figure 6. Effect of Sparganii Rhizome extract on ECAR and OCR of A375 cells (x±s, n=5)

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图 7 三棱提取物对A375细胞葡萄糖摄取、乳酸、丙酮酸、ATP生成的影响

注：与对照组相比, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001。x±s，n=4。

Figure 7. Effects of Sparganii Rhizome extract on glucose uptake, lactic acid, pyruvate and ATP production in A375 cells

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图 8 三棱提取物对A375细胞糖酵解关键激酶活性的影响

注：与对照组相比, ^**P < 0.01, ^***P < 0.001。x±s，n=3。

Figure 8. Effect of Sparganii Rhizome extract on glycolytic key kinase activity in A375 cells

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图 9 三棱提取物对A375细胞糖酵解关键激酶表达的影响

注：与对照组相比, ^**P < 0.01, ^***P < 0.001。x±s，n=3。

Figure 9. Effect of Sparganii Rhizome extract on expression of glycolytic key kinases in A375 cells

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表 1 中药三棱主要活性成分

Table 1. Main active ingredients in Sparganii Rhizome

Mol ID	化合物	OB/%	DL
MOL000296	常春藤皂苷元(Hederagenin)	36.91	0.75
MOL000358	β-谷甾醇(β-Sitosterol)	36.91	0.75
MOL000392	刺芒柄花素(Formononetin)	69.67	0.21
MOL000449	豆甾醇(Stigmasterol)	43.83	0.76
MOL001297	反式-11-二十碳烯酸 (trans-Gondoic acid)	30.70	0.20

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