基于UPLC-Q-TOF-MS技术鉴定金钗石斛生物碱类成分及其活性研究Identification of alkaloid components in Dendrobium nobile Lindl. based on UPLC-Q-TOF-MS technique and its bioactivity analysis
陈洁,洪梦杰,蒋成功,李翔,于白音,刘羽佳
CHEN Jie,HONG Mengjie,JIANG Chenggong,LI Xiang,YU Baiyin,LIU Yujia
摘要(Abstract):
目的 采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)技术,快速而全面分析金钗石斛化学成分,初步探究金钗石斛生物碱(Dendrobium nobile Lindl. Alkaloid,DNAL)对三阴性乳腺癌细胞的作用及分子机制。方法 采用UPLC-Q-TOF-MS,分别在正、负离子扫描模式下对金钗石斛化学成分进行分析;采用CCK-8、划痕和小室侵袭实验检测DNAL对MDA-MB-231细胞的作用;采用RT-qPCR和Western blot方法探究DNAL对MDA-MB-231细胞作用的分子机制。结果 共鉴定出230种化学成分,其中生物碱类相对含量最高,共16种,占比33.73%。细胞实验结果表明,DNAL能够显著抑制MDA-MB-231细胞的增殖、侵袭和转移。经DNAL处理的MDA-MB-231细胞中Claudin-1的表达显著上调,EGFR、STAT3、MMP-2、MMP-9、PIK3CA和AKT1的表达显著下调; EGFR和STAT3磷酸化水平显著降低,转移相关蛋白MMP-2、MMP-9和VEGFA的表达量显著降低。结论 DNAL可能通过调控EGFR/STAT3信号通路,降低转移相关蛋白MMP-2、MMP-9和VEGFA的表达来抑制三阴性乳腺癌细胞的增殖、侵袭和转移。
Objective To rapidly identify and analyze the chemical constituents of Dendrobium nobile Lindl.,and to explore the effects and underlying mechanism of total alkaloids of Dendrobium nobile( DNAL) in the treatment of Triple-negative Breast Cancer( TNBC) through in vitro cell experiments. Methods The chemical constituents of Dendrobium nobile Lindl. w ere analyzed and identified by ultra-high-performance liquid-chromatography coupled w ith quadrupole time-of-flight mass spectrometry( UPLC-Q-TOF-M S). CCK-8,w ound healing and transw ell assays w ere performed to evaluate the effects of Dendrobium nobile Lindl. alkaloids on M DA-M B-231 cells' proliferation,migration and invasion respectively. RT-q PCR and w estern blot w ere carried out to explore the potential underlying mechanism. Results A total of 230 chemical components of Dendrobium nobile Lindl. w ere analyzed and identified,including 16 alkaloids,represents approximately 33. 73% as the highest relative content. In vitro cell experiments exhibited significantly inhibitory effects of DNAL on M DA-M B-231 cell proliferation,migration and invasion. The expression level of Claudin-1 w as significantly upregulated,w hereas the expression levels of EGFR,STAT3,M M P-2,M M P-9,PIK3CA and AKT1 w ere significantly dow nregulated in M DA-M B-231 cells after DNAL treatment. The expressions of p EGFR and p STAT3,together w ith the migration related protein M M P2,M M P9 and VEGFA w ere significantly dow nregulated after DNAL treatment. Conclusion The inhibitory effects of DNAL on M DA-M B-231 cells' proliferation,migration and invasion may carried out though EGFR/STAT3 signaling pathw ay by dow nregulating M M P2,M M P9 and VEGFA expressions.
关键词(KeyWords):
金钗石斛;生物碱;三阴性乳腺癌;增殖;侵袭;转移;信号通路
Dendrobium nobile Lindl.;alkaloid;triple negative breast cancer;proliferation;invasion;migration;signaling pathway
基金项目(Foundation): 广东省基础与应用基础研究自然科学基金面上项目(2024A1515013167);; 广东省普通高校重点领域专项(2022ZDZX4044,2024ZDZX2023);; 韶关市科技计划项目(220606154533798,230615178031000)
作者(Author):
陈洁,洪梦杰,蒋成功,李翔,于白音,刘羽佳
CHEN Jie,HONG Mengjie,JIANG Chenggong,LI Xiang,YU Baiyin,LIU Yujia
DOI: 10.14066/j.cnki.cn21-1349/r.2024.0055
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- 金钗石斛
- 生物碱
- 三阴性乳腺癌
- 增殖
- 侵袭
- 转移
- 信号通路
Dendrobium nobile Lindl. - alkaloid
- triple negative breast cancer
- proliferation
- invasion
- migration
- signaling pathway