基于UHPLC-Q-Orbitrap MS技术的仙鹤草干预H1299细胞增殖和凋亡的代谢组学分析  

Metabolomic analysis of Agrimonia pilosa intervention in proliferation and apoptosis of H1299 cells based on UHPLC-Q-Orbitrap MS technology

作　　者：同泽华 郭文军 李萌 徐雅娟[2] 张洪铭 窦泽宇 解生旭[2] 王卫芳 TONG Ze-hua;GUO Wen-jun;LI Meng;XU Ya-juan;ZHANG Hong-ming;DOU Ze-yu;XIE Sheng-xu;WANG Wei-fang(College of Pharmacy,Changchun University of Chinese Medicine,Changchun 130117,China;Jilin Academy of Chinese Medicine Sciences,Changchun 130021,China;School of Clinical Medicine,Changchun University of Chinese Medicine,Changchun 130117,China)

机构地区：[1]长春中医药大学药学院,吉林长春130117 [2]吉林省中医药科学院,吉林长春130021 [3]长春中医药大学临床医学院,吉林长春130117

出　　处：《中国药理学通报》2025年第5期970-978,共9页Chinese Pharmacological Bulletin

基　　金：吉林省科技发展计划项目(No YDZJ202201ZYTS181);吉林省教育厅科学研究项目(No JJKH20241084KJ);吉林省中医药管理局科研项目(No 2020053);吉林省博士后科研资助项目(No 201410)。

摘　　要：目的采用非靶向代谢组学等方法,探讨仙鹤草(Agrimonia pilosa,AP)对非小细胞肺癌(non-small cell lung cancer,NSCLC)H1299细胞增殖、凋亡的影响及分子机制。方法以H1299细胞为研究对象,通过CCK-8法、集落形成、LDH、Hoechst 33258染色、AO/EB染色、流式细胞检测、RT-qPCR等实验,检测AP对细胞的增殖和凋亡的作用。采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱仪(Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry,UHPLC-Q-Orbitrap MS)进行代谢组学分析,检测主要差异代谢物,并分析其相关代谢通路。结果与对照组相比,AP给药组能够明显抑制H1299细胞的增殖及集落形成,同时LDH的释放量增大,且呈剂量依赖性。荧光显微镜和流式细胞仪及RT-qPCR分析发现,AP给药后H1299细胞发生皱缩、核碎片增多现象,阻滞于G 0/G 1期,上调凋亡基因caspase-3、Bax,下调Bcl-2诱导细胞凋亡作用。代谢组学分析筛选出35个差异代谢物,分别为PC(O-30∶1)、D-Glutamic acid、PE(18∶0/15∶0)等,主要涉及代谢通路包括氨基酸代谢、甘油磷脂代谢和嘌呤代谢等。结论AP可能通过干扰H1299细胞的多条代谢通路,抑制细胞增殖和促进细胞凋亡,而发挥药理作用。Aim To investigate the effects of Agrimonia pilosa(AP)on the proliferation and apoptosis of non-small cell lung cancer(NSCLC)H1299 cells using non-targeted metabolomics and other methods,and to explore the underlying molecular mechanisms.Methods Taking H1299 cells as the research object,the effect of AP on cell proliferation and apoptosis was detected through CCK-8 method,colony formation,LDH,Hoechst 33258 staining,AO/EB staining,flow cytometry detection,RT qPCR and other experiments.The main differential metabolites were detected by the metabolomics method of ultra-high phase liquid chromatography and mass spectrometry(UHPLC-Q-Orbitrap MS),and related metabolic pathways were analyzed.Results Compared with the control group,AP treatment was able to significantly inhibit the proliferation and colony formation of H1299 cells,while the release of LDH increased in a dose-dependent manner.Fluorescence microscopy and flow cytometry and RT-qPCR analysis revealed that H1299 cells underwent crumpling and increased nuclear fragmentation after AP administration,blocked in G 0/G 1 phase,up-regulated apoptotic genes caspase-3 and Bax,and down-regulated apoptosis-inducing effects of Bcl-2.Metabolomics analysis screened 35 differential metabolites,which were PC(O-30∶1),D-Glutamic acid,PE(18∶0/15∶0),etc.The main metabolic pathways involved included amino acid metabolism,glycerophospholipid metabolism and purine metabolism so on.Conclusions AP may exert its pharmacological effects by interfering with multiple metabolic pathways in H1299 cells,inhibiting cell proliferation and promoting apoptosis.

关 键 词：仙鹤草 H1299细胞 代谢组学 细胞凋亡 非小细胞肺癌 细胞增殖 

分 类 号：R282.71[医药卫生—中药学] R329.25[医药卫生—中医学] R329.28R734.2