陆苑;王艳丽;宋忠军;朱晓青;刘春花;陈际宇;李勇军;何艳;
LU Yuan;WANG Yan-li;SONG Zhong-jun;ZHU Xiao-qing;LIU Chun-hua;CHEN Ji-yu;LI Yong-jun;HE Yan;Clinical Trial Research Center, the Affiliated Hospital of Guizhou Medical University;Provincial Key Laboratory of Pharmaceutics in Guizhou Province, State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University;School of Pharmaceutical Sciences, Guizhou Medical University;Engineering Research Center for the Development and Application of Ethnic Medicine and Traditional

• 1:贵州医科大学附属医院临床试验研究中心
• 2:贵州医科大学贵州省药物制剂重点实验室/省部共建药用植物功效与利用国家重点实验室
• 3:贵州医科大学药学院
• 4:贵州医科大学民族药与中药开发应用教育部工程研究中心

摘要(Abstract)：

该研究基于高效液相色谱-四极杆/静电场轨道阱高分辨质谱联用仪探究银杏黄酮苷元(ginkgo flavone aglycone, GA)联合阿霉素(doxorubicin, DOX)对H22肝癌细胞的抑制作用及作用机制。首先考察不同浓度GA和DOX对H22细胞存活率的影响，以组合指数(combination index, CI)对两者的联合用药进行评价。将实验分为对照组(CON)、DOX组、GA组、联合用药组(GDOX),通过代谢组学策略探寻联合用药较单独用药后显著改变的代谢标志物，分析可能的代谢通路和作用机制。结果显示，以30μg·mL^(-1) GA与0.5μmol·L(-1) GA与0.5μmol·L^(-1) DOX作为联合给药浓度，CI为0.808,表明两者联合发挥协同抗肿瘤作用。代谢组学分析鉴定出L-精氨酸、L-酪氨酸和L-缬氨酸等23种代谢标志物，以氨基酸类物质为主。与CON组相比，DOX治疗后22种代谢标志物被显著下调，GA治疗后17种代谢标志物被显著下调。相较于DOX和GA组，GDOX联合用药进一步下调了这些代谢标志物在肝癌中的水平，这可能是两者联合起到增效作用的原因。通路富集分析中找到5条关键代谢通路，包括谷胱甘肽代谢，苯丙氨酸代谢，精氨酸和脯氨酸代谢，β-丙氨酸代谢，缬氨酸、亮氨酸和异亮氨酸降解。这表明GA联合DOX的协同抗肿瘤作用，可能是通过干预多种氨基酸的代谢，进而影响肿瘤细胞的能量供应实现。
Ultra-high-performance liquid chromatography-Q exactive orbitrap tandem mass spectrometry(UHPLC-QEOrbitrap-MS/MS) was used to explore the inhibitory effect and mechanism of ginkgo flavone aglycone(GA) combined with doxorubicin(DOX) on H22 cells. The effects of different concentrations of GA and DOX on the viability of H22 cells were investigated, and combination index(CI) was used to evaluate the effects. In the experiments, control(CON) group, DOX group, GA group, and combined GA and DOX(GDOX) group were constructed. Then the metabolomics strategy was employed to explore the metabolic markers that were significantly changed after combination therapy on the basis of single medication treatment, and by analyzing their biological significance, the effect and mechanism of the anti-tumor effect of GA combined with DOX were explained. The results revealed that when 30 μg·mL(-1) DOX作为联合给药浓度，CI为0.808,表明两者联合发挥协同抗肿瘤作用。代谢组学分析鉴定出L-精氨酸、L-酪氨酸和L-缬氨酸等23种代谢标志物，以氨基酸类物质为主。与CON组相比，DOX治疗后22种代谢标志物被显著下调，GA治疗后17种代谢标志物被显著下调。相较于DOX和GA组，GDOX联合用药进一步下调了这些代谢标志物在肝癌中的水平，这可能是两者联合起到增效作用的原因。通路富集分析中找到5条关键代谢通路，包括谷胱甘肽代谢，苯丙氨酸代谢，精氨酸和脯氨酸代谢，β-丙氨酸代谢，缬氨酸、亮氨酸和异亮氨酸降解。这表明GA联合DOX的协同抗肿瘤作用，可能是通过干预多种氨基酸的代谢，进而影响肿瘤细胞的能量供应实现。
Ultra-high-performance liquid chromatography-Q exactive orbitrap tandem mass spectrometry(UHPLC-QEOrbitrap-MS/MS) was used to explore the inhibitory effect and mechanism of ginkgo flavone aglycone(GA) combined with doxorubicin(DOX) on H22 cells. The effects of different concentrations of GA and DOX on the viability of H22 cells were investigated, and combination index(CI) was used to evaluate the effects. In the experiments, control(CON) group, DOX group, GA group, and combined GA and DOX(GDOX) group were constructed. Then the metabolomics strategy was employed to explore the metabolic markers that were significantly changed after combination therapy on the basis of single medication treatment, and by analyzing their biological significance, the effect and mechanism of the anti-tumor effect of GA combined with DOX were explained. The results revealed that when 30 μg·mL^(-1) GA and 0.5 μmol·L(-1) GA and 0.5 μmol·L^(-1) DOX was determined as the co-administration concentration, the CI value was 0.808, indicating that the combination of GA and DOX had a synergistic anti-tumor effect. Metabolomics analysis identified 23 metabolic markers, including L-arginine, L-tyrosine and L-valine, mostly amino acids. Compared with the CON group, 22 and 17 metabolic markers were significantly down-regulated after DOX treatment and GA treatment, respectively. Compared with the DOX and GA groups, the treatment of GA combined with DOX further down-regulated the levels of these metabolic markers in liver cancer, which might contribute to the synergistic effect of the two. Five key metabolic pathways were found in pathway enrichment analysis, including glutathione metabolism, phenylalanine metabolism, arginine and proline metabolism, β-alanine metabolism, and valine, leucine and isoleucine degradation. These findings demonstrated that the combination of GA and DOX remarkably inhibited the viability of H22 cells and exerted a synergistic anti-tumor effect. The mechanism might be related to the influence of the energy supply of tumor cells by interfering with the metabolism of various amino acids.

关键词(KeyWords)： 银杏黄酮苷元;阿霉素;H22细胞;肝细胞癌;细胞代谢组学;协同作用
ginkgo flavone aglycone;doxorubicin;H22 cell;hepatocellular carcinoma;cell metabolomics;synergy

Abstract:

Keywords:

基金项目(Foundation): 贵州省科技厅项目(黔科合基础-ZK[2022]一般374,黔科合基础[2020]1Y381);; 贵州医科大学项目(新苗计划19NSP056);; 贵州医科大学附属医院项目(NFSC培育计划GYFYNSFC-2021-56);; 贵州省普通高等学校科技拔尖人才项目(KY[2021]033);; 贵州省优秀青年科技人才项目(黔科合平台人才[2021]5619号)

作者(Authors): 陆苑;王艳丽;宋忠军;朱晓青;刘春花;陈际宇;李勇军;何艳;
LU Yuan;WANG Yan-li;SONG Zhong-jun;ZHU Xiao-qing;LIU Chun-hua;CHEN Ji-yu;LI Yong-jun;HE Yan;Clinical Trial Research Center, the Affiliated Hospital of Guizhou Medical University;Provincial Key Laboratory of Pharmaceutics in Guizhou Province, State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University;School of Pharmaceutical Sciences, Guizhou Medical University;Engineering Research Center for the Development and Application of Ethnic Medicine and Traditional

DOI: 10.19540/j.cnki.cjcmm.20220506.401

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