柴胡皂苷D对小鼠乳腺癌治疗过程中的药物代谢影响研究

Metabolomics of Mice Serum Analysis to Assess the Effects of Saikosaponin D on Breast Cancer

本工作建立了一种代谢组学方法用于研究柴胡皂苷D(SsD)对乳腺癌的治疗作用及其治疗过程中药物作用对机体代谢物产生的影响。采用不同浓度的SsD对小鼠乳腺癌肿瘤进行治疗,SsD给药不同天数后,采集小鼠血液,提取血清,进行超高效液相色谱-串联静电场轨道阱质谱(UPLC-Orbitrap MS)测定;筛查对照组、SsD低和高剂量给药组不同给药天数后小鼠血清内的代谢物变化,并对其进行定性分析。实验结果表明,随给药天数的增加,对照组、SsD低和高剂量给药组的组内和组间均有明显的差异代谢物变化。对代谢物变化进行筛查,组内筛查获得12个目标代谢物,这12个代谢物在肿瘤生长过程中趋势变化显著,可为肿瘤发展演化研究提供理论参考;组间筛查获得7个显著变化的差异代谢物离子,经鉴定分别为3-亚甲基吲哚、苯乙胺、吡哌酸、L-犬尿氨酸、吲哚乙醛、5-乙酰氨基-6-氨基-3-甲基尿嘧啶和脯氨酸甜菜碱,前5种化合物离子与组内筛查所得的差异离子重合,且不同SsD浓度对代谢产物种类存在影响。

The implementation of targeted therapies for breast cancer has been a challenge.Saikosaponin D(SsD),a triterpene saponin derived from Bupleurum,exhibits potential therapeutic properties for cancer therapy.Here,the role of SsD in broad anti-proliferation effects in breast cancer was investigated,and the SsD pharmaceutical efficacy was evaluated by metabolomics analysis.Low(0.4 mg/kg)and high(2.0 mg/kg)concentrations of SsDs were used to treat breast cancer tumors in mice by intraperitoneal injection.Blood samples of mice were collected and the serum samples were extracted after SsD administration for different days.Subsequently,the serum samples were analyzed by ultra-high performance liquid chromatography tandem Orbitrap mass spectrometry(UPLC-Orbitrap MS).According to the“80%rule”,UPLC-Orbitrap MS data were processed and proper ions were screened out for further analysis.Statistical methods including one-way analysis of variance(One-way ANOVA),principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)were performed to cross validate the reasonable and accuracy of the models.Representational metabolite ions present significant differences acquired by comparison within/between control group,SsD low concentration group and SsD high concentration group.The results of animal survival experiments demonstrated that SsD had obvious therapeutic effect on tumor.Mice treated with low and high concentrations of SsD showed 60%and 80%survival rate,respectively,with a significant increase in survival compared to untreated control mice.Metabonomic results showed that 12 target metabolite ions including pipecolic acid,N-acryloylglycine,3-methyleneindolenine,L-kynurenine,phenylethylamine,DL-2-aminooctanoic acid,N(6)-methyllysine,indoleacetaldehyde,3-methylhistamine,hexanoylglycine,hypoxanthine and hexanoylcarnitine were obtained by intra-group(in control group)screening.The signal intensities of these 12 ions changed significantly in the process of tumor growth,which provided theoretical basis for the study of tumor development and evolution.Meanwhile,7 important metabolite ions including 3-methylene-indolenine,phenylethylamine,pipecolic acid,L-kynurenine,indoleacetaldehyde,5-acetylamino-6-formylamino-3-methyluracil and proline betaine were obtained by analyzing inter-group data(compared among three groups),and the first 5 metabolite ions were overlapped with the ions in the list of intra-group screening.Moreover,the biological significances of the selected ions with significant difference were investigated,and two highly correlated manifested metabolic pathways including amino acid metabolism and nucleotide metabolism were discussed.Overall,the results derived from intra-group metabolomic analysis were highly correlated with the inter-group results,and the metabolites along the relevant pathways deserve further attention.This study provides a practical strategy for targeting metabolic analysis using natural product to improve the survival of patients with breast cancer.