半枫荷调控RA模型的非靶向代谢组学研究

Untargeted metabonomics study of Semiliquidambar cathayesis in treatment of rheumatoid arthritis

关键词:  超高效液相色谱联用四极杆飞行时间质谱, 血浆, 差异代谢物, 代谢通路, 壮药

Key words:  UPLC-QTOF/MS, plasma, differential metabolites, metabolic pathways, Zhuang medicine

摘要:

为探讨半枫荷干预类风湿性关节炎(rheumatoid arthritis, RA)模型大鼠血浆内容物代谢轮廓的变化和特征,该研究以半枫荷正丁醇提取物给药前后RA模型大鼠血浆为研究对象,借助超高效液相色谱联用四极杆飞行时间质谱(UPLC-QTOF/MS)技术进行非靶向代谢组学检测,并用SIMCA-P软件对代谢物测定结果进行多元变量统计分析,筛选差异代谢物并作通路富集分析。结果表明:(1)给药前后大鼠血浆代谢轮廓存在显著差异,与模型组相比,给药组在正负离子模式合并后筛选出321种差异代谢物,其中负离子模式鉴定到174种代谢物,正离子模式鉴定到192种代谢物。(2)鉴定到的所有代谢物根据其化学分类归属信息归为12种类型,有机酸及其衍生物和脂类及类脂分子这2类代谢物数量占比较高。(3)通路富集获得37个代谢通路且呈显著性差异(P<0.05),给药组中蛋白质的消化和吸收、肿瘤胆碱代谢通路和ABC转运蛋白通路出现较大扰动且富集到的差异代谢物数量最多,所有通路均显著上调(P<0.05)。这对阐明半枫荷调控RA症状的变化机制具有一定指导价值和理论意义。

Abstract:

In order to explore the changes and characteristics of plasma content metabolic profile in rheumatoid arthritis(RA)model rats after the intervention of effective parts of Semiliquidambar cathayesis. Based on the Ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)technique, the differences of plasma metabolite profiles in rat arthritis models before and after the administration from the perspective of nontargeted metabolomics were analysed. Chromatographic experiments were performed on a HILIC column(100 mm×2.1 mm, 1.7 μm)using a mobile phase that consisted of 25 mmol·L^-1 ammonium acetate and 25 mmol·L^-1 ammonia in water and acetonitrile. Mass spectrometry was conducted in the positive and negative modes by electrospray ionization(ESI). Metabolic information about the plasma was acquired using a multivariate statistical analysis model. Principal component analysis(PCA)and partial least square discriminant analysis(PLS-DA)were conducted for pattern recognition and difference analysis. PCA was performed for data variables in the positive and negative ion modes, respectively. The metabolic compounds were divided into different groups on the basis of their chemical taxonomy. A permutation test(n=200)was conducted to verify the fit of the model. The differential metabolites were screened on the basis of variable importance in project(VIP>1), analysis of variance(ANOVA, P<0.05)and maximum fold change(>1.5)by using the PLS-DA model. The compounds were identified based on the data retrieved from the METLIN and HMDB databases according to the quality information of percursor ions and fragment ions. Plasma metabolic profile before and after administration showed significant differences. Metabolite determination results were screened by SIMCA-P software, followed by t-test and fold change analysis, screening differential metabolites and pathway enrichment analysis. The results were as follows:(1)Compared with the model group, after the combination of positive and negative ion mode, 321 different metabolites were selected, 174 metabolites were identified in the negative ion pattern, 192 metabolites were identified in positive ion pattern.(2)All metabolites identified were classified into 12 types according to their chemical classification attribution information, organic acids and derivatives, lipids and lipid-like molecules accounted for a high number of metabolites.(3)A total of 37 metabolic pathways were obtained by pathway enrichment and showed significant difference(P<0.05), digestion and absorption of proteins, tumor choline metabolism pathways and ABC transporters enriched the largest number of differential metabolites, all pathways were significantly upregulated(P<0.05). Accordingly, a theoretical reference has been presented for the transformation mechanism of S. cathayesis regulating RA.