基于非靶向代谢组学的缺血性脑卒中生物标志物的分析

doi:10.3724/SP.J.1123.2024.02015

色谱 ›› 2025, Vol. 43 ›› Issue (2): 139-147.DOI: 10.3724/SP.J.1123.2024.02015

基于非靶向代谢组学的缺血性脑卒中生物标志物的分析

徐菲³, 刘天平³, 关雅今³, 郝伟超⁴, 温鼎声⁵, 谢水林²^,^*(), 别亚男¹^,^*()

1.广东药科大学基础医学院, 广东广州 510006
2.华南理工大学生物科学与工程学院, 广东广州 510006
3.广东明珠生物技术有限公司, 广东佛山 528500
4.广东药科大学附属第一医院肿瘤科, 广东广州 510062
5.广东药科大学药学院, 广东广州 510006

收稿日期:2024-02-21 出版日期:2025-02-08 发布日期:2025-01-23
通讯作者: *E-mail:bieyanan0624@outlook.com(别亚男);
E-mail:xsl@scut.edu.cn(谢水林).
基金资助:
广东省基础与应用基础研究基金项目(2022A1515110078);广州市基础与应用基础研究项目(202201010358);广州市基础与应用基础研究项目(202201010139);佛山市高新区高技术产业化与创业团队项目(2120197000014);国家自然科学基金项目(82203847);国家自然科学基金项目(82301476)

Analysis of ischemic stroke biomarkers based on non-targeted metabolomics

XU Fei³, LIU Tianping³, GUAN Yajin³, HAO Weichao⁴, WEN Dingsheng⁵, XIE Shuilin²^,^*(), BIE Yanan¹^,^*()

1. School of Basic Medical Sciences, Guangdong Pharmaceutical University, Guangzhou 510006, China
2. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
3. Guangdong Mingzhu Biotechnology Co., Ltd., Foshan 528500, China
4. Department of Oncology, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou 510062, China
5. College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China

Received:2024-02-21 Online:2025-02-08 Published:2025-01-23
Supported by:
Guangdong Basic and Applied Basic Research Fund Project(2022A1515110078);Basic and Applied Basic Research Project of Guangzhou(202201010358);Basic and Applied Basic Research Project of Guangzhou(202201010139);High Tech Industrialization and Entrepreneurship Team Project Funding of Gaoxin District, Foshan City(2120197000014);National Natural Science Foundation of China(82203847);National Natural Science Foundation of China(82301476)

摘要/Abstract

摘要：

缺血性脑卒中(ischemic stroke, IS)生物标志物的探索仍未满足临床需求。本研究应用非靶向代谢组学方法,探索IS后血浆和脑组织中的差异代谢物和代谢通路,寻找新的潜在生物标志物和治疗靶点。将12头西藏小型猪随机分为模型组和假手术组,采用额颞入路电凝闭塞大脑中动脉构建缺血性脑卒中模型。术后36 h收集血浆及脑组织样本,采用液相色谱-质谱联用技术进行检测,在p<0.05的条件下进行主成分分析与偏最小二乘判别分析,筛选差异代谢物并剔除外源性代谢物,最后进行HMDB(Human Metabolome Database)化合物分类、KEGG通路分析及VIP分析。血浆代谢组学结果显示,86个差异代谢物上调,149个下调,显著差异代谢产物包括(Z)-3-氧代-2-(2-戊烯基)-1-环戊乙酸、反式肉桂酸和肉桂酰甘氨酸。脑组织代谢组学结果显示58个差异代谢物上调,53个下调,显著差异代谢物包括2,3-二氢黄酮-3-醇、胍基乙酸(GAA)、N-乙酰-D-色氨酸、氧化型谷胱甘肽、2-羟基喹啉和N-乙酰-L-天冬氨酸(NAA)。在血浆和脑组织中,前五类化合物中共同化合物类别为有机酸及其衍生物、脂质和脂质样分子、有机杂环化合物以及有机氧化合物。血浆和脑组织的共同代谢通路涉及氨基酸代谢、消化系统、癌症概述和脂类代谢通路。(Z)-3-氧代-2-(2-戊烯基)-1-环戊乙酸、GAA、氧化型谷胱甘肽及NAA可能是潜在的生物标志物。本研究为IS的早期筛查和临床治疗方法的开发提供了理论基础。

关键词: 缺血性脑卒中, 非靶向代谢组学, 生物标志物, 血浆, 脑组织

Abstract:

Biomarkers for ischemic stroke (IS) are yet to fulfill clinical requirements. This study used non-targeted metabolomics to investigate differential metabolites and metabolic pathways in plasma and brain tissue following IS, with the aim of identifying new potential biomarkers and therapeutic targets. Twelve Tibetan miniature pigs were randomly assigned to a model- or sham-operation group. An electrocoagulation-based anterior temporal approach was employed to occlude the middle cerebral artery, thereby creating a model for IS. Plasma and brain tissue samples were collected 36 h post-surgery and analyzed using liquid chromatography-mass spectrometry. Principal component and partial least squares discriminant analyses were used to screen for differential metabolites and exclude exogenous metabolites at p<0.05. Compounds were classified according to the HMDB (Human Metabolome Database), and subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway and VIP (variable importance in projection) analyses. Plasma metabolomics revealed that 86 metabolites were upregulated while 149 were downregulated, with (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentylacetic acid, trans-cinnamic acid and cinnamoylglycine determined to be significant metabolites. Fifty-eight differential metabolites were upregulated in brain tissue and 53 were downregulated, with 2,3-dihydroflavon-3-ol, guanidinoacetic acid (GAA), N-acetyl-D-tryptophan, oxidized glutathione, 2-hydroxyquinoline, and N-acetyl-L-aspartate (NAA) identified as significant metabolites. Organic acids and derivatives, lipids and lipid-like molecules, organoheterocyclic compounds, and organic oxygen compounds were found to be common compound categories among the top five types of compound in both plasma and brain tissue. Common metabolic pathways in plasma and brain tissue include amino acid metabolism, digestive system, cancer overview, and lipid metabolism pathways, with the (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentylacetic acid, GAA, oxidized glutathione, and NAA metabolites serving as potential biomarkers. This study provides a theoretical foundation for the early screening and development of clinical treatment strategies for IS.

Key words: ischemic stroke (IS), non-target metabolomics, biomarkers, plasma, brain tissue

中图分类号:

O658

徐菲, 刘天平, 关雅今, 郝伟超, 温鼎声, 谢水林, 别亚男. 基于非靶向代谢组学的缺血性脑卒中生物标志物的分析[J]. 色谱, 2025, 43(2): 139-147.

XU Fei, LIU Tianping, GUAN Yajin, HAO Weichao, WEN Dingsheng, XIE Shuilin, BIE Yanan. Analysis of ischemic stroke biomarkers based on non-targeted metabolomics[J]. Chinese Journal of Chromatography, 2025, 43(2): 139-147.

图/表 6

图 1 MCAO与Sham组猪脑大体观察与右脑HE染色图

Fig. 1 Gross anatomical observation and hematoxylin-eosin (HE) staining of the right hemisphere in the MCAO and Sham groups a. gross anatomical observation of the brain; b. HE staining of ischaemic stroke injury. Sham: the sham operation group; MCAO: the middle cerebral artery occlusion model group.

图 2 MCAO与Sham组的PCA与PLS-DA分析

Fig. 2 Analysis of PCA and PLS-DA of MCAO and Sham groups a. PCA score plot for plasma; b. PLS-DA analysis for plasma; c. permutation testing for plasma, where the horizontal axis shows the permutation retention of the test (the dashed line parallel to the horizontal axis indicates the zero scale line), and the vertical axis displays the values of the R2 and Q2 permutation tests, and the two dashed lines represent the regression lines of R2 and Q2, respectively (excluding those outside the zero scale line); d. PLS-DA analysis for brain tissue; e. permutation testing for brain tissue. MCAOR: the right hemisphere of the MCAO group; ShamR: the right hemisphere of the Sham group.

图 3 MCAO与Sham组的差异代谢火山图

Fig. 3 Volcano plots of differential metabolism in MCAO and Sham groups a. plasma; b. bain tissue. up: upregulated; nosig: not significantly different; down: downregulated.

图 4 血浆和脑组织的HMDB化合物分类(super class)

Fig. 4 HMDB compound classification at the super class level a. plasma; b. brain tissue.

图 5 差异代谢物的KEGG功能通路

Fig. 5 KEGG functional pathway of differential metabolites a. plasma; b. brain tissue.

图 6 差异代谢物的聚类热图和VIP条形图

Fig. 6 Differential metabolite clustering heat maps and VIP bar charts a. plasma; b. brain tissue. C: Sham group; M: MCAO group. C19H37N5O5: N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-t-butyl-alanyl-L-alanine, 2-aminoethyl amide; C21H18O11: (2S,3S,4S,5R)-3,4,5-trihydroxy-6-(5-hydroxy-3-(4-hydroxyphenyl)-4-oxochromen-7-yl)oxyoxane-2-carboxylic acid; C16H29N3O4: ethyl-3-((5-methyl-4-oxo-1,5-diazacycloundecane-1-carbonyl)amino)propanoate; C25H20Cl2N2O3: (4-chlorophenyl) (1R)-6-chloro-1-(4-methoxyphenyl)-1,3,4,9-tetrahydropyrido(3,4-b)indole-2-carboxylate.

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基于非靶向代谢组学的缺血性脑卒中生物标志物的分析

Analysis of ischemic stroke biomarkers based on non-targeted metabolomics

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