基于GC-MS技术的脑缺血再灌注损伤大鼠脑组织与血清代谢谱研究

张春兵; 李鹏飞; 朱小飞; 高峰

基于GC-MS技术的脑缺血再灌注损伤大鼠脑组织与血清代谢谱研究

南京中医药大学附属医院，江苏南京 210029

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出版历程
- 刊出日期: 2018-11-10

GC-MS-Based Technique Identifies Metabolite Profile of Brain Tissues and Serum from Brain Ischemia/Reperfusion Injury Rats

摘要

摘要: 目的探讨脑缺血再灌注损伤大鼠脑组织和血清中差异表达的代谢物。方法采用Zea Longa改良栓线法构建大鼠脑中动脉栓塞（MCAO）模型，收集脑组织和血清，采用气相色谱-质谱（GC-MS）方法检测代谢物表达谱。利用OmicShare工具对代谢物进行聚类分析。运用主成分分析法（PCA）、最小二乘法-判别分析法（PLS-DA）结合t检验筛选得到差异表达的代谢物。进一步利用Metabo Analyst数据库对差异表达的代谢物进行通路分析。结果总共鉴定出105个血清代谢物以及95个脑组织代谢物。PLS-DA分析表明，假手术组和MCAO模型组血清和脑组织区分较好。假手术组相比，MCAO大鼠血清中尿素、缬氨酸、亮氨酸、异亮氨酸、甘露糖、尿素、苯丙氨酸以及蛋氨酸代谢物显著升高（P<0.05），与脯氨酸、丙氨酸、谷氨酸、肌醇、丝氨酸、丙三醇、亚油酸以及4-羟基脯氨酸显著降低（P<0.05）。MCAO模型大鼠脑组织丙氨酸、乳酸、尿素、亮氨酸、脯氨酸、缬氨酸、2-羟基异丁酸、异亮氨酸显著升高（P<0.05）。代谢通路分析结果表明，这些差异代谢物可参与部分氨基酸合成及代谢通路、亚油酸代谢通路、甲烷代谢通路、甘油酯类代谢通路及氨酰-tRNA生物合成等。结论脑缺血再灌注损伤脑组织与血清中存在差异表达的代谢物，可能参与脑卒中发病机制，为研发脑卒中的早期诊断标志物提供依据。
- 代谢组学 /
- 脑缺血再灌注损伤 /
- 缺血性脑卒中 /
- 生物标志物
Abstract: OBJECTIVE In the present study， we aimed to investigate the metabolite changes in the brain tissues and serum from middle cerebral artery occlusion (MCAO) model rats using gas chromatography-mass spectrometry (GC-MS)-based metabolomics technique. METHODS The transient model of middle cerebral artery occlusion (MCAO) rats was established using Zea Longa thread embolism methods. GC-MS-based metabolomics technique were performed to investigate the metabolites in brain tissues and serum from MCAO and Sham group rats. OmicShare tools were to perform the heatmap and hierarchical cluster analysis of the metabolites. Principal component analysis (PCA) were used to detect the difference between both group rats. A combination of VIP value of Partial least squares discriminant analysis (PLS-DA) with p value of t test were to screen differentially expressed metabolites. The pathway database MetaboAnalyst were applied to analyse the pathway that these differentially expressed metabolites participated in. RESULTS We identified and quantified 95 metabolites in brain tissues and 105 in serum， respectively. Compared with the sham group， the concentrations of urea， valine， leucine， isoleucine， mannose， urea， phenylalanine， and methionine in serum from MCAO group rats were significantly increased（P<0.05）. However， the concentrations of proline， alanine， glutamic acid， myo-inositol， serine， glycerol， linoleic acid， and 4-hydroxyproline were markedly decreased（P<0.05）. In the brain tissues from MCAO group rats， the concentrations of alanine， lactic acid， urea， leucine， proline， valine， 2-hydroxyisobutyric acid， and isoleucine were significantly increased（P<0.05）. The metabolic pathway results showed that these metabolites were associated wih some amino acid biosynthesis and metabolism， linoleic acid metabolism， methane metabolism， glycerolipid metabolism， and aminoacyl-tRNA biosynthesis， etc. CONCLUSION Our findings demonstrate the presence of characteristic changes in metabolites in the brain tissues and serum from MCAO rats and highlights the utility of metabolomics in identifying elements of stroke disease pathogenesis and for the development of early diagnostic biomarker of the disease.
- metabolomics /
- cerebral ischemia reperfusion injury /
- ischemic stroke /
- biomarker

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参考文献(20)

[1]	ADAMSKI MG， LI Y， WAGNER E， et al. Expression profile based gene clusters for ischemic stroke detection [J]. Genomics， 2014， 104(3):163-169.
[2]	LI P， TENG F， GAO F， et al. Identification of circulating microRNAs as potential biomarkers for detecting acute ischemic stroke [J]. Cell Mol Neurobiol， 2015， 35(3):433-447.
[3]	MISRA S， KUMAR A， KUMAR P， et al. Blood-based protein biomarkers for stroke differentiation: A systematic review [J]. Proteomics Clin Applicat， 2017，11(9/10):1700007.
[4]	GRAHAM SF， REY NL， YILMAZ A， et al. Biochemical profiling of the brain and blood metabolome in a mouse model of prodromal Parkinson's disease reveal distinct metabolic profiles [J]. J Proteome Res， 2018， 17(7):2460-2469.
[5]	QURESHI MI， VORKAS PA， COUPLAND AP， et al. Lessons from metabonomics on the neurobiology of stroke [J]. Neuroscientist， 2016， 23(4):374-382.
[6]	WANG D， KONG J， WU J， et al. GC-MS-based metabolomics identifies an amino acid signature of acute ischemic stroke [J]. Neurosci Lett， 2017， 642:7-13.
[7]	WANG Y， WANG YG， MA TF， et al. Dynamic metabolites profile of cerebral ischemia/reperfusion revealed by ¹H NMR-based metabolomics contributes to potential biomarkers [J]. Int J Clin Exp Pathol， 2014，7(7): 4067-4075.
[8]	WANG Y， WANG Y， LI M， et al. ¹H NMR-based metabolomics exploring biomarkers in rat cerebrospinal fluid after cerebral ischemia/reperfusion[J]. Mol Biosyst， 2013， 9 (3):431-439.
[9]	LI P， SHEN M， GAO F， et al. An antagomir to microRNA-106b-5p ameliorates cerebral ischemia and reperfusion injury in rats via inhibiting apoptosis and oxidative stress [J]. Mol Neurobiol， 2017， 54(4):2901-2921.
[10]	孙润彬，俞晓忆，毛勇，等. 昆布-甘草合用对SD 大鼠基础代谢干扰的代谢组学研究 [J]. 药学学报， 2015， 50 (3): 312-318.
[11]	CLOONAN L， FITZPATRICK KM， KANAKIS AS， et al. Metabolic determinants of white matter hyperintensity burden in patients with ischemic stroke [J]. Atherosclerosis， 2015， 240(1):149-153.
[12]	LIU P， LI R， ANTONOV AA， et al. Discovery of metabolite biomarkers for acute ischemic stroke progression [J]. J Proteome Res， 2017， 16(2):773-779.
[13]	ORMSTAD H， VERKERK R， SANDVIK L， et al. Serum phenylalanine， tyrosine， and their ratio in acute ischemic stroke: on the trail of a biomarker?[J]. J Mol Neurosci， 2016， 58(1):102-108.
[14]	HUANG Q， LI C， XIA N， et al. Neurochemical changes in unilateral cerebral hemisphere during the subacute stage of focal cerebral ischemia-reperfusion in rats: An ex vivo ¹H magnetic resonance spectroscopy study [J]. Brain Res， 2018， 1684:67-74.
[15]	RUIZ-CANELA M， TOLEDO E， CLISH CB， et al. Plasma branched-chain amino acids and incident cardiovascular disease in the predimed trial [J]. Clin Chem， 2016， 62(4):582-592.
[16]	KIMBERLY WT， WANG Y， PHAM L， et al. Metabolite profiling identifies a branched chain amino acid signature in acute cardioembolic stroke [J]. Stroke， 2013， 44 (5)， 1389-1395.
[17]	RUAN L， WANG Y， CHEN SC， et al. Metabolite changes in the ipsilateral and contralateral cerebral hemispheres in rats with middle cerebral artery occlusion [J]. Neur Regen Res， 2017， 12(6):931-937.
[18]	CAMBRAY S， PORTERO-OTIN M， JOVE M， et al. Metabolomic estimation of the diagnosis and onset time of permanent and transient cerebral ischemia [J]. Mol Neurobiol， 2018， 55(7):6193-6200.
[19]	ZHU Y， GUO Z， ZHANG L， et al. System-wide assembly of pathways and modules hierarchically reveal metabolic mechanism of cerebral ischemia [J]. Sci Rep， 2015， 5:17068.
[20]	IRIE M， FUJIMURA Y， YAMATO M， et al. Integrated MALDI-MS imaging and LC-MS techniques for visualizing spatiotemporal metabolomic dynamics in a rat stroke model [J]. Metabolomics， 2014， 10(3):473-483.