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

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

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。

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.