基于小分子代谢标志物探讨支气管哮喘寒热证型的辨证特点

Investigation of differences between cold and heat syndromes of bronchial asthma based on biomarker of small molecular metabolites

目的采用代谢组学方法探索支气管哮喘慢性持续期虚寒证和虚热证患者的小分子代谢物差异,揭示哮喘临床证候分型与小分子代谢物之间的相关性,寻找哮喘证型的潜在生物标志物。方法选取北京地区汉族哮喘慢性持续期患者20例(虚寒证组与虚热证组各10例),健康志愿者10例为对照组,取得受试者知情同意后,收集空腹外周抗凝血2 mL,抗凝静置,离心取血清,-80℃冰箱保存,进行气相色谱-质谱联用(GC-MS)分析,采用多维分析(O)PLS-DA和单维分析(t检验)相结合的办法,筛选组间差异代谢物(VIP>1,P<0.05)。通过MBRole与KEGG进行差异代谢物通路富集分析。结果慢性持续期哮喘患者与对照组相比,共发现40种差异代谢物;虚寒证组与虚热证组相比,共发现18种差异代谢物;既是哮喘相关小分子代谢物,又可以区分虚寒证和虚热证两种不同证型的小分子代谢物有4种,分别为:二十四碳烷、4-氨基丁酸、苯甲酰甲酸、异肌醇。经MBRole与KEGG分析哮喘虚寒证组和虚热证组差异代谢物,发现β-丙氨酸代谢通路受到扰动。结论支气管哮喘慢性持续期患者与对照组相比小分子代谢物有显著差异,哮喘虚寒证与虚热证患者小分子代谢物有显著差异,4-氨基丁酸可能是潜在鉴别支气管哮喘慢性持续期虚寒证和虚热证型的生物标记物,哮喘虚寒证组和虚热证组β-丙氨酸代谢通路受到扰动。

Objective To explore the difference in small molecular metabolites between deficiency cold syndrome and deficiency heat syndrome of chronic bronchial asthma and the correlation between the clinical syndromes and the metabolites to identity potential biomarkers that could differentiate asthma syndromes based on the metabonomics. Methods Thirty subjects(ten of deficiency cold syndrome, ten of deficiency heat syndrome, and ten healthy volunteers) were recruited in the study. With informed consent from the subjects, 2 mL of blood was collected containing anticoagulant and kept in a static and anticoagulant condition, and then serum was separated by centrifugation and stored at-80 ℃ in refrigerator. It was analyzed with GC-MS and multidimensional analysis(O) PLS-DA and one-dimensional analysis(t test) were made to identify different metabolites between the groups(VIP>1, P<0.05). Then enrichment analysis of pathways of differential metabolites was done with MBRole and KEGG. Results Forty different metabolites were found between chronic asthma patients and healthy controls, and eighteen such metabolites between patients of cold and heat deficiency syndromes. Among them, four small molecular metabolites can tell both asthma patients from healthy controls and deficiency cold syndrome from deficiency heat syndrome, i.e. tetracosane, 4-aminobutyric acid, benzoylformic acid, and allo-Inositol. MBRole and KEGG pathway analysis of the metabolites of the deficiency cold syndrome group and deficiency heat syndrome group found disturbances of pathways of beta-alanine metabolism. Conclusion The small molecular metabolites are significantly different between chronic bronchial asthma patients and healthy controls, and between patient of cold deficiency syndrome and those of heat deficiency syndrome. 4-aminobutyric acid might be the potential biomarkers to differentiate deficiency cold syndrome from deficiency heat syndrome of chronic bronchial asthma patients, and beta-alanine metabolic pathway disturbance was found between deficiency cold syndrome group and deficiency heat syndrome group.