Metabolic characteristics of Qi-Yin deficiency and heat stagnation in liver meridian patterns of dry eye based on tear metabolomics

基于泪液代谢组学研究气阴两虚型与肝经郁热型干眼的代谢特征

Objective To explore the metabolic differences between dry eye patients with Qi-Yin defi-ciency and heat stagnation in liver meridian patterns,and clarify their metabolic characteris-tics.Methods Patients with dry eye who were treated in the Ophthalmology Ward and Outpatient Department of the First Hospital of Hunan University of Chinese Medicine from October 1,2020,to October 30,2021 were enrolled as the research participants in the study.They were assigned to two groups based on traditional Chinese medicine(TCM)syndrome types:heat stagnation in liver meridian pattern group and Qi-Yin deficiency pattern group.Healthy vol-unteers who underwent health check-ups in the Health Management Department were in-cluded as healthy group following the random number table method.The tears of the pa-tients and the healthy volunteer participants were tested by high-performance liquid chro-matography-mass spectrometry(LC-MS).The differential metabolites were screened out by multivariate statistical analysis,and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment was performed on the differential metabolites.Finally,the association analysis of differential proteins and metabolites was conducted to verify and supplement the metabolites.Results A total of 32 dry eye patients were enrolled,including 16 cases with heat stagnation in liver meridian pattern and 16 cases with Qi-Yin deficiency pattern.Fourteen healthy volun-teers were included as healthy group.There were no significant differences in baseline char-acteristics among the three groups(P>0.05).A total of 412 biomarkers were determined in Qi-Yin deficiency pattern group,mainly including lipids,lipid-like molecules,organic acids and their derivatives,organic heterocyclic compounds,and nucleosides and their analogues.For heat stagnation in liver meridian pattern group,112 metabolites were determined,main-ly including organic acids and their derivatives,lipids,and lipid-like molecules.The KEGG enrichment results of pathways and the relative content analysis of differential markers de-monstrate that purine metabolism and caffeine metabolism pathways are common metabol-ic characteristics of all dry eyes.Among them,deoxyinosine monophosphate(dIMP)and 2-(formamido)-N1-(5-phospha-D-ribosyl)acetamidine can serve as their biomarkers.The main characteristics of Qi-Yin deficiency syndrome pattern were the significant enhancement of metabolic pathways such as lysine degradation,ovarian steroidogenesis,cholesterol metabolism,pyrimidine metabolism,and bile secretion(P<0.05).Dry eye associated with the heat stagnation in liver meridian pattern is mainly characterized by inhibition of the valine,leucine,and isoleucine biosynthesis pathways(P<0.05).Conclusion Metabolomics can be used as an effective basis for TCM syndrome classification.Different patterns of dry eye syndrome exhibit typical characteristics in the types and concen-trations of metabolites,which correspond to the syndrome classification in TCM.This study initially confirms the rationality of TCM syndrome classification and provides significant ref-erence for the mechanism of dry eye and drug development.

目的探究肝经郁热型和气阴两虚型干眼患者的代谢差异,明确两者的代谢特征。方法选取2020年10月1日至2021年10月30日于湖南中医药大学第一附属医院眼科病房及门诊就诊的干眼患者为研究对象,依据中医证型分为肝经郁热型组和气阴两虚型组;按照随机数字表法纳入健康管理科体检的健康志愿者为健康组。采用高分辨率液相色谱-质谱技术(LC-MS)分别对肝经郁热型和气阴两虚型干眼患者及健康志愿者泪液进行检测,通过多元统计分析方法筛选出差异代谢物,并对差异的代谢物进行京都基因与基因组百科全书(KEGG)通路富集,最后对差异蛋白和代谢物进行关联分析,以验证和补充代谢物。结果研究最终共纳入干眼症患者32例,其中肝经郁热型16例,气阴两虚型16例;14名健康志愿者作为健康组。三组患者基线特征比较差异无统计学意义(P>0.05)。气阴两虚型组共确认了412种标志物,主要包括脂质及脂质样分子、有机酸及其衍生物、有机杂环化合物、核苷、核苷酸和类似物;肝经郁热型组共确认了112种代谢物,主要包括有机酸及衍生物和脂质及脂质样分子;结合KEGG通路富集结果和差异标志物的相对含量分析可得,嘌呤代谢及咖啡因代谢途径是所有干眼的共有代谢特征。其中脱氧肌苷酸(dIMP)和2-甲酰胺基-N1-(5-磷酸-D-核糖基)乙脒可作为其生物标志物。而气阴两虚型中以赖氨酸降解、卵巢类固醇生成、胆固醇代谢、嘧啶代谢和胆汁的分泌等代谢途径的显著增强为主要特征(P<0.05)。肝经郁热型干眼以缬氨酸、亮氨酸和异亮氨酸途径的抑制为主要特征(P<0.05)。结论代谢组学可以作为中医证型分类的有效依据,不同证型干眼症在代谢物的种类和含量上均具有典型的特征,且与中医的证候分型的表型特征相对应。本研究初步证实了中医证型分类的合理性,并对干眼病机制和药物开发提供了重要参考。