基于高通量测序技术的2型糖尿病湿热困脾证患者肠道菌群结构特点与功能差异的研究

Study on Structural Characteristics and Functional Differences of Intestinal Flora in Patients with Type 2 Diabetes Mellitus of Dampness Heat Trapping Spleen Syndrome Based on High-Throughput Sequencing Technology

目的探讨2型糖尿病湿热困脾证与健康人群肠道菌群结构差异及功能变化。方法选择2019年1月—2020年1月就诊于南京市中医院2型糖尿病湿热困脾证患者30例,另选择同期健康体检者30例作为对照组,通过16Sr DNA高通量测序技术,获得样本的生物学信息,并进行两组间物种及功能注释的分析比较。结果两组共有OTUs为433个,组间比较对照组有83个,湿热困脾证有96个;门水平下,湿热困脾证患者拟杆菌门、变形门的相对丰度显著上升;纲水平下,湿热困脾证患者拟杆菌纲、β-变形菌纲的相对丰度显著上升;目水平下,湿热困脾证患者拟杆菌目、伯克氏菌目、脱硫弧菌目、食物谷菌目的相对丰度较对照组显著上升;科水平下,普氏菌科、产碱菌科、脱硫弧菌科在湿热困脾证患者中的相对丰度上升;属水平下湿热困脾证患者普氏菌属、梭杆菌属、萨特菌属相对丰度较对照组显著上升。基于NMDS、ANOSIM的β多样性分析和以shannon、simpson指数代表的α多样性分析,提示与正常人群相比,湿热困脾证患者肠道菌群多样性存在显著差异(P<0.01,或P<0.05);LEfSe分析可用以筛选湿热困脾证显著高丰度关键标志物为变形菌门、梭杆菌属、拟杆菌门、普雷沃氏菌科、普雷沃菌属等7种菌群;在KEGG数据库富集到湿热困脾证患者涉及的主要代谢通路有9个(P<0.05,或P<0.01),包括丙氨酸、天冬氨酸、谷氨酸代谢、丁酸代谢、三羧酸循环通路、脂肪酸生物合成、叶酸合成、嘌呤代谢、脂多糖生物合成等通路。结论与健康人群相比,2型糖尿病湿热困脾患者在肠道菌群多样性、菌群组成结构上及功能变异等方面存在显著差异,可为2型糖尿病中医辨证提供参考。

Objective To investigate the structural differences and functional changes of intestinal microflora between type 2 diabetes mellitus(T2DM)with dampness heat trapping spleen syndrome and healthy people.Methods From January 2019 to January 2020,30 patients with dampness heat trapping spleen syndrome of type 2 diabetes mellitus in Nanjing hospital of traditional Chinese medicine were selected,and another 30 healthy people were selected as the control group.The biological information of the samples was obtained by 16sr DNA high-throughput sequencing technology,and the species and function annotation between the two groups were analyzed and compared.Results There were 433 OTUs in the two groups,83 in the control group and 96 in the damp heat syndrome;At the portal level,the relative abundance of Bacteroides and Deformable hilum increased significantly in patients with spleen dampness heat syndrome;At the level of class,Bacteroides and Betaproteobacteria relative abundance of proteus increased significantly;Compared with the control group,the relative abundance of Bacteroides,Burkholderia,Desulfovibrionales and Victivallales in patients with dampness heat syndrome increased significantly;At the family level,the relative abundance of Prevotellaceae,Alcaligenes and Desulfovibrionaceae increased in patients with damp heat syndrome;The relative abundance of Prevotella,Fusobacterium and Sutterella in patients with spleen dampness heat syndrome was significantly higher than that in the control group.Design and implementation of network management system based on NMDS and anosimβdiversity analysis and the index of Shannon and Simpsonαdiversity analysis showed that there was a significant difference in intestinal flora diversity between patients with damp heat syndrome and normal people(P<0.01,or P<0.05);LEfSe analysis can be used to screen 7 bacterial groups with high abundance of dampness heat syndrome,including Proteobacteria,Fusobacterium,Bacteroides,Prevotellaceae and Prevotella etc;In KEGG database,there were 9 major metabolic pathways involved in patients with dampness heat syndrome(P<0.05,or P<0.01),including alanine,aspartic acid and glutamic acid metabolism,butyric acid metabolism and tricarboxylic acid cycle pathway,fatty acid biosynthesis,folate biosynthesis,purine metabolism,lipopolysaccharide biosynthesis and so on.Conclusion Compared with healthy people,there are significant differences in intestinal flora diversity,flora composition and functional variation in type 2 diabetes patients with dampness heat trapping spleen syndrome,which can provide reference for TCM differentiation of type 2 diabetes.