基于高通量测序探析2型糖尿病腹泻患者不同证型肠道菌群的特征

Changes of Intestinal Microbiota in Type 2 Diabetic Diarrhea Patients with Different Syndromes Based on High-throughput Sequencing

目的:探讨湿热证、脾肾两虚证2型糖尿病腹泻(T2DD)患者肠道菌群的群落结构变化。方法:选择2020年3月至2021年5月就诊于陕西中医药大学附属医院内分泌一科的2型糖尿病(T2DM)患者14例(T2DM组),湿热证T2DD患者12例(湿热证T2DD组)、脾肾两虚证T2DD患者13例(脾肾两虚证T2DD组),以及健康体检者12例(对照组)为研究对象,比较各组患者体质量指数(BMI)、空腹血糖(FPG)、餐后2 h血糖(2 h PBG)、糖化血红蛋白(HbA1c)水平,收集粪便样本,粪便样本提取DNA建库,采用高通量16S rDNA测序技术,对4组患者肠道菌群群落组成及差异菌进行比较。结果:与健康组比较,其余各组患者FPG,2 h PBG,HbA1c水平均明显增高(P<0.05);Alpha多样性显示各组肠道菌群物种丰富度、均匀度和多样性差异无统计学意义;Beta多样性表明各组样本内部肠道菌群趋于一致,组间差异无统计学意义;在门水平下相对丰度前5的菌门分别为拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和梭杆菌门(Fusobacteria),其中拟杆菌门、厚壁菌门和变形菌门为主要优势菌门。与对照组比较,3组T2DM患者的拟杆菌门的相对丰度均出现升高的趋势,厚壁菌门有下降的趋势;脾肾两虚T2DD组放线菌门的相对丰度显著高于其余各组;湿热证T2DD组厚壁菌门、梭杆菌门相对丰度显著低于其余各组;在属水平下相对丰度前10的菌属为Phocaeicola、拟杆菌属(Bacteroides)、Pseudescherichia、普氏菌属(Prevotella)、双歧杆菌属(Bifidobacterium)、粪杆菌属(Faecalibacterium)、梭杆菌属(Fusobacterium)、罗氏菌属(Roseburia)、柠檬酸杆菌属(Citrobacter)、鲸杆菌属(Cetobacterium);LEfSe分析结果显示在属水平,糖尿病患者的普氏菌属(Prevotella)、Mediterraneibacter、副拟杆菌属(Parabacteroides)、Fusicatenibacter相对丰度显著高于对照组;拟杆菌属(Bacteroides)、萨特氏菌属(Sutterella)可能是湿热证T2DD的特征肠道菌;Faecalibacterium、Limosilactobacillus、优杆菌属(Eubacterium)、Gemmiger、Enterocloster、别样杆菌属(Alistipes)、Parasutterella、Oscillibacter可能是脾肾两虚证T2DD的特征肠道菌。结论:Bacteroides可能是湿热证T2DD的特征肠道菌,Parasutterella可能是脾肾两虚证T2DD的特征肠道菌,可为T2DD湿热证和脾肾两虚证患者的中医现代化机制研究、诊断及治疗提供参考。

Objective:To investigate the changes of intestinal microbiota in type 2 diabetic diarrhea(T2DD)patients with dampness-heat syndrome and spleen-kidney deficiency syndrome.Method:T2DD patients who were admitted to the Department of EndocrinologyⅠof the Affiliated Hospital of Shaanxi University of Chinese Medicine from March 2020 to May 2021 were selected,including 14 patients with type 2diabetes mellitus(T2DM),12 T2DD patients with dampness-heat syndrome,and 13 T2DD patients with spleenkidney deficiency syndrome.Twelve healthy subjects receiving medical examination were selected as control group.Their body mass index(BMI),fasting plasma glucose(FPG),2 h postprandial blood glucose(2 h PBG)and glycosylated hemoglobin(HbA1c)were compared.Fecal samples were collected for DNA extraction to build a database.High-throughput 16S rDNA sequencing was used to compare the composition of intestinal microbiota and the differential bacteria among the four groups.Result:Compared with the conditions in control group,the levels of FPG,2 h PBG and HbA1c in the other groups were increased(P<0.05).Alpha diversity showed no significant difference in species richness,evenness and diversity of intestinal microbiota among the groups.Beta diversity indicated that intestinal microbiota tended to be consistent in each group,and there was no marked difference between groups.The top 5 phylum by relative abundance were Bacteroidetes,Proteobacteria,Firmicutes,Actinobacteria,and Fusobacteria,among which,Bacteroidetes,Proteobacteria and Firmicutes were dominant.Compared with the control group,the three diabetic groups had elevated relative abundance of Bacteroidetes while decreased relative abundance of Firmicutes.The relative abundance of Actinomycetes in spleen-kidney deficiency T2DD group was significantly higher than that in the other groups,and the relative abundance of Firmicutes and Fusobacteria in the dampness-heat T2DD group was significantly lower than that in the other groups.At the genus level,the top 10 bacteria by relative abundance were Phocaeicola,Bacteroides,Pseudescherichia,Prevotella,Bifidobacterium,Faecalibacterium,Fusobacterium,Roseburia,Citrobacter,and Cetobacterium.LEfSe analysis revealed that the relative abundance of Prevotella,Mediterraneibacter,Parabacteroides,and Fusicatenibacter in diabetic patients was remarkably higher than that in healthy patients.Bacteroides and Sutterella might be the characteristic microbiota of T2DD patients with dampness-heat syndrome,while Faecalibacterium,Limosilactobacillus,Eubacterium,Gemmiger,Enterocloster,Alistipes,Parasutterella and Oscillibacter might be the characteristic microbiota of T2DD patients with spleen-kidney deficiency syndrome.Conclusion:Bacteroides and Parasutterella might be the characteristic microbiota of T2DD patients with dampness-heat syndrome and spleen-kidney deficiency syndrome,respectively.This paper provided reference for studying the mechanism,diagnosis and treatment of modern traditional Chinese medicine for T2DD of dampness-heat type and spleen-kidney deficiency type.