Characteristics and Functional Differences of Intestinal Flora in Type 2 Diabetes Mellitus Patients with Dampness Heat Trapping Spleen Syndrome and Qi-Yin Deficiency Syndrome： An Analysis Based on High-throughput Sequencing Technology

Ying-zhuo RAN; Xin SHAO; gang HU; Yuan-yuan LU; Ming LI; Bao-hua SHEN; Wen-wen KONG; Yan-hua GUAN; Xin YANG; Jia FANG; Jing LIU

doi:10.13422/j.cnki.syfjx.20220298

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Characteristics and Functional Differences of Intestinal Flora in Type 2 Diabetes Mellitus Patients with Dampness Heat Trapping Spleen Syndrome and Qi-Yin Deficiency Syndrome： An Analysis Based on High-throughput Sequencing Technology

更新时间：2021-12-14

- Characteristics and Functional Differences of Intestinal Flora in Type 2 Diabetes Mellitus Patients with Dampness Heat Trapping Spleen Syndrome and Qi-Yin Deficiency Syndrome： An Analysis Based on High-throughput Sequencing Technology
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 2, Pages: 139-146(2022)
- 作者机构：
  
  南京市中医院，南京 210012
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220298
  CLC： R22;R242;R2-031;R255.4
- Received：09 June 2021，
  
  Published Online：20 November 2021，
  
  Published：20 January 2022
- 稿件说明：
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冉颖卓,邵鑫,胡钢等.基于高通量测序技术的2型糖尿病湿热困脾证、气阴两虚证患者肠道菌群特征及功能差异[J].中国实验方剂学杂志,2022,28(02):139-146.

RAN Ying-zhuo,SHAO Xin,HU gang,et al.Characteristics and Functional Differences of Intestinal Flora in Type 2 Diabetes Mellitus Patients with Dampness Heat Trapping Spleen Syndrome and Qi-Yin Deficiency Syndrome： An Analysis Based on High-throughput Sequencing Technology[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(02):139-146.
冉颖卓,邵鑫,胡钢等.基于高通量测序技术的2型糖尿病湿热困脾证、气阴两虚证患者肠道菌群特征及功能差异[J].中国实验方剂学杂志,2022,28(02):139-146. DOI： 10.13422/j.cnki.syfjx.20220298.

RAN Ying-zhuo,SHAO Xin,HU gang,et al.Characteristics and Functional Differences of Intestinal Flora in Type 2 Diabetes Mellitus Patients with Dampness Heat Trapping Spleen Syndrome and Qi-Yin Deficiency Syndrome： An Analysis Based on High-throughput Sequencing Technology[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(02):139-146. DOI： 10.13422/j.cnki.syfjx.20220298.

摘要

目的

探讨2型糖尿病（T2DM）湿热困脾证、气阴两虚证患者肠道菌群的结构特征和功能差异。

方法

选择2018年6月至2020年1月就诊于南京中医药大学附属南京中医院的T2DM湿热困脾证患者62例，气阴两虚证患者60例，收集其血清及粪便样本，比较两证型患者体质量指数（BMI），糖脂代谢，空腹胰岛素（FINS），空腹C肽（FCP），胰岛素抵抗指数（HOMA-IR），粪便样本提取DNA建库，采用高通量16S rDNA测序技术，对2组肠道菌群及菌群代谢通路分析比较。

结果

①与气阴两虚证患者比较，湿热困脾证患者的BMI，空腹血糖（FPG），餐后2 h血糖（2 h PBG），总胆固醇（TC），甘油三酯（TG），低密底脂蛋白（LDL），FINS，FCP，HOMA-IR均明显升高，HDL明显降低（

0.05，

0.01）。②两组菌群物种组成及差异，在纲水平上，以拟杆菌纲，梭状芽胞杆菌纲，

蛋白杆菌纲等为主，气阴两虚证的梭状芽胞杆菌、柔膜菌纲、疣微菌纲的相对丰度明显高于湿热困脾证（

0.05）；目水平下以拟杆菌目、梭菌目、肠杆菌目等为主，气阴两虚证的梭菌目、丹毒丝菌目、疣微菌目的相对丰度明显高于湿热困脾证，而气单胞菌目则在湿热困脾证高于气阴两虚证（

0.05）；科水平下以拟杆菌科、普氏菌科、瘤胃球菌科等为主，其中气阴两虚证的瘤胃球菌科、紫单胞菌科、丹毒丝菌科等的相对丰度明显高于湿热困脾证（

0.05）；属水平下以拟杆菌属、普氏菌属、副拟杆菌属等为主，其中气阴两虚证的副拟杆菌属、丁酸弧菌属、瘤胃梭菌属等的相对丰度显著高于湿热困脾证，而在湿热困脾组的克雷伯菌属、巨球型菌属则高于气阴两虚证（

0.05）。③基于运算分类单位（OTU）的Venn分析发现湿热困脾证和气阴两虚证患者独有的OUT分别49，47个。④基于OTU的

多样性和

分析，结果Shannon，Simpson指数差异均具有统计学意义，Ace，Chao指数差异无统计学意义，气阴两虚证患者的肠道微生物多样性高于湿热困脾证患者（

0.05）；Anosim分析提示两组

多样性差异具有统计学意义（

0.05）。⑤线性判别效应（LEfSe）分析发现湿热困脾证可选择克雷伯菌、巨球型菌属、气单胞菌目3种菌群作为关键的生物标志物，气阴两虚证可选择瘤胃梭菌属、伯克氏菌科、劳氏菌属、丁酸弧菌属、丹毒丝菌目等14种菌群作为关键的生物标志物。⑥功能注释及分析可见湿热困脾证涉及精氨酸和脯氨酸代谢、脂多糖生物合成、烟酸和烟酰胺代谢等9个代谢通路，气阴两虚证涉及阿卡波糖和缬草霉素生物合成，胰高血糖素信号通路，NOD样受体信号通路等10个代谢通路。

结论

T2DM湿热困脾证和气阴两虚证患者存在肠道菌群明显的差异和功能差异，可作为T2DM中医辨证客观化的参考和中医药治疗的靶点。

Abstract

Objective

To explore the structural characteristics and functional differences of intestinal flora in patients with type 2 diabetes mellitus （T2DM） of dampness heat trapping spleen（DHTS） syndrome and Qi-Yin deficiency（QYD） syndrome.

Method

From June 2018 to January 2020，62 T2DM patients with DHTS syndrome and 60 with QYD syndrome were selected from Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine. Serum and fecal samples were collected to compare body mass index（BMI），glucose and lipid metabolism，fasting insulin （FINS） and fasting C-peptide （FCP） levels，and homeostasis model assessment of insulin resistance（HOMA-IR） of the two syndrome types. Fecal samples were extracted for DNA database construction，and 16S rDNA high-throughput sequencing was used to analyze and compare the intestinal flora and metabolic pathways.

Result

① The BMI，fasting plasma glucose（FPG），2-hour postprandial blood glucose （2 h PBG），total cholesterol（TC），triglyceride（TG），low density lipoprotein（LDL），FINS，FCP，and HOMA-IR were higher in patients with DHTS syndrome than in patients with QYD syndrome，and the high density lipoprotein（HDL） of the former was lower than that of the latter，（

0.05，

0.01）. ② In terms of species composition and differences，Bacteroidetes， Clostridia and Gammaproteobacteria were dominant at the class level，and the relative abundance of Clostridia，Mollicutes and Verrucomicrobiae in QYD syndrome group was higher than that in DHTS syndrome group. At the order level，Bacteroidales，Clostridiales and Enterobacteriales were mainly found. The relative abundance of Clostridiales，Erysipelotrichales and Verrucomicrobiales in QYD syndrome group was obviously higher than that in DHTS syndrome group，while Aeromonadales in the former was lower than that in the latter （

0.05）. At the family level，Bacteroidaceae，Prevotellaceae and Ruminococcaceae were predominant. The relative abundance of Ruminococcaceae，Porphyromonadaceae and Erysipelotrichaceae in QYD syndrome group was higher than that in DHTS syndrome group（

0.05）. At the genus level，

Bacteroides

，

Prevotella

and

Parabacteroide

s were mainly found. The relative abundance of

Parabacteroides

，

Butyrivibrio

and

Ruminiclostridium

in QYD syndrome group was higher than that in DHTS syndrome group，while that of

Klebsiella

and

Megasphaera

in DHTS syndrome group was higher than that in QYD syndrome group（

0.05）. ③ Through Venn analysis of operational taxonomic units（OTU），it was found that there were 49 OTUs in patients with DHTS syndrome patients and 47 OTUs in QYD syndrome patients. ④ The results of OTU

diversity and

analysis showed that Shannon and Simpson indexes had statistical differences，while Ace and Chao indexes had no statistical differences. The intestinal microbial diversity of patients with QYD syndrome was higher than that of patients with DHTS syndrome（

0.05）. The analysis of similarities （ANOSIM） showed that the difference of

diversity between the two groups was significant（

0.05）. ⑤ Linear discriminant analysis Effect Size（LEfSe） results demonstrated that

Klebsiella

，

Megasphaera

and Aeromonadales could be selected as the key biomarkers for DHTS syndrome； 14 bacteria such as

Ruminiclostridium

，Burkholderiaceae，

Lautropia

，

Butyrivibrio

，Erysipelotrichales can be selected as the key biomarkers for QYD syndrome. ⑥Functional annotation and analysis showed that the DHTS syndrome involved 9 metabolic pathways，including arginine and proline metabolism，lipopolysaccharide biosynthesis，nicotinic acid and nicotinamide metabolism，while the QYD syndrome involved 10 metabolic pathways，including acarbose and valinomycin biosynthesis，glucagon signaling pathway and NOD-like receptor signaling pathway.

Conclusion

There are obvious differences in intestinal flora and functions in T2DM patients of DHTS syndrome and QYD syndrome，which can be used as reference for traditional Chinese medicine （TCM） syndrome differentiation and the target of TCM treatment.

关键词

Keywords

references

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