Roux‑en‑Y胃旁路术对单纯肥胖症和肥胖症合并糖尿病患者肠道菌群的调控作用

Regulation of intestinal microbiota by Roux‑en‑Y gastric bypass on patients with obesity or obesity combined with diabetes

目的探讨Roux‑en‑Y胃旁路术(RYGB)对单纯肥胖症和肥胖症合并糖尿病患者肠道菌群的调控作用。方法采用回顾性描述性研究方法。收集2016年7月至2017年8月暨南大学附属第一医院收治的20例肥胖症(9例单纯肥胖症、11例肥胖症合并糖尿病)行RYGB患者的手术前后粪便样本和临床资料;男11例,女9例;年龄为(33±11)岁。观察指标:(1)肠道菌群的组成和结构变化。(2)单纯肥胖症患者术后肠道菌群的属水平改变情况。(3)肥胖症合并糖尿病患者术后肠道菌群的属水平改变情况。采用电话或门诊方式进行随访,了解患者的体质量、抗菌药物使用和血糖控制等情况。按统一培训要点,收集粪便样本,储存于DNA稳定剂中,45 h内行实验室分析。随访时间截至2018年11月。正态分布的计量资料以x±s表示,组间比较采用独立样本t检验,组内比较采用配对样本t检验;偏态分布的计量资料以M(Q_(1),Q_(3))表示,组间比较采用两个独立样本比较的Wilcoxon秩和检验。计数资料以绝对数表示,组间比较采用χ^(2)检验、ANOSIM分析、线性判别(LEfSe)分析和Metastats分析。结果(1)肠道菌群的组成和结构变化。单纯肥胖症患者和肥胖症合并糖尿病患者术前肠道菌群α多样性Shannon指数分别为4.37±0.69和4.47±0.85,两者比较,差异无统计学意义(t=0.28,P>0.05)。术前LEfSe分析结果显示:单纯肥胖症患者和肥胖症合并糖尿病患者术前厚壁菌门和拟杆菌门的菌属丰度存在差异,单纯肥胖症患者Parasutterella的丰度为0.0001130(0,0.0043782),肥胖症合并糖尿病患者的丰度为0.0084640(0.0013257,0.0349831);两者比较,差异有统计学意义(Z=2.12,P<0.05)。术前PCoA分析结果显示:单纯肥胖症患者和肥胖症合并糖尿病患者术前主成分1、主成分2、主成分3贡献率分别为24.98%、22.24%、16.33%;ANOSIM比较结果显示:两者术前肠道菌群差异性比较,差异无统计学意义(r=-0.11,P>0.05)。单纯肥胖症患者和肥胖症合并糖尿病患者术后肠道菌群α多样性Shannon指数分别为4.60±0.65和4.66±0.40,两者比较,差异无统计学意义(t=0.24,P>0.05)。术后LEfSe分析结果显示:单纯肥胖症患者和肥胖症合并糖尿病患者术后拟杆菌门、变形菌门和厚壁菌门的菌属丰度存在差异,单纯肥胖症患者Morganella和Coprococcus_2的丰度分别为0.0001920(0.0000119,0.0015690)和0(0,0);肥胖症合并糖尿病患者上述指标分别为0(0,0)和0.0000541(0,0.0004190);两者比较,差异均有统计学意义(Z=2.70,2.29,P<0.05)。(2)单纯肥胖症患者术后肠道菌群的属水平改变情况。单纯肥胖症患者术后肠道菌群10个菌属发生变化,其中术后增加菌属7种:Veillonella、Morganella、Granulicatella、Aeromonas、Streptococcus、Rothia和Megasphaera,属于厚壁菌门和变形菌门;术后减少的菌属为Ruminococcus_torques_group、Romboutsia和Erysipelotrichaceae_UCG‑003,属于厚壁菌门和放线菌门。LEfSe分析结果显示:单纯肥胖症患者术前显著富集的菌属为Ruminococcus_torques_group、Romboutsia、Erysipelotrichaceae_UCG‑003,属于厚壁菌门;术后显著富集的菌属为Rothia、Granulicatella、Enterococcus、Streptococcus、Megasphaera、Veillonella、Aeromonas、Morganella,属于放线菌门、厚壁菌门和变形菌门。(3)肥胖症合并糖尿病患者术后肠道菌群的属水平改变情况。肥胖症合并糖尿病患者术后的肠道菌群16个菌属水平增加:Streptococcus、Veillonella、Haemophilus、Pluralibacter、Gemella、Lachnospiraceae_NC2004_group、Granulicatella、Aeromonas、uncultured_bacterium_f_Saccharimonadaceae、Ruminiclostridium_9、Butyricicoccus、Fusobacterium、Anaerotruncus、Fusicatenibacter、Klebsiella和Eubacterium_eligens_group,属于厚壁菌门和变形菌门。LEfSe分析结果显示:肥胖症合并糖尿病患者术前显著富集的菌属为Fusicatenibacter、Tyzzerella_3、Butyricicoccus,属于厚壁菌门;术后显著富集的菌属的Gemella、Granulicatella、Enterococcus、Streptococcus、Lachnospiraceae_NC2004_group、Eubacterium_eligens_group、Anaerotruncus、Ruminiclostridium_9、Anaeroglobus、Veillonella、Fusobacterium、uncultured_bacterium_f_Saccharimonadaceae、Aeromonas、Klebsiella、Pluralibacter、Proteus、Haemophilus,属于变形菌门和厚壁菌门。结论RYGB可以显著提高单纯肥胖症和肥胖症合并糖尿病患者的肠道菌群丰度;在属水平,两类患者各有特异性改变的肠道菌群。

Objective To investigate the regulation of intestinal microbiota by Roux-en-Y gastric bypass(RYGB)on patients with obesity or obesity combined with diabetes.Methods The retrospective and descriptive study was conducted.The stool samples before and after surgery and clinical data of 20 patients with obesity,including 9 simple obesity cases and 11 obesity combined with diabetes cases,who underwent RYGB in the First Affiliated Hospital of Ji′nan University from July 2016 to August 2017 were collected.There were 11 males and 9 females,aged(33±11)years.Observation indicators:(1)changes in composition and structure of intestinal microflora;(2)changes of intestinal microflora in simple obesity patients after operation;(3)changes of intestinal microflora in obesity combined with diabetes patients after operation.Follow up was conducted using telephone interview or outpatient examinations to detect the body mass,the application of antimicrobial agent and the blood glucose control of patients.According to the unified training points,the stool samples were collected and stored into the DNA stabilizer,and then conducted to laboratory analysis within 45 hours.The follow up was up to November 2018.Measurement data with normal distribution were represented as Mean±SD,and independent-samples t test was used for inter-group comparison and paired-samples t test was used for intra-group comparison.Measurement data with skewed distribution were represented as M(Q_(1),Q_(3)),and Wilcoxon signed rank test of two independent samples was used for inter-group comparison.Count data were described as absolute numbers,and the chi-square test,ANOSIM analysis,linear discriminant(LEfSe)analysis and the Metastats analysis were used for inter-group comparison.Results(1)Changes in composition and structure of intestinal microflora.The Shannon index ofαdiversity of preoperative intestinal microflora in simple obesity patients and obesity combined with diabetes patients was 4.37±0.69 and 4.47±0.85,respectively,showing no significant difference between them(t=0.28,P>0.05).Results of preoperative LEfSe analysis showed that there were differences in the bacterial abundance of Firmicutes and Bacteroidea between simple obesity patients and obesity combined with diabetes patients.The abundances of Parasutterella in simple obesity patients and obesity combined with diabetes patients was 0.0001130(0,0.0043782)and 0.0084640(0.0013257,0.0349831),respectively,showing a significant difference between them(Z=2.12,P<0.05).Results of preoperative PCoA analysis showed that the contribution rates of principal component 1,principal component 2 and principal component 3 were 24.98%,22.24%and 16.33%in simple obesity patients and obesity combined with diabetes patients and results of ANOSIM comparison showed that there was no significant difference in preoperative intestinal microflora between them(r=‒0.11,P>0.05).The Shannon index ofαdiversity of postoperative intestinal microflora in simple obesity patients and obesity combined with diabetes patients was 4.60±0.65 and 4.66±0.40,respectively,showing no significant difference between them(t=0.24,P>0.05).Results of postoperative LEfSe analysis showed that there were differences in the bacterial abundance of Bacteroidea,Proteus and Firmicutes between simple obesity patients and obesity combined with diabetes patients.The abundances of Morganella and Coprococcus_2 in simple obesity patients and obesity combined with diabetes patients were 0.0001920(0.0000119,0.0015690),0(0,0)and 0(0,0),0.0000541(0,0.0004190),showing significant differences between them(Z=2.70,2.29,P<0.05).(2)Changes of intestinal microflora in simple obesity patients after operation.There were 10 genera of bacteria of intestinal bacteria changing after surgery,including 7 species of bacteria increasing in the Firmicutes and the Proteobacteria as Veillonella,Morganella,Granulicatella,Aeromonas,Streptococcus,Rothia and Megasphaera and the bacteria decreasing in the Firmicutes and the Actinobacteria as Ruminococcus_torques_group,Romboutsia and Erysipelotrichaceae_UCG-003.Results of LEfSe analysis showed that the bacteria significantly enriched in simple obesity patients before surgery were Ruminococcus_torques_group,Romboutsia and Erysipelotrichaceae_UCG‑003,belonging to Firmicutes,and the bacteria significantly enriched in simple obesity patients after surgery were Rothia,Granulicatella,Enterococcus,Streptococcus,Megasphaera,Veillonella,Aeromonas and Morganella,belonging to Actinobacteria,Firmicutes and Proteobacteria.(3)Changes of intestinal microflora in obesity combined with diabetes patients after operation.There were 16 bacteria of intestinal bacteria increasing after surgery,including Streptococcus,Veillonella,Haemophilus,Pluralibacter,Gemella,Lachnospiraceae_NC2004_group,Granulicatella,Aeromonas,uncultured_bacterium_f_Saccharimonadaceae,Ruminiclostridium_9,Butyricicoccus,Fusobacterium,Anaerotruncus,Fusicatenibacter,Klebsiella and Eubacterium_eligens_group,which belonged to the Firmicutes and the Proteobacteria.Results of LEfSe analysis showed that the bacteria significantly enriched in obesity combined with diabetes patients before surgery were Fusicatenibacter,Tyzzerella_3 and Butyricicoccus,belonging to the Firmicutes,and the bacteria significantly enriched in obesity combined with diabetes patients after surgery were Gemella,Granulicatella,Enterococcus,Streptococcus,Lachnospiraceae_NC2004_group,Eubacterium_eligens_group,Anaerotruncus,Ruminiclostridium_9,Anaeroglobus,Veillonella,Fusobacterium,uncultured_bacterium_f_Saccharimonadaceae,Aeromonas,Klebsiella,Pluralibacter,Proteus and Haemophilus,belonging to the Firmicutes and the Proteobacteria.Conclusions RYGB can significantly increases the intestinal microflora abundance in simple obesity patients and obesity combined with diabetes patients.The two types of patients have specific changes in intestinal microflora at the genus level.