高通量测序分析六味地黄汤对肝肾阴虚型妊娠期便秘患者肠道菌群的影响

Effects of Liuweidihuang Decoction on Intestinal Flora of Constipation Patients with Deficient Liver and Kidney during Pregnancy by High Throughput Sequencing

目的:采用高通量测序技术探讨六味地黄汤对肝肾阴虚型妊娠期便秘患者肠道菌群的影响。方法:选取肝肾阴虚型妊娠期便秘患者28例,分别于治疗前和服用六味地黄汤2周后收集其粪便标本各50 g,采用QIAamp DNA Stool Mini Kit试剂盒提取治疗前后粪便样本的总基因组脱氧核糖核酸(DNA),通过聚合酶链反应(PCR)扩增获得16S rRNA基因V4标签片段,构建Illumina平台文库并测序;采用定量分析微生物生态学(QIIME)软件分析肠道菌群变化,绘制稀释曲线,分析治疗前后粪便样本生物信息学,进行Alpha多样性指数分析,并分别使用Chao1、Shannon及Simpson指数计算肠道菌群丰度和菌群多样性;采用LefSe技术分析治疗前后粪便样本中菌群组成中的差异性菌属。结果:稀释曲线表明测序数据量合理,基本能覆盖样本中所有的细菌种类;六味地黄汤治疗后患者肠道菌群的Chao1指数高于治疗前(P<0.05),但Shannon指数和Simpson指数在治疗前后比较,差异均无统计学意义(P>0.05);治疗前后患者肠道菌群在门水平中均以bacteroidetes、firmicutes和proteobacteria为优势菌群,治疗后在属水平肠道菌群共有10个菌属丰度发生明显改变,其中escherichia、alistipes、parabacteroides、bacteroides fragilis、coprococcus菌属丰度较治疗前明显降低,faecalibacterium、roseburia、bifidobacterium、prevotella、lactobacillus菌属丰度较治疗前升高(P<0.05)。结论:妊娠期便秘患者服用六味地黄汤后,肠道内菌群结构改变显著,尤其在属水平有明显差异。

Objective:To explore the effect of liuweidihuang decoction on intestinal flora of patients of constipation with deficient liver and kidney during pregnancy by high throughput sequencing technology.Methods:Twenty-eight patients of constipation during pregnancy with deficiency of liver and kidney were selected,and 50 g of their fecal samples were collected before treatment in 2 weeks after taking liuweidihuang decoction.The QIAamp DNA Stool Mini Kit was used to extract the total genomic deoxyribonucleic acid(DNA)of fecal samples before and after treatment,and the 16S rRNA gene V4 tag fragment was obtained by polymerase chain reaction(PCR)amplification,and the Illumina platform library was constructed and sequenced;Quantitative insights into microbial ecology(QIIME)software was used to analyze the changes of intestinal flora,draw dilution curves and detect bioinformatics of fecal samples before and after treatment.Alpha diversity index analysis was performed,and then intestinal flora abundance Diversity and diversity of flora were calculated through Chao1,Shannon and Simpson indexes.LefSe technology was used to analyze the difference in bacterial flora in fecal samples before and after treatment.Results:The dilution curves showed reasonable amount of sequencing data,which basically covered all bacterial species in the samples.The Chao1 index of the intestinal flora of the patients was higher after treatment(P<0.05),but the difference between Shannon and Simpson indexes was not statistically significant(P>0.05);After treatment with liuweidihuang decoction,there was significant difference in the abundance(P<0.05),before and after treatment,bacteroidetes,firmicutes and proteobacteria were used as dominant flora at the portal level,but there was no significant difference in comparison(P>0.05).At the genus level,the abundance of 10 bacteria was obviously changed after treatment of pregnant women.Among them,the abundance of escherichia,alistipes,parabacteroides,bacteroides fragilis,and coprococcus genus were significantly lower,and the abundance of faecalibacterium,roseburia,bifidobacterium,prevotella,and lactobacillus bacteria were higher than before treatment,and the differences were statistically significant(P<0.05).Conclusion:After liuweidihuang decoction is taken,the structure of intestinal flora of pregnant patients with constipation can be changed significantly,especially at the genus level.