目的:基于宏基因组测序的方法比较紫草市场药材正伪品对小鼠肠道菌群调节作用的异同。方法:首先将24只清洁级雌性BLAB/C小鼠随机分为3组,分别为空白对照组、A1(新疆紫草)组、A2(紫草非药典品)组,灌胃到达指定时间后,提取结肠内容物(粪便)、回肠内容物(粪便)、小肠内容物(粪便,除去回肠部位)用于肠道菌群分析;对提取的小鼠肠道内容物进行基因组DNA提取和PCR扩增,对PCR产物进行混样和纯化,构建文库并上机测序;对测序数据进行质控,并去除其中的嵌合体序列后,得到最终的有效数据;对获得的有效数据进行分类操作单元(operational taxonomic unit,OTU)聚类和物种注释,并进行样本多样性分析。结果:本研究中A1、A2组均降低了小鼠结肠菌群多样性;在门水平上,A1组显著提高了小鼠小肠和回肠中Firmicutes的丰度,A1、A2组显著提高了小鼠结肠中Bacteroidetes的相对丰度;在属水平上,A1组显著提高了小鼠小肠中Lactobacillus的相对丰度,A2组显著提高了小鼠回肠中Lactobacillus的相对丰度;A1组提高了小鼠结肠中优势菌之一Lactobacillus的相对丰度,A2组提高了结肠中Bacteroides的相对丰度;病原菌Alistipes主要存在于结肠中,A2实验组显著降低了小鼠结肠中Alistipes的相对丰度,而A1实验组则扶植了小鼠结肠中的Alistipes。结论:根据肠道菌群调节作用的结果,紫草药材中药典收载品和市场混淆品2个实验组的肠道菌群调节作用不一致,本研究结果可为深入探索其作用机制提供理论基础。
Objective: To compare the regulatory effects of authentic and counterfeit Arnebiae Radix on intestinal flora in mice based on metagenomic sequencing. Methods: Firstly, 24 clean grade female BLAB/C mice were randomly divided into 3 groups:blank control group, A1 (Arnebia euchroma) group and A2 (Arnebiae Radix whose origins were not included in Chinese Pharmacopoeia) group. After gavage arrived at the specified time, colon contents (feces), ileal contents (feces) and small intestinal contents (feces, except ileal parts) were extracted for intestinal flora analysis. Genomic DNA was extracted and amplified by PCR from the extracted mouse intestinal contents. The PCR products were mixed and purified. Then library was constructed and sequenced. After quality control of sequencing data and removal of chimera sequence, the final effective data was obtained. Operational taxonomic unit(OTU) clustering and species annotation were performed on the obtained valid data, and sample diversity analysis was conducted. Results: In this study, both A1 (Arnebia euchroma) group and A2 (Arnebiae Radix non-pharmacopoeia) group reduced the diversity of mice colon microbiota. At the phylum level, group A1 significantly increased the abundance of Firmicutes in the small intestine and ileum, and groups A1 and A2 significantly increased the relative abundance of Bacteroidetes in the colon. At the genus level, group A1 significantly increased the relative abundance of Lactobacillus in the small intestine of mice, and group A2 significantly increased the relative abundance of Lactobacillus in the ileum of mice. Group A1 increased the relative abundance of Lactobacillus, one of the dominant bacteria in the colon of mice, and group A2 increased the relative abundance of Bacteroides. Alistipes mainly existed in the colon, A2 group significantly reduced the relative abundance of Alistipes in the colon of mice, while Alistipes in the A1 group was cultivated. Conclusion: According to the results of the regulation effect of intestinal flora, the intestinal flora regulation effect in the two experimental groups of the authentic Arnebiae Radix and its confusion products from markets are not consistent, the results of this study can provide a theoretical basis for further exploring the mechanism of Arnebiae Radix.
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