基于网络药理学联合16S rDNA高通量测序技术分析丹参对感染大肠杆菌小鼠肠道菌群的影响

doi:10.11843/j.issn.0366-6964.2022.10.040

摘要/Abstract

摘要： 本研究旨在通过网络药理学联合16S rDNA高通量测序技术分析丹参对感染大肠杆菌小鼠肠道菌群的影响。利用传统中药系统药理学数据库和分析平台（TCMSP）筛选丹参有效成分及靶点基因。使用基因数据库（Genecards）获得E.coli靶点基因。两者靶点基因取交集，通过STRING平台进行蛋白质相互作用分析，构建PPI网络，并运用Cytoscape 3.8.2软件构建“丹参活性成分-潜在作用靶点”网络。利用DAVID在线工具进行基因本体论（GO）功能富集分析，Metascape数据库进行京都基因与基因组百科全书（KEGG）通路富集分析。试验选用110只雄性昆明小鼠，随机分为对照组、大肠杆菌组和低、中、高剂量丹参大肠杆菌感染组，每组22只。连续灌胃丹参液28 d后，用E.coli O₁₀₁进行腹腔注射，对照组注射无菌生理盐水，观察24 h后，收集各组粪便。应用16S rDNA高通量测序技术对各组小鼠肠道菌群进行分析。结果显示：1）网络药理学丹参有效成分36个，作用靶点669个，Genecards数据库搜索E.coli靶点8 902个，对两者取交集去除孤立点获得丹参治疗E.coli潜在靶点51个；GO、KEGG分析，获得关键靶点18个、关键通路47条，其中生物过程（BP）、分子功能（MF）和细胞成分（CC）各为174、76、45条；2）通过Illumina Miseq测序共获得950 855条有效序列，2 537个操作分类单元（OTUs）；3） Alpha多样性分析结果表明，不同剂量丹参组肠道菌群多样性与对照组具有显著差异，大肠杆菌组肠道菌群多样性显著低于对照组（P<0.05）；4）相比于大肠杆菌感染组，在门水平上，低、中、高剂量丹参大肠杆菌感染组Bacteroidetes （拟杆菌门）丰度极显著增高（P<0.01），Firmicutes （厚壁菌门）丰度显著增高（P<0.05），Proteobacteria （变形菌门）极显著降低（P<0.01），在属水平上Bacteroides（拟杆菌属）丰度极显著增高（P<0.01）；5）基于LEfSe分析，对照组、大肠杆菌感染组、丹参高剂量大肠杆菌感染组共发现14种显著差异菌群；6） PICRUSt功能预测分析发现，丹参对大肠杆菌感染小鼠肠道菌群调节功能主要富集的途径是氨基酸、碳水化合物运输与代谢，翻译、核糖体结构和生物转化，细胞壁/膜/生物合成等方面。综上，基于网络药理学联合16S rDNA高通量测序技术全面揭示了丹参多成分、多靶点、多途径调节相关菌群生物丰富度，对大肠杆菌感染小鼠的肠道菌群紊乱有缓解作用，为临床应用丹参治疗大肠杆菌感染提供理论依据。

关键词: 丹参, 网络药理学, 肠道菌群, 高通量测序, 小鼠

Abstract: This study was conducted to investigate the effects of Radix Salviae Miltiorrhizae on the intestinal flora of mice injected with Escherichia coli(E. coli) based on network pharmacology and 16S rDNA high-throughput sequencing technology. The active components and corresponding targets of Radix Salviae Miltiorrhizae were screened by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and the E. coli targets were searched by Genecards database. Protein interaction analysis of the intersection of the target genes was performed through SRTING database to construct the protein-protein interaction(PPI) network, and the "Radix Salviae Miltiorrhizae active ingredient-potential action target" network was constructed by Cytoscape 3.8.2 software. DAVID online tools were used to analyze the function of gene ontology (GO), Metascape online tools were used to analyze the enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. One hundred and ten male Kunming mice were randomly divided into control group, E. coli group, and low-, medium- and high-dose Radix Salviae Miltiorrhizae injected with E. coli group, with 22 mice in each group. E.coli O₁₀₁ was injected intraperitoneally after continuously gavaged with Radix Salviae Miltiorrhizae solution for 28 days, while sterile normal saline was injected into the control group. After observation for 24 h, feces were collected from each group. The 16S rDNA high-throughput sequencing technology was used to analyze the intestinal flora of mice in each group. The results were showed as follows:1) The network pharmacological analysis showed that the active components of Radix Salviae Miltiorrhizae were 36, and the number of action targets was 669. The Genecards database searched for 8 902 E. coli targets, and the intersection between the two was selected and isolated points were removed, finally, 51 potential targets for E. coli treatment with Radix Salviae Miltiorrhizae were obtained. Through GO and KEGG analysis, 18 key targets and 16 key pathways were identified, including 174, 76 and 45 biological processes (BP), molecular functions (MF) and cellular components (CC), respectively; 2) A total of 950 855 valid sequences and 2 537 operational classification units (OTUs) were obtained by Illumina Miseq sequencing; 3) Alpha diversity analysis results showed that the intestinal flora diversity of Radix Salviae Miltiorrhizae groups was significantly different from that of the control group, and the intestinal flora diversity of the E. coli group was significantly lower than that of the control group (P<0.05); 4) Compared with E. coli infection group, at the phylum level, the abundance of Bacteroides was extremely significant increased(P<0.01) and Firmicutes was significantly increased(P<0.05) and Proteobacteria was extremely significant decreased (P<0.01) in the low-, medium- and high-dose Radix Salviae Miltiorrhizae injected with E. coli groups, while at genus level, the abundance of Bacteroides was extremely significant increased (P<0.01); 5) Based on LEfSe analysis, a total of 14 significantly different microflora were found in the control group, E. coli infection group, and high-dose Radix Salviae Miltiorrhizae injected with E. coli group. 6) PICRUSt functional prediction analysis showed that the main functional pathways of Radix Salviae Miltiorrhizae for intestinal flora of mice infected with E. coli were amino acids, carbohydrate transport and metabolism; Translation, ribosomal structure and biotransformation; Cell wall/membrane/biosynthesis, etc. In summary, network pharmacology combines with 16S rDNA high-throughput sequencing technology has revealed that Radix Salviae Miltiorrhizae regulates the relative microflora biological abundance through multi-component, multi-target, and multi-pathway, which alleviates the intestinal flora damage in mice with flora imbalance caused by E. coli infection, and provides theoretical basis for clinical application of Radix Salviae Miltiorrhizae in the treatment of E. coli infection.

Key words: Radix Salviae Miltiorrhizae, network pharmacology, intestinal flora, high-throughput sequencing, mice

中图分类号:

S853.74

王乐, 陈泓岑, 张永红, 吴琼, 侯佳佳, 王天祎, 卢天航, 黄传发, 张华, 崔德凤. 基于网络药理学联合16S rDNA高通量测序技术分析丹参对感染大肠杆菌小鼠肠道菌群的影响[J]. 畜牧兽医学报, 2022, 53(10): 3695-3711.

WANG Le, CHEN Hongcen, ZHANG Yonghong, WU Qiong, HOU Jiajia, WANG Tianyi, LU Tianhang, HUANG Chuanfa, ZHANG Hua, CUI Defeng. Effects of Radix Salviae Miltiorrhizae on Intestinal Microflora of Escherichia coli Infected Mice Based on Network Pharmacology and 16S rDNA High-throughput Sequencing Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3695-3711.

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