Exploring on Mechanism of Gegen Qinliantang in Interventing Antibiotic-associated Diarrhea Based on 16S rRNA Sequencing and Network Pharmacology

SU Gang; YANG Guangyong; ZHANG Gengxin; SHEN Junxi; HAN Huizi; TIAN Weiyi; WANG Wenjia; WANG Ping; TU Xiaohua; HE Guangzhi

doi:10.13422/j.cnki.syfjx.20230662

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Exploring on Mechanism of Gegen Qinliantang in Interventing Antibiotic-associated Diarrhea Based on 16S rRNA Sequencing and Network Pharmacology

更新时间：2023-11-07

- Exploring on Mechanism of Gegen Qinliantang in Interventing Antibiotic-associated Diarrhea Based on 16S rRNA Sequencing and Network Pharmacology
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 23, Pages: 81-88(2023)
- 作者机构：
  
  贵州中医药大学基础医学院，贵阳 550025
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230662
  CLC： R22;R574.4;R28;Q939.121
- Published：05 December 2023，
  
  Published Online：09 February 2023，
  
  Received：23 November 2022，
- 稿件说明：
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苏钢,杨光勇,张庚鑫等.基于16S rRNA测序与网络药理学探讨葛根芩连汤干预抗生素相关性腹泻的作用机制[J].中国实验方剂学杂志,2023,29(23):81-88.

SU Gang,YANG Guangyong,ZHANG Gengxin,et al.Exploring on Mechanism of Gegen Qinliantang in Interventing Antibiotic-associated Diarrhea Based on 16S rRNA Sequencing and Network Pharmacology[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(23):81-88.
苏钢,杨光勇,张庚鑫等.基于16S rRNA测序与网络药理学探讨葛根芩连汤干预抗生素相关性腹泻的作用机制[J].中国实验方剂学杂志,2023,29(23):81-88. DOI： 10.13422/j.cnki.syfjx.20230662.

SU Gang,YANG Guangyong,ZHANG Gengxin,et al.Exploring on Mechanism of Gegen Qinliantang in Interventing Antibiotic-associated Diarrhea Based on 16S rRNA Sequencing and Network Pharmacology[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(23):81-88. DOI： 10.13422/j.cnki.syfjx.20230662.

摘要

目的

通过16S rRNA测序与网络药理学探究葛根芩连汤（GQT）对抗生素相关性腹泻（AAD）肠道菌群的作用机制。

方法

将60只SD大鼠随机平均分为空白组、模型组、丽珠肠乐组（0.15 g·kg

-1

）、葛根芩连汤高、中、低剂量组（10.08、5.04、2.52 g·kg

-1

），除空白组外，各组每日均给予克林霉素（250 mg·kg

-1

）灌胃造模，连续7 d。造模成功后，各给药组按剂量灌胃对应药物，1次/d，持续14 d，空白组与模型组给予等体积生理盐水灌胃。通过中药系统药理学数据库与分析平台（TCMSP）筛选GQT活性成分及作用靶点，利用人类基因数据库GeneCards、在线人类孟德尔遗传（OMIM）数据库、药物遗传学与药物基因组学知识库（PharmGKB）、药品生物信息学和化学信息学数据库DrugBank、疾病相关的基因与突变位点数据库DisGeNET检索AAD疾病靶点，通过R软件分析获得“药物-疾病”共有靶点。利用STRING数据库分析靶点蛋白质-蛋白质相互作用关系，并进行京都基因和基因组百科全书（KEGG）通路富集分析。通过苏木素-伊红（HE）染色观察大鼠结肠病理学改变，并结合16S rRNA测序AAD结肠内容物菌群结构验证网络药理学结果。

结果

通过网络药理学从GQT中筛选出238个活性成分作用于276个成分靶点，其中槲皮素、葛根素、汉黄芩素、芹黄素为GQT主要核心成分，AAD疾病靶点1 097个，药物-疾病交集靶点127个。蛋白质-蛋白质相互作用网络主要包括蛋白激酶B1（Akt1）、白细胞介素-6（IL-6）及IL-1

等核心靶点，主要富集于IL-17信号通路。通过动物实验验证发现，与空白组比较，模型组结肠结构严重异常，肠道上皮柱状细胞损伤、杯状细胞减少、大量炎症细胞浸润；与模型组比较，GQT高剂量组结肠结构有所改善，但仍存在异常；GQT中、低剂量组和丽珠肠乐组结肠结构明显改善，达到正常水平。GQT可改善AAD肠道菌群结构多样性，在门水平增加厚壁菌门Firmicutes丰度，降低拟杆菌门Bacteroidetes丰度，在属水平增加乳酸杆菌属

Lactobacillus

丰度，降低普雷沃氏菌属

Prevotella

与拟杆菌属

Bacteroides

丰度，其中乳酸球菌属

Lactococcus

可作为GQT治疗AAD的生物标记物，通过菌群功能代谢预测发现GQT可促进肠道中醋酸与乳酸代谢途径。

结论

GQT可能通过槲皮素、汉黄芩素等关键成分作用于Akt1及IL-6等靶点，激活IL-17信号通路，同时改善肠道乳酸球菌属丰度及醋酸、乳酸代谢途径，从而发挥修复肠道屏障的作用来治疗AAD。

Abstract

Objective

To investigate the mechanism of Gegen Qinliantang（GQT） on the intestinal flora of antibiotic-associated diarrhea（AAD） by 16S rRNA sequencing and network pharmacology.

Method

Sixty SD rats were randomly divided into six groups（

=10）， including blank group， model group， GQT high-， medium- and low-dose groups（10.08， 5.04， 2.52 g·kg

-1

） as well as Lizhu Changle group（0.15 g·kg

-1

）， except for the blank group， each group was given clindamycin（250 mg·kg

-1

） by gavage once a day for 7 consecutive days. After successful modeling， the blank group and the model group were given equal volumes of normal saline by gavage. The other groups were given corresponding doses of drugs by gavage for 14 days. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） was used to screen the active components and targets of GQT， GeneCards， Online Mendelian Inheritance in Man（OMIM） database， Pharmacogenetics and Pharmacogenomics Knowledge Base（PharmGKB）， DrugBank and DisGeNET were used to search for AAD disease targets. The drug-disease common targets were obtained by R software. STRING was applied to analyze the target protein-protein interaction， and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis was performed. Then hematoxylin-eosin（HE） staining was used to observe the pathological changes of the colon， and 16S rRNA sequencing of AAD colon content flora structure further verified the results of network pharmacology.

Result

Through network pharmacology， it was found that 238 active components were screened from GQT and acted on 276 component targets， among which quercetin， puerarin， wogonin and apigenin were the main core components of GQT， 1 097 AAD disease targets and 127 drug-disease intersection targets. The protein-protein interaction network mainly included core targets such as protein kinase B1（Akt1）， interleukin（IL）-6 and IL-1

， which were mainly enriched in the IL-17 signaling pathway. It was verified through animal experiments that compared with the blank group， the colon structure of the model group was seriously abnormal， the intestinal epithelial columnar cells were damaged， the goblet cells were reduced， and a large number of inflammatory cells were infiltrated. Compared with the model group， the colon structure of the GQT high-dose group improved， but there were still abnormalities， the colon structure of GQT medium- and low- dose groups and Lizhu Changle group improved significantly and reached the normal level. GQT could improve the structural diversity of AAD intestinal flora. At the phylum level， the abundance of Firmicutes was increased and the abundance of Bacteroidetes was decreased. At the genus level， the abundance of Lactobacillus was increased， and the abundances of

Prevotella

and

Bacteroides

were decreased. Among them，

Lactococcus

could be used as a biomarker for AAD treatment with GQT， and the prediction of functional metabolism of intestinal flora revealed that GQT could promote acetate and lactate metabolic pathways in the intestine.

Conclusion

GQT may activate IL-17 signaling pathway by acting on the targets of Akt1 and IL-6 through key components such as quercetin and wogonin， and improve the abundance of

Lactococcus

in the intestinal tract as well as acetate and lactate metabolic pathways， so as to play a role in repairing the intestinal barrier for the treatment of AAD.

关键词

葛根芩连汤抗生素相关性腹泻（AAD）肠道菌群网络药理学肠道屏障炎症因子白细胞介素（IL）

Keywords

Gegen Qinliantangantibiotic-associated diarrhea（AAD）intestinal floranetwork pharmacologyintestinal barrierinflammatory factorinterleukin（IL）

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