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

苏钢; 杨光勇; 张庚鑫; 沈俊希; 韩慧子; 田维毅; 王文佳; 王平; 涂小华; 何光志

doi:10.13422/j.cnki.syfjx.20230662

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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

药理 | 更新时间：2023-11-07

- 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
  增强出版
- Exploring on Mechanism of Gegen Qinliantang in Interventing Antibiotic-associated Diarrhea Based on 16S rRNA Sequencing and Network Pharmacology
- 中国实验方剂学杂志 2023年29卷第23期页码：81-88
- 作者机构：
  
  贵州中医药大学基础医学院，贵阳 550025
- 作者简介：
  
  苏钢，在读硕士，从事中医基础研究，E-mail：754932057@qq.com
  何光志，博士，教授，硕士生导师，从事中西医结合基础研究，E-mail：39272454@qq.com
- 基金信息：
  
  国家自然科学基金项目(82060796);贵州省科学技术基金项目(黔科合基础［2020］1Y392);贵州省科技创新人才团队项目(黔科合平台人才［2020］5010);贵州省发改委工程研究中心建设项目(黔发改高技（2020）896号)
- DOI：10.13422/j.cnki.syfjx.20230662
  中图分类号： R22;R574.4;R28;Q939.121
- 纸质出版日期：2023-12-05，
  
  网络出版日期：2023-02-09，
  
  收稿日期：2022-11-23，
- 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）

references

LARCOMBE S，HUTTON M L，LYRAS D.Involvement of bacteria other than clostridium difficile in antibiotic-associated diarrhoea［J］.Trends Microbiol，2016，24（6）：463-476.

SHARON T，WELSH R M，ADAMS E H，et al.Public health response to US cases of Candida auris，a globally emerging，multidrug-resistant yeast，2013—2017［J］.Open Forum Infect Dis，2017，doi：10.1093/ofid/ofx163.002http://dx.doi.org/10.1093/ofid/ofx163.002.

FITZGERALD J，PATEL S，ECKENBERGER J，et al.Improved gut microbiome recovery following drug therapy is linked to abundance and replication of probiotic strains［J］.Gut Microbes，2022，14（1）：2094664.

SHAN Y，SENTHIL-KUMAR P，ZEBLEY M，et al.Clostridium difficile infection：No longer the infection of antibiotic associated diarrhea［J］.Gastroenterology，2017，doi：10.1016/S0016-5085（17）34343-3http://dx.doi.org/10.1016/S0016-5085（17）34343-3.

HÖGENAUER C，HAMMER H F，KREJS G J，et al.Mechanisms and management of antibiotic-associated diarrhea［J］.Clin Infect Dis，1998，27（4）：702-710.

赵明禄，郑佳，郭玉婷.葛根芩连汤及其加减方治疗溃疡性结肠炎的研究进展［J］.中国实验方剂学杂志，2023，29（20）：239-246.

续畅，钟萌，马致洁，等.葛根芩连汤的现代研究进展［J］.吉林中医药，2015（6）：629-631，632.

CUI H，CAI Y，WANG L，et al.Berberine regulates Treg/Th17 balance to treat ulcerative colitis through modulating the gut microbiota in the colon［J］.Front Pharmacol，2018，9：571.

XU J，LIAN F，ZHAO L，et al.Structural modulation of gut microbiota during alleviation of type 2 diabetes with a Chinese herbal formula［J］.ISME J，2015，9（3）：552-562.

LI Q，CUI Y，XU B，et al.Main active components of Jiawei Gegen Qinlian decoction protects against ulcerative colitis under different dietary environments in a gut microbiota-dependent manner［J］.Pharmacol Res，2021，170：105694.

张庚鑫，杜海洋，王平，等.基于16S rRNA测序研究葛根芩连汤对菌群失调性腹泻大鼠肠道菌群结构的影响［J］.中国实验方剂学杂志，2021，27（11）：19-26.

FURUKAWA K.Towards cross fertilization of multiple disciplines［J］.New Generat Comput，2002，20（2）：141-142.

招志辉.葛根芩连汤传统煎煮与十功能煎药机煎煮一致性评价的初步研究［D］.广州：广州中医药大学，2018.

张文娣.抗生素相关性腹泻模型的构建［D］.广州：南方医科大学，2015.

SHAO H，ZHANG C，XIAO N，et al.Gut microbiota characteristics in mice with antibiotic-associated diarrhea［J］.BMC Microbiol，2020，20（1）：313.

刘茜明，杨光勇，何光志，等.ERIC-PCR技术分析葛根芩连汤对抗生素相关性腹泻模型肠道菌群结构的影响［J］.黑龙江畜牧兽医，2016（6）：23-27，285.

ISLAM M S，QUISPE C，HOSSAIN R，et al.Neuropharmacological effects of quercetin：A literature-based review［J］.Front Pharmacol，2021，12：665031.

MI W，HU Z，XU L，et al.Quercetin positively affects gene expression profiles and metabolic pathway of antibiotic-treated mouse gut microbiota［J］.Front Microbiol，2022，13：983358.

LI J，ZHANG L，LI Y，et al.Puerarin improves intestinal barrier function through enhancing goblet cells and mucus barrier-ScienceDirect ［J］.J Funct Foods，2020，75：104246.

CUI L，GUAN X，DING W，et al.Scutellaria baicalensis Georgi polysaccharide ameliorates DSS-induced ulcerative colitis by improving intestinal barrier function and modulating gut microbiota［J］.Int J Biol Macromol，2021，166：1035-1045.

TANG B，LI X，REN Y，et al.MicroRNA-29a regulates lipopolysaccharide （LPS）-induced inflammatory responses in murine macrophages through the Akt1/ NF-κB pathway［J］.Exp Cell Res，2017，360（2）：74-80.

JORDAN S C，AMMERMAN N，CHOI J，et al.Interleukin-6：An important mediator of allograft njury［J］.Transplantation，2020，104（12）：2497-2506.

HUANG J，LEE H Y，ZHAO X，et al.Interleukin-17D regulates group 3 innate lymphoid cell function through its receptor CD93［J］.Immunity，2021，54（4）：673-686.

PHYO L Y，SINGKHAMANAN K，LAOCHAREONSUK W，et al.Fecal microbiome alterations in pediatric patients with short bowel syndrome receiving a rotating cycle of gastrointestinal prophylactic antibiotics［J］.Pediatr Surg Int，2021，37（10）：1371-1381.

KOMAROFF A L.The Microbiome and risk for obesity and diabetes［J］.JAMA，2017，317（4）：355-356.

BURRELLO C，GARAVAGLIA F，CRIBIÙ F M，et al.Therapeutic faecal microbiota transplantation ontrols intestinal inflammation through IL10 secretion by immune cells［J］.Nat Commun，2018，9（1）：5184.

CAPURSO L.Thirty Years of Lactobacillus rhamnosus GG： A Review［J］.J Clin Gastroenterol，2019，53（Suppl 1）：S1-S41.

KOSLER S，STRUKELJ B，BERLEC A，et al.Lactic acid bacteria with concomitant IL-17，IL-23 and TNF-α binding ability for the treatment of inflammatory bowel disease［J］.Curr Pharm Biotechnol，2017，18（4）：318-326.

ASHBY K H，MENDOZA V.Lactobacillus GG for the treatment and cure of ulcerative colitis—A case report：731［J］.Am J Gastroenterol，2006，101（9）：S298.

HUANG Y，TANG J，CAI Z，et al.Prevotella induces the production of Th17 cells in the colon of mice［J］.J Immunol Res，2020，2020：9607328.

LIU J B，CHEN K，LI Z F，et al.Glyphosate-induced gut microbiota dysbiosis facilitates male reproductive toxicity in rats［J］.Sci Total Environ，2022，805：150368.

HU C，XU B，WANG X，et al.Gut microbiota-derived short-chain fatty acids regulate group 3 innate lymphoid cells in HCC［J］.Hepatology，2023，77（1）：48-64.

PUCINO V，CERTO M，BULUSU V，et al.Lactate buildup at the site of chronic inflammation promotes disease by inducing CD4+ T cell metabolic rewiring［J］.Cell Metab，2019，30（6）：1055-1074.

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