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  

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

作　　者：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 SU Gang;YANG Guangyong;ZHANG Gengxin;SHEN Junxi;HAN Huizi;TIAN Weiyi;WANG Wenjia;WANG Ping;TU Xiaohua;HE Guangzhi(School of Basic Medicine,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China)

机构地区：[1]贵州中医药大学基础医学院,贵阳550025

出　　处：《中国实验方剂学杂志》2023年第23期81-88,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：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)。

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1097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-1β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,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(n=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,1097 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-1β,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 mode

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) 

分 类 号：R22[医药卫生—中医基础理论] R574.4[医药卫生—中医学] R28[生物学—微生物学] Q939.121