基于网络药理学和细胞实验探究补肾清透方治疗慢性乙型肝炎病毒携带状态的作用机制  

Based on Network Pharmacology and Cell Experiment to Discuss the Mechanism of Bushen Qingtou Prescription in the Treatment of the Chronic Hepatitis B Virus Carrier State

作　　者：黄锦桢 王宇新 陈团团 赖剑萍 廖勉勉 邢宇锋[3] 韩志毅[3] 童光东[1,3] HUANG Jinzhen;WANG Yuxin;CHEN Tuantuan;LAI Jianping;LIAO Mianmian;XING Yufeng;HAN Zhiyi;TONG Guangdong(The Fourth Clinical Medical School of Guangzhou University of Chinese Medicine,Shenzhen 518033 Guangdong,China;The Affiliated Dongguan Traditional Chinese Medicine Hospital of Guangzhou University of Chinese Medicine,Dongguan 523000 Guangdong,China;Department of Hepatology,Shenzhen Traditional Chinese Medicine Hospital,Shenzhen 518033 Guangdong,China)

机构地区：[1]广州中医药大学第四临床医学院,广东深圳518033 [2]广州中医药大学附属东莞市中医院,广东东莞523000 [3]深圳市中医院肝病科,广东深圳518033

出　　处：《中药新药与临床药理》2022年第12期1654-1664,共11页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：“十三五”国家科技重大专项(2018ZX10725505-002)。

摘　　要：目的 以网络药理学的方法探讨补肾清透方(Bushen Qingtou Prescription)治疗慢性乙型肝炎病毒(hepatitis B virus,HBV)携带状态的核心靶点及信号通路等,进一步采用细胞实验验证预测靶点。方法 通过TCMSP、BATMAN和GeneCards数据库分别得到补肾清透方的药物活性成分、药物与疾病靶点,并将药物与疾病的交集靶点上传至STRING数据库以获取蛋白质-蛋白质相互作用图。运用DAVID6.7数据库对交集靶点富集分析,Cytoscape 3.7.2软件构建“药物-成分-共同靶点-疾病”网络图以获得核心活性成分及核心靶点,并通过AutoVina软件对核心靶点及活性成分进行分子对接。此外,观察补肾清透方对HepG2-HBx细胞系中HBx的影响,并使用蛋白免疫印迹法验证核心靶点蛋白的表达水平。结果 共得到补肾清透方治疗HBV携带状态的191个活性成分,478个药物靶点、1 705个疾病靶点,213个交集靶点。富集分析共涉及734个生物过程,86个细胞组分,141个分子功能,138条KEGG通路,通路主要与TNF、HIF-1、TOLL样受体等信号通道有关。网络图表明核心活性成分为槲皮素、木犀草素、山柰酚和β-谷甾醇,核心靶点为COX-2、COX-1及AR。分子对接结果显示核心成分及核心靶点的大多数结合能≤-5.0 kcal·mol^(-1),展现出较好的亲和力。细胞实验结果表明与空白组比较,HBx、COX-2表达在补肾清透方50μg·mL^(-1)组稍下降(P<0.05),在100、200μg·mL^(-1)两组中明显下降(P<0.01),COX-1表达在用药组中均明显升高(P<0.01),AR表达则无明显变化(P>0.05)。结论 补肾清透方可以通过多成分、多靶点及多通路防治HBV携带状态,而COX-2表达的下调及COX-1表达的上调可能是其发挥抗病毒作用的主要机制。Objective To explore core targets and the mechanism of signal pathways of Bushen Qingtou Prescription(BSQT) in the treatment of chronic hepatitis B virus(HBV) carrier state from the perspective of network pharmacology,and the results were further verified by cell experiment. Methods The active components of BSQT,drugs and disease targets were obtained from TCMSP, BATMAN and GeneCards databases, and the intersection targets were uploaded to STRING database to obtain protein-protein interaction network map. DAVID 6.7 database was used to conduct the enrichment analysis of intersection targets, and Cytoscape 3.7.2 software was used to construct the network diagram of "drug-component-common target-disease" to obtain the core active components and targets. Then AutoVina software was used for molecular docking between core target and active component. In addition,the effect of BSQT on HBx in HepG2-HBx cell line was observed,and the expression level of core target protein was verified by Western Blotting. Results A total of 191 active components,478 drug targets,1 705 disease targets and 213 intersection targets were obtained. Enrichment analysis showed that 734 biological processes,86 cellular components, 141 molecular functions and 138 KEGG pathways were involved. KEGG pathway was mainly related to TNF, HIF-1, Toll-like receptors, and other signal pathways. The results of network diagram showed that the core active components are quercetin,luteolin,kaempferol and β-sitosterol. The core targets are COX-2,COX-1 and AR. And the molecular docking showed that most of the binding energies of active components connected with core targets were less than or equal to-5.0 kcal·mol^(-1),showing a good affinity. The results of cell experiments showed that compared with the blank group,the expressions of HBx and COX-2 were slightly decreased in the 50 μg·mL^(-1)BSQT group(P<0.05),and significantly decreased in the 100 and 200 μg·mL^(-1)BSQT groups(P<0.01). And the expression of COX-1 was significantly increased in all BSQT gr

关 键 词：补肾清透方 慢性乙型肝炎病毒(HBV) 网络药理学 HepG2-HBx 细胞实验 作用机制 

分 类 号：R285.5[医药卫生—中药学]