基于网络药理学和细胞实验探讨阿魏酸抗肝纤维化的作用机制  

To Explore the Mechanism of Ferulic Acid Against Liver Fibrosis Based on Network Pharmacology and Cell Experiment

作　　者：孙墨晗 张秋菊[1] 赵哲 靳玉秋 田萌媛 陈光顺[1] Sun Mohan;Zhang Qiuju;Zhao Zhe;Jin Yuqiu;Tian Mengyuan;Chen Guangshun(Gansu University of Traditional Chinese Medicine,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学,兰州730000

出　　处：《世界科学技术-中医药现代化》2023年第12期3908-3919,共12页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：甘肃省教育厅教育科技创新项目(2021CXZX-772):基于“土得木达”法探讨参苓白术散在TGF-β/Smads信号通路中抗肝纤维化的作用机制,负责人:孙墨晗;国家自然科学基金重大课题(81660772):基于TGF-β1/smad与JAK/STAT多信号转导通路的舒肝和络醒脾法抗肝纤维化作用机制研究,负责人:赵鲲鹏。

摘　　要：目的基于网络药理学研究阿魏酸(Ferulic acid,FA)对肝纤维化(hepatic fibrosis,HF)疾病的作用机制,并根据结果建立大鼠肝星状细胞(Hepatic stellate cell-T6,HSC-T6)体外模型进行验证。方法通过有机小分子生物活性数据库(PubChem)、小分子药物靶点预测在线平台(SwissTargetPrediction)和药效预测靶点数据库(PharmMapper)筛选出FA的潜在靶点,与突变位点数据库(DisGeNET)、人类基因数据库(GeneCards)和人类孟德尔遗传病数据库(OMIM)中筛选的FA靶点进行重合取交集,随后利用STRING平台进行蛋白相互作用分析(Protein-protein interaction,PPI)、采用R644.0.3对关键靶点进行基因本体论(Gene ontology,GO)和京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析,Cytoscape 3.7.2软件构建“成分-靶点-疾病”网络图。基于此,通过细胞活性检测(Cell counting kit-8,CCK-8)法检测HSC-T6的增殖情况并以此确定分组:空白组、低剂量组(100μg·mL^(-1)FA)、中剂量组(200μg·mL^(-1)FA)、高剂量组(400μg·mL^(-1)FA)和阳性对照组(200μg·mL^(-1)秋水仙碱),划痕实验检测HSC-T6的迁移能力,酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA)法检测HSC-T6内α-平滑肌肌动蛋白(Alpha-smooth muscle actin,α-SMA)的表达情况,流式细胞仪检测HSC-T6的周期变化,聚合酶链式反应(Quantitative real time polymerase chain reaction,qRT-PCR)法检测JAK2、STAT3 mRNA相对表达,蛋白质免疫印迹(Western blot)法检测JAK2、STAT3蛋白的分子表达量。结果获得FA与HF的交集靶点254个,核心靶点有信号转导和转录激活因子3(STAT3)、白蛋白(Albumin,ALB)、蛋白激酶B1(Protein kinase B,Akt1)、肿瘤抑制蛋白(Tumor protein P53,TP53),表皮生长因子受体(Epidermal growth factor receptor,EGFR)和胱天蛋白酶3(Caspase-3,CASP3)等。KEGG分析发现FA对HF的作用通路主要涉及磷脂酰肌醇3激酶-蛋白激酶B(Phosphatidylinositol 3 kinase-protein kinaseObjective To study the mechanism of ferulic acid(FA)on hepatic fibrosis(HF)based on network pharmacology,and establish an in vitro model of rat hepatic stellate Cell-T6(HSC-T6)according to the results.Methods The potential targets of FA were screened through PubChem,swisstargetprediction and pharmmapper,and overlapped with the FA targets screened in disgenet,genecards and OMIM.Then,protein protein interaction(PPI)was analyzed by using string platform.Gene ontology(go)and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were carried out for key targets by using R644.0.3,and the"component target disease"network diagram was constructed by Cytoscape 3.7.2 software.Based on this,the proliferation of HSC-T6 was detected by cell counting kit-8(CCK-8)method,and the grouping was determined:blank group and low-dose group(100μg·mL^(-1) FA),medium dose group(200μg·mL^(-1) FA),high dose group(400μg·mL^(-1) FA)and positive control group(200μg·mL^(-1) colchicine),the migration ability of HSC-T6 was detected by scratch test,and the content of HSC-T6 was detected by enzyme linked immunosorbent assay(ELISA)α-Alpha smooth muscle actin,α-Flow cytometry was used to detect the changes of HSC-T6 cycle,quantitative real time polymerase chain reaction(qRT-PCR)was used to detect the relative expression of JAK2 and STAT3 mRNA,and Western Blot was used to detect the molecular expression of JAK2 and STAT3 protein.Results 254 intersection targets of FA and HF were obtained.The core targets were signal transducer and activvator of Transcription(STAT3),albumin(ALB),protein kinase B(AKT1),tumor suppressor protein p53(TP53),epidermal growth factor receptor(EGFR)and caspase-3(CASP3).KEGG analysis showed that the action pathway of FA on HF mainly involved phosphatidylinositol 3 kinase protein kinase B(PI3K-Akt),vascular endothelial growth factor(VEGF),Janus kinase/signal transducer and activator of transcription(JAK/STAT)Tumor necrosis factor(TNF)and other pathways.The experimental results showed that in CCK-8 expe

关 键 词：阿魏酸 肝纤维化 网络药理学 HSC-T6 JAK2 STAT3 

分 类 号：R285[医药卫生—中药学]