基于消斑通脉方抗动脉粥样硬化作用机制的网络药理学分析和体外实验验证  

Network pharmacological analysis and in vitro experimental verification based on anti-atherosclerosis mechanism of Xiaoban Tongmai Formula

作　　者：曹珊[1] 张艺嘉 白杨[3] 陈芳[4] 谢莎 韩倩倩[5] CAO Shan;ZHANG Yijia;BAI Yang;CHEN Fang;XIE Sha;HAN Qianqian(Department of Formula,School of Traditional Chinese Medicine,Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China;Department of Academic Affairs,Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China;Graduate School,Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China;Department of Pathophysiology,School of Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450046,China;Basic Experimental Center,Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学中医学院方剂教研室,河南郑州450046 [2]河南中医药大学教务处,河南郑州450046 [3]河南中医药大学研究生院,河南郑州450046 [4]河南中医药大学医学院病理生理学教研室,河南郑州450046 [5]河南中医药大学基础实验中心,河南郑州450046

出　　处：《吉林大学学报（医学版）》2024年第4期925-938,共14页Journal of Jilin University：Medicine Edition

基　　金：河南省科技厅科技攻关项目(232102310434);河南省科技厅面上科学基金项目(242300421295);河南省卫健委中医药科学研究重大专项项目(2022ZYZD20);河南省卫健委中医药科学研究重点项目(2023ZY1031);崔应民全国名老中医药专家传承工作室建设项目(国中医药人教函[2022]75号)。

摘　　要：目的:利用网络药理学分析方法初步预测消斑通脉方抗动脉粥样硬化(AS)的潜在作用通路和靶点,联合体外细胞实验对其可能机制进行验证。方法:采用中药系统药理学数据库与分析平台(TCMSP)、GeneCards、Swiss Target Prediction和Uniprot等数据库,收集消斑通脉方中活性化合物及对应靶点信息,构建“成分-靶点-疾病”网络,通过蛋白-蛋白互作(PPI)网络预测可能的作用靶点和通路,对交集靶点进行基因本体论(GO)功能富集分析和京都基因与基因组百科全书(KEGG)信号通路富集分析。体外培养人主动脉血管平滑肌细胞(HA-VSMCs)并鉴定,采用氧化低密度脂蛋白(ox-LDL)诱导HA-VSMCs异常增殖并进行鉴定。MTT法检测不同浓度消斑通脉方作用后各组HA-VSMCs增殖活性,确定消斑通脉方安全性。HA-VSMCs分为空白组、模型组(诱导HA-VSMCs异常增殖)、瑞舒伐他汀组(诱导HA-VSMCs异常增殖后采用4μmol·L^(−1)瑞舒伐他汀干预)及低、中和高剂量消斑通脉方组(诱导HA-VSMCs异常增殖后分别采用0.025、0.050和0.100 mg·L^(−1)消斑通脉方干预)。酶联免疫吸附试验(ELISA)法检测各组HA-VSMCs培养上清中人单核细胞趋化蛋白1(MCP-1)、白细胞介素6(IL-6)和白细胞介素8(IL-8)水平,实时荧光定量PCR(RT-qPCR)法检测各组HA-VSMCs中核因子κB(NF-κB)p65 mRNA和成纤维细胞生长因子2(FGF2)mRNA表达水平,Western blotting法检测各组HA-VSMCs中NF-κB p65和FGF2蛋白表达水平。结果:消斑通脉方中含有103种活性成分,可通过作用于189个靶基因发挥抗AS作用,潜在作用靶点包括IL-6、IL-8、血管内皮生长因子A(VEGFA)、核因子κB1(NF-κB1)和RELA(NF-κB p65)等。GO功能分析和KEGG信号通路富集分析,消斑通脉方通过调节脂质、缺氧诱导因子1(HIF-1)、表皮生长因子(EGF)、磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)和NF-κB等信号通路发挥抗AS作用。细胞形态表现和免疫荧光染色结果证明细�Objective:To preliminarily predict the potential pathways and targets of Xiaoban Tongmai Formula in anti-atherosclerosis(AS)by network pharmacology analysis,and to verify its possible mechanism combined with in vitro cell experiment.Method:The databases including Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),GeneCards,Swiss Target Prediction,and Uniprot were used to collect the information on active compounds and corresponding targets of Xiaoban Tongmai Formula to construct the“compound-target-disease”network.The potential targets and pathways were predicted by protein-protein interaction(PPI)network,and the intersection targets were subjected to Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The human aortic vascular smooth muscle cells(HA-VSMCs)were cultured and identified in vitro,and the abnormal proliferation of HA-VSMCs were induced by oxidized low-density lipoprotein(ox-LDL)and identified;MTT method was used to detect the proliferation activities of the HA-VSMCs in various groups after treated with different concentrations of Xiaoban Tongmai Formula;the safety of Xiaoban Tongmai Fang was confirmed.The HA-VSMCs were divided into blank group,model group(the abnormal proliferation of HA-VSMCs was induced),rosuvastatin group(treated with 4μmol·L^(−1) rosuvastatin after inducing the abnormal proliferation of HA-VSMCs),and low,medium,and high doses of Xiaoban Tongmai Formula groups(treated with 0.025,0.050,and 0.100 mg·L^(−1) Xiaoban Tongmai Formula after inducing the abnormal proliferation of HA-VSMCs);enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of monocyte chemotactic protein^(−1)(MCP−1),interleukin-6(IL-6),and interleukin-8(IL-8)in supernatant of the HA-VSMCs in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of nuclear factor kappa-B(NF-κB)p65 mRNA and fibroblast growth factors 2(FGF2)mR

关 键 词：消斑通脉方 动脉粥样硬化 血管平滑肌细胞 网络药理学 细胞增殖 

分 类 号：R285.5[医药卫生—中药学] R541.4[医药卫生—中医学]