Mechanism of Dengzhan Shengmai capsule in treating coronary heart disease based on network pharmacology and molecular docking technology
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摘要:目的
基于网络药理学结合分子对接技术探讨灯盏生脉胶囊(DZSM)治疗冠心病(CHD)的潜在作用靶点及机制。
方法采用TCMSP和ETCM数据库检索DZSM的化学成分; 采用Swiss ADME数据库进行活性成分筛选, Swiss Target Prediction数据库获取活性成分的潜在靶点。检索GeneCards和DisGeNET数据库获取CHD靶点, 构建"DZSM-活性成分-CHD靶点"网络。对关键活性成分和核心靶点进行分子对接, 验证结合特性。于DAVID数据库进行基因本体(GO)和京都基因和基因组百科全书(KEGG)富集分析。采用氧化型低密度脂蛋白(ox-LDL)诱导的小鼠巨噬细胞系(RAW264.7细胞)模型体外验证野黄芩苷对CHD的治疗作用。采用Griess反应测定细胞上清液中一氧化氮(NO)生成量。采用实时定量聚合酶链反应(qRT-PCR)检测丝氨酸/苏氨酸激酶(AKT)表达水平; 采用蛋白质免疫印迹试验(Western Blot)检测AKT蛋白表达和磷酸化水平。
结果共获得DZSM的56个活性化合物, 通过作用于136个靶点调控CHD进展。其中山奈酚、槲皮素、木犀草素、芹菜素、野黄芩素、6-羟基山奈酚、野黄芩苷、对壬基酚、麦冬皂苷D和人参皂苷Rb1能够调控共113个CHD靶点。AKT1、SRC、PPARG、EGFR、ESR1、PTGS2、SIRT1、MAPK1、MMP9和PPARA基因为DZSM治疗CHD的核心靶点。分子对接结果显示, 关键活性成分与核心靶点具有良好的结合特性。体外实验结果表明, 野黄芩苷可减少巨噬细胞一氧化氮生成, 增加AKT mRNA、AKT蛋白表达和磷酸化水平(P < 0.05)。KEGG富集分析显示, DZSM主要通过调控癌症通路、内分泌抵抗、糖尿病并发症中的AGE-RAGE信号通路、流体剪切应力与动脉粥样硬化、脂质与动脉粥样硬化、松弛素信号通路等途径治疗CHD。
结论DZSM通过多成分、多靶点、多途径发挥治疗CHD的作用。
Abstract:ObjectiveTo explore the potential target and mechanism of Dengzhan Shengmai capsule (DZSM) in the treatment of coronary heart disease (CHD) based on network pharmacology and molecular docking technology.
MethodsTCMSP and ETCM databases were employed to search the chemical components of DZSM. Swiss ADME database was used to screen active ingredients, and Swiss Target Prediction database was used to obtain potential targets of active ingredients. The CHD target was obtained by searching GeneCards and DisGeNET databases, and the DZSM-active ingredient-CHD target network was constructed. Molecular docking of key active ingredients and core targets was performed to verify binding properties. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis were performed in the DAVID database. A mouse macrophage cell line (RAW264.7 cells) model induced by oxidized low density lipoprotein (ox-LDL) was used to test the therapeutic effect of scutellarin on CHD in vitro. The production of nitric oxide (NO) in cell supernatant was measured by Griess reaction. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of serine/threonine kinase (AKT); The expression and phosphorylation of AKT protein were detected by Western Blot.
ResultsA total of 56 active compounds of DZSM were obtained to regulate CHD progression by acting on 136 targets. Among them, kaempferol, quercetin, luteolin, apigenin, scutellarein, 6-hydroxykaempferol, scutellarin, nonylphenol, Ophiopogonin D, and Ginsenoside Rb1 could regulate 113 CHD targets. AKT1, SRC, PPARG, EGFR, ESR1, PTGS2, SIRT1, MAPK1, MMP9 and PPARA genes were the core targets of DZSM therapy for CHD. Molecular docking showed that the key active ingredients and core targets had good binding properties. The results of in vitro experiments showed that scutellarin could reduce the production of nitric oxide and increase the level of AKT, protein expression and phosphorylation in macrophages (P < 0.05). KEGG enrichment analysis showed that DZSM treated CHD mainly by regulating cancer pathways, endocrine resistance, AGE-RAGE signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, lipid and atherosclerosis, and relaxin signaling pathway.
ConclusionDZSM plays a role in the treatment of CHD through multi-component, multi-target and multi-pathway.
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图 1 “DZSM-活性成分-CHD靶点”网络图
深蓝色节点为中药，紫色节点为灯盏细辛活性化合物，黄色节点为人参活性化合物，粉色节点为五味子活性化合物，浅蓝色节点为麦冬活性化合物，绿色节点为靶点，红色字体为多味中药共有成分。
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图 5 野黄芩苷体外缓解CHD的靶点验证
A: NO含量; B: AKT mRNA表达; C: AKT蛋白表达; D: p-AKT蛋白表达。与模型组比较, *P < 0.05, **P < 0.01; 与空白组比较, ## P < 0.01。
表 1 qRT-PCR引物序列
基因 上游引物(5′→3′) 下游引物(5′→3′) AKT GGACTACTTGCACTCCGAGAAG CATAGTGGCACCGTCCTTGATC GAPDH GTGGGAATGGGTCAGAAGGA CTTCTCCATGTCGTCCCAGT AKT: 丝氨酸/苏氨酸激酶。 
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表 2 DZSM活性化合物
英文名 中文名 CAS号 植物 2, 6-Dimethoxybenzoic acid 2, 6-二甲氧基苯甲酸 1466-76-8 灯盏细辛 6-Hydroxykaempferol 6-羟基山奈酚 4324-55-4 灯盏细辛 Apigenin 芹菜素 520-36-5 灯盏细辛 Baicalein 黄芩素 491-67-8 灯盏细辛 Caffeic acid 咖啡酸 331-39-5 灯盏细辛 Catechol 邻苯二酚 120-80-9 灯盏细辛 Cedar acid 丁香酸 530-57-4 灯盏细辛 Cinnamic Acid 肉桂酸(反式) 621-82-9 灯盏细辛 Esculetin 秦皮乙素 305-01-1 灯盏细辛 Ferulic acid 阿魏酸 1135-24-6 灯盏细辛 Formononetin 芒柄花黄素 485-72-3 灯盏细辛 Gallic acid 没食子酸 149-91-7 灯盏细辛 Isoliquiritigenin 异甘草素 961-29-5 灯盏细辛 Isoscopoletin 异东莨菪内酯 776-86-3 灯盏细辛 Kaempferol 山奈酚 520-18-3 灯盏细辛、人参 Luteolin 木犀草素 491-70-3 灯盏细辛 Naringenin 柚皮素 480-41-1 灯盏细辛 p-Coumaric acid 对香豆酸 501-98-4 灯盏细辛 p-Hydroxybenzoic acid 对羟基苯甲酸 99-96-7 灯盏细辛 Protocatechuic acid 原儿茶酸 99-50-3 灯盏细辛、五味子 Quercetin 槲皮素 117-39-5 灯盏细辛 Scopoletol 东莨菪内酯 92-61-5 灯盏细辛 Scutellarin 野黄芩苷 27740-01-8 灯盏细辛 Scutellarein 野黄芩素 529-53-3 灯盏细辛 N-p-Coumaroyltyramine N-对反式香豆酰酪胺 36417-86-4 麦冬 Oleanolic Acid 齐墩果酸 508-02-1 麦冬 Ophiopogonin D 麦冬皂苷D 945619-74-9 麦冬 9-Hexadecenoic acid 9-十六烯酸 10030-73-6 人参 Cedrol 雪松醇 77-53-2 人参 Ginsenoside Rb1 人参皂苷Rb1 41753-43-9 人参 Ginsenoside Rb2 人参皂苷Rb2 11021-13-9 人参 Ginsenoside Re 人参皂苷Re 51542-56-4 人参 Ginsenoside Rg1 人参皂苷Rg1 22427-39-0 人参 Ginsenoside Rg3 人参皂苷Rg3 38243-03-7 人参 Ginsenoside Rg5 人参皂苷Rg5 186763-78-0 人参 Ginsenoside Rk1 人参皂苷Rk1 494753-69-4 人参 Malvic acid — 503-05-9 人参 Nepetin 泽兰黄酮 520-11-6 人参 Niacin 烟酸 59-67-6 人参 Palmitic acid 棕榈酸 57-10-3 人参 Pentadecylic acid 十五烷酸 1002-84-2 人参 Sallcylic acid 水杨酸 69-72-7 人参 Succinic Acid 琥珀酸 110-15-6 人参 Vitamin H 吡哆素 22879-79-4 人参 Tridecanoic acid 十三烷酸 638-53-9 人参 Deoxygomisin A 戈米辛N 69176-52-9 五味子 Gomisin L1 R(+)-戈米辛M1 82467-50-3 五味子 Gomisin L2 — 82425-44-3 五味子 Longispinogenin — 465-94-1 五味子 Nonylphenol 对壬基酚 104-40-5 五味子 Nordihydroguaiaretic Acid 马索罗酚 27686-84-6 五味子 Psilostachyin A — 3533-47-9 五味子 Schisandrin 五味子醇甲 7432-28-2 五味子 Schisandrin B 五味子乙素 61281-37-6 五味子 Schisanhenol B 五味子酚乙 102681-52-7 五味子 Schizandrin C 五味子丙素 61301-33-5 五味子
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表 3 DZSM治疗CHD的核心靶点
基因名 节点度值 中介中心性 接近中心性 邻域连通性 AKT1 78 0.157 674 9 0.683 673 5 21.54 SRC 56 0.054 896 3 0.598 214 3 25.89 PPARG 55 0.071 025 9 0.595 555 6 23.16 EGFR 54 0.046 872 0 0.590 308 4 25.48 ESR1 52 0.083 235 2 0.585 152 8 23.73 PTGS2 52 0.053 084 9 0.590 308 4 25.54 SIRT1 50 0.043 275 1 0.580 086 6 25.84 MAPK1 44 0.034 526 5 0.558 333 3 26.55 MMP9 43 0.038 211 2 0.565 400 8 28.19 PPARA 40 0.036 245 2 0.556 016 6 24.53 AKT1: AKT丝氨酸/苏氨酸激酶1; SRC: 原癌基因SRC; PPARG: 过氧化物酶体增殖物激活受体γ; EGFR: 表皮生长因子受体; ESR1: 雌激素受体1; PTGS2: 前列腺素内过氧化物合成酶2; SIRT1: 沉默信息调节因子1; MAPK1: 丝裂原活化蛋白激酶1; MMP9: 基质金属蛋白酶9; PPARA: 过氧化物酶体增殖物激活受体α。
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