Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 9: 6055 - 6071

DOI: https://doi.org/10.12418/CJAN2023.556

RESEARCH PAPER

Network Pharmacology Study on Antibacterial Active Ingredients and Mechanism of Sophorae flavescentis Radix

  • WU Yalin , 1 ,
  • CAO Zhigang 1 ,
  • SUN Panpan 1 ,
  • ZHANG Hua 1 ,
  • YIN Wei 1 ,
  • FAN Kuohai 2 ,
  • SUN Yaogui 1 ,
  • LI Hongquan 1 ,
  • SUN Na , 1, *
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  • 1 Shanxi Key Laboratory of Chinese Veterinary Medicine Modernization, Collage of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China
  • 2 Laboratory Animal Center, Shanxi Agricultural University, Taigu 030801, China
* professor, E-mail:

Received date: 2022-12-14

  Online published: 2023-09-13

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Abstract

Based on network pharmacology and molecular docking technology, the main antibacterial active ingredients and pathways of Sophorae flavescentis Radix were explored to provide a theoretical basis for developing as a new feed additive. The active ingredients of Sophorae flavescentis Radix and their related potential targets were screened out by the TCMSP. The antibacterial related targets were obtained from the OMIM and GeneCards databases. The intersection of Sophorae flavescentis Radix and antibacterial targets were obtained through the Venny platform, and the intersection targets were imported into the STRING online analysis platform to construct a protein-protein interaction (PPI) network map and core target analysis. GO functional enrichment and KEGG pathway enrichment analysis were performed through the DAVID database. The affinity between key medicinal ingredients and key antibacterial targets was confirmed by molecular docking method, and the docking results were plotted. There were 24 medicinal ingredients of Sophorae flavescentis Radix, 197 drug targets, 907 antibacterial targets, and 75 intersection targets of the drugs and antibacterial effects were obtained. The protein interaction network analysis showed that 32 proteins including serine/threonine protein kinase (AKT1), interleukin-6 (IL-6), tumor necrosis factor (TNF), interleukin-1β (IL-1β), tumor protein P53 (TP53) may be the key targets of the antibacterial effect of Sophorae flavescentis Radix. The GO function enrichment analysis of the key antibacterial targets of Sophorae flavescentis Radix yielded 616 entries, and the KEGG pathway enrichment analysis yielded 156 pathways. Molecular docking results showed that the top 5 active ingredients (quercetin, luteolin, formononetin, 8-isopentenyl-kaempferol, phaseolin) had good affinity with AKT1, TNF, TP53, and AKT1, TNF, TP53 may be the main targets of antibacterial effect of Sophorae flavescentis Radix. The main medicinal ingredients of Sophorae flavescentis Radix quercetin, luteolin, and formononetin acted on AKT1, TNF, TP53 and other targets, and exerted antibacterial effect through signaling pathways including IL-17, TNF, and C-type lectin receptors. The results of this study show that Sophorae flavescentis Radix achieves antibacterial effect through multiple ingredients, targets, and pathways.

Key words： Sophorae flavescentis Radix; network pharmacology; antibacterial activity; feed additives

Cite this article

WU Yalin , CAO Zhigang , SUN Panpan , ZHANG Hua , YIN Wei , FAN Kuohai , SUN Yaogui , LI Hongquan , SUN Na . Network Pharmacology Study on Antibacterial Active Ingredients and Mechanism of Sophorae flavescentis Radix[J]. Chinese Journal of Animal Nutrition, 2023 , 35(9) : 6055 -6071 . DOI: 10.12418/CJAN2023.556

2020年以前,在畜禽养殖业中广泛用抗生素作为饲料添加剂来提高畜禽生长性能、免疫力和预防细菌性疾病[1]。据报道,每年家禽抗生素销量远远超过人用抗生素,造成抗生素滥用[2]。抗生素的滥用带来了很多负面影响,包括产生耐药性、破坏动物肠道微生态环境和抗生素药物污染[3]。为了减少抗生素带来的负面影响,农业农村部发布“禁抗令”。在这个大背景下,中草药植物开发为新型饲料添加剂成为研究热点。
许多中草药植物提取物口服利用度低[4],进入动物体内只有少部分能被吸收进入血液,而大部分会与肠道菌群接触,从而改变肠道菌群的多样性[5]。中草药植物提取物作为饲料添加剂添加至饲料中已用于养殖生产上,现国内外研究表明,中草药植物提取物作为饲料添加剂的作用机制主要集中在增强免疫力、促生长、调控肠道菌群、缓解应激等方面[6]。目前,博落回散、山花黄芩提取物散、裸花紫珠末等中草药植物提取物已被注册为新兽药饲料添加剂,这些饲料添加剂具有抗菌、消炎、开胃、促生长等功能。
苦参,豆科槐属植物,其性味苦、寒,归心脏、肝脏、胃、大肠、膀胱经,具有利尿、杀虫、清热燥湿和抗菌等功效。有研究证明,苦参提取物有抑菌及促生长作用[7-8],但苦参具体的抑菌药效成分及其作用机制尚未知。所以本研究将从抑菌方面入手,探究苦参提取物的关键抑菌靶点及作用机制,并为将其开发为新型饲料添加剂提供理论基础。本研究使用中药系统药理数据库与分析平台(TCMSP)预测苦参药效成分及靶点,从疾病数据库收集抑菌潜在靶点,利用基因本体(GO)功能和京都基因与基因组百科全书(KEGG)富集分析来寻找潜在作用靶点和代谢通路,利用分子对接来验证苦参核心药效成分与核心靶点之间的相互结合的稳定性,最终确定苦参抑菌的核心成分、核心靶点与通路。

1 材料与方法

1.1 数据库平台

网络药理学研究所用数据库网址见表1。
表1 本研究所用数据库网址

Table 1 Database website used in this study

数据库 Databases 网址 Website
中药系统药理数据库与分析平台 TCMSP https://lsp.nwu.edu.cn/tcmsp.php
PubChem https://pubchem.ncbi.nlm.nih.gov/
Uniprot https://www.uniprot.org/
Genecards https://www.genecards.org/
OMIM https://omim.org/
Venny https://bioinfogp.cnb.csic.es/tools/venny/
String https://www.string-db.org/
DAVID https://david.ncifcrf.gov/tools.jsp

1.2 试验方法

1.2.1 苦参药效成分筛选

苦参的化学成分通过TCMSP进行搜索,依据口服生物利用度(OB)≥30%和类药性(DL)≥0.18筛选苦参药效成分,并获取所筛选苦参药效成分的潜在靶点,苦参药效成分的关键信息通过PubChem数据库搜索。

1.2.2 苦参药效成分-靶点网络图的构建

将筛选到的苦参药效成分的潜在靶点转换为统一的基因名称,并且利用Cytoscape 3.7.0软件将苦参药效成分及其作用靶点构建成苦参药效成分-靶点网络图。

1.2.3 抑菌相关基因检索

利用GeneCards和OMIM数据库筛选抑菌相关靶点,合并2个数据库中所检索的基因后,删除重复项,即完成抑菌相关基因的收集。

1.2.4 苦参抑菌作用的基因靶点筛选

将抑菌相关靶点与苦参潜在作用靶点上传到Venny 2.1软件分析得到两者的交叉靶点并绘制成韦恩图,筛选与苦参抗菌相关的基因,利用Cytoscape 3.7.0软件将其可视化构建药物-成分-疾病-靶点网络图。

1.2.5 苦参抑菌的关键靶点蛋白相互作用(PPI)网络图的构建

将交集的靶点导入至STRING数据库,进行PPI预测分析,将获得PPI关系网络图导入Cytoscape 3.7.0软件,进行拓扑学分析。以2倍中位数的度(degree)、节点紧密度(closeness)与节点连接度(betweeness)的中位数作为筛选标准,以筛选关键靶点。

1.2.6 苦参抑菌作用靶点GO功能和KEGG富集分析

使用DAVID数据库对苦参抑菌靶点进行GO功能和KEGG富集分析,调整筛选标准为P≤0.05,将GO功能富集分析排名前10的条目制成统计柱状图,选取KEGG富集分析排名前20的信号通路绘制富集显著性气泡图,以探讨苦参抑菌靶点在基因功能和信号通路中的作用。

1.2.7 分子对接

选取苦参药效成分中的5个抑菌关键靶点和5个苦参关键药效成分进行对接。从PBD数据库下载关键抑菌靶点蛋白结构。苦参的关键药效成分的分子结构从PubChem数据库中下载。靶点蛋白和关键药效成分分子的前期处理利用PyMol 2.4软件和Autodock Tools 1.5.7软件完成。之后将关键抑菌靶点和药效成分结构导入Autodock Tools 1.5.7软件中进行分子对接验证试验,其活性口袋位置取决于原配体位置,结合能越小代表对接越稳定,若结合能<0 kcal/mol,表示蛋白与小分子能够自发地结合;结合能≤-5.0 kcal/mol,则表示结合活性良好。选取结合能≤-5.0 kcal/mol的蛋白-小分子对接,通过PyMol 2.4软件进行对接结果可视化。

2 结果与分析

2.1 苦参所含药效成分

运用TCMSP数据库搜索得到113个化学成分、以OB≥30%、DL≥0.18为前提,筛选出24个药效成分,如表2所示。药效成分主要为黄酮类化合物、生物碱类化合物等。
表2 苦参主要药效成分

Table 2 Main medicinal ingredients of Sophorae flavescentis Radix

编号
ID		 分子名称
Molecular name		 口服生物利用度
OB/%		 类药性
DL		 度
Degree
MOL000098 槲皮素 Quercetin 46.43 0.28 148
MOL000006 木犀草素 Luteolin 36.16 0.25 56
MOL000392 刺芒柄花素 Formononetin 69.67 0.21 36
MOL003542 8-异戊烯基山奈酚8-isopentenyl-kaempferol 38.04 0.39 26
MOL000456 菜豆素 Phaseolin 78.20 0.73 23
MOL003673 Wighteone 42.80 0.36 17
MOL003648 (+)-高丽槐素 Inermin 65.83 0.54 16
MOL006596 大豆抗毒素 Glyceollin 97.27 0.76 16
MOL001484 (-)-高丽槐素 Inermine 75.18 0.54 15
MOL004941 甘草素(2R)-7-hydroxy-2-(4-hydroxyphenyl)chroman-4-one 71.12 0.18 13
MOL006613 苦参醇 Ckushenin 47.62 0.38 13
MOL006604 (2S)-7-hydroxy-2-(4-hydroxyphenyl)-5-
methoxy-8-(3-methylbut-2-enyl)chroman-4-one		 48.09 0.39 12
MOL005944 苦参碱 Matrine 63.77 0.25 11
MOL001040 柚皮素
(2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one		 42.36 0.21 8
MOL005100 二氢黄酮甙
5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one		 47.74 0.27 8
MOL006626 里查酮G Leachianone G 60.97 0.40 8
MOL004580 花旗松素 Cis-dihydroquercetin 66.44 0.27 6
MOL006620 苦参醇J Kushenol J 50.86 0.24 6
MOL003347 贯叶金丝桃素 Hyperforin 44.03 0.60 5
MOL006623 苦参醇T Kushenol T 51.28 0.64 5
MOL006630 桑叶活性黄酮 Norartocarpetin 54.93 0.24 4
MOL003627 槐果碱 Sophocarpine 64.26 0.25 3
MOL003680 槐定碱 Sophoridine 60.07 0.25 3
MOL006650 (-)-maackiain-3-O-glucosyl-6'-O-malonate 48.69 0.52 2

2.2 苦参药效成分-靶点网络图的构建

苦参药效成分的作用靶点利用TCMSP数据库收集并去除重复项,共获得药效成分作用靶点197个。苦参药效成分-靶点网络图的构建通过Cytoscape 3.7.0软件完成,如图1所示。
图1 苦参药效成分-靶点网络图

PSMD3:蛋白酶体26S非腺嘌呤核苷三磷酸酶调节亚基3 26S proteasome non-ATPase regulatory subunit 3;HSD3B1:3β羟基类固醇脱氢酶/δ5-4异构酶1型 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase type 1;HSD3B2:3β羟基类固醇脱氢酶/δ5-4异构酶2型 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase type 2;HTR3A:5-羟色胺受体3A 5-hydroxytryptamine receptor 3A;MMP2:基质金属蛋白酶2 matrix metalloproteinase 2;HSPA5:热休克蛋白5 heat shock protein 5;ACHE:乙酰胆碱酯酶 acetylcholinesterase;ACACA:乙酰辅酶A羧化酶 acetyl-CoA carboxylase;AHSA1:热休克90 kDa蛋白ATP酶同源物1(酵母)激活因子 activator of 90 kDa heat shock protein ATPase homolog 1;APC2:腺瘤性多发性大肠杆菌蛋白2 adenomatous polyposis coli protein 2;ADCY2:腺苷酸环化酶2 adenylate cyclase type 2;AKR1C3:醛固酮类还原酶家族1成员C3 Aldo-keto reductase family 1 member C3;AKR1B1:醛糖还原酶 aldose reductase;AAGAB:α-γ衔接蛋白p34 alpha- and gamma-adapt in-binding protein p34;ADRA1A:α-1A肾上腺素能受体 alpha-1A adrenergic receptor;ADRA1B:α-1B肾上腺素能受体 alpha-1B adrenergic receptor;ADRA1D:α-1D肾上腺素能受体 alpha-1D adrenergic receptor;ADRA2A:α-2A肾上腺素能受体 alpha-2A adrenergic receptor;ADRA2B:α-2B肾上腺素能受体 alpha-2B adrenergic receptor;ADRA2C:α-2C肾上腺素能受体 alpha-2C adrenergic receptor;MAOA:胺氧化酶A amine oxidase [flavin-containing] A;MAOB:胺氧化酶B amine oxidase [flavin-containing] B;APP:β淀粉样蛋白A4 amyloid beta A4 protein;AR:雄激素受体 androgen receptor;BAX:细胞凋亡调节因子 apoptosis regulator;BCL2:细胞凋亡调节因子2 apoptosis regulatory factor 2;ALOX5:花生四烯酸5-脂氧合酶 arachidonate 5-lipoxygenase;AHR:芳香烃受体 aryl hydrocarbon receptor;ATP5F1B:ATP合酶β,线粒体 ATP synthase subunit beta, mitochondrial;ABCG2:腺苷三磷酸结合盒转运蛋白G2 ATP-binding cassette sub-family G member 2;XIAP:抑制细胞凋亡样蛋白 baculoviral IAP repeat-containing protein;BIRC5:抑制细胞凋亡样蛋白5 baculoviral IAP repeat-containing protein 5;BAD:BCL2细胞死亡拮抗剂 BCL2 antagonist of cell death;BCL2L1:BCL-2样蛋白1 BCL-2-like protein 1;ADRB1:肾上腺素β1受体 beta-1 adrenergic receptor;ADRB2:肾上腺素β2受体 beta-2 adrenergic receptor;KCNMA1:钙激活钾通道蛋白α1 calcium-activated potassium channel subunit alpha 1;PKIA:cAMP环磷酸腺苷依赖的蛋白激酶抑制因子α cAMP-dependent protein kinase inhibitor alpha;CA2:碳酸酐酶2 carbonic anhydrase 2;CASP3:半胱氨酸-天冬氨酸蛋白酶3 caspase 3;CASP7:半胱氨酸-天冬氨酸蛋白酶7 caspase 7;CASP8:半胱氨酸-天冬氨酸蛋白酶8 caspase 8;CASP9:半胱氨酸-天冬氨酸蛋白酶9 caspase 9;CTSD:组织蛋白酶D cathepsin D;CAV1:微囊蛋白1 caveolin 1;CCL2:CC基序趋化因子配体2 CC motif chemokine 2;CD40LG:CD40配体 CD40 ligand;CD44:CD44抗原 CD44 antigen;CDK2:细胞分裂蛋白激酶2 cell division protein kinase 2;CDK4:细胞分裂蛋白激酶4 cell division protein kinase 4;CDK6:细胞分裂蛋白激酶6 cell division protein kinase 6;TP53:肿瘤蛋白p53 cellular tumor antigen p53;CTRB1:胰凝乳蛋白酶B1 chymotrypsinogen B1;CLDN4:紧密链接蛋白4 claudin 4;F7:凝血因子7 coagulation factor Ⅶ;COL1A1:1型α-1胶原蛋白链 collagen alpha-1(I) chain;COL3A1:3型α-1胶原蛋白链 collagen alpha-1(Ⅲ) chain;CRP:C反应蛋白 C-reactive protein;CXCL10:趋化因子10(C-X-C基序) C-X-C motif chemokine 10;CXCL11:趋化因子11(C-X-C基序) C-X-C motif chemokine 11;CXCL2:趋化因子2(C-X-C基序) C-X-C motif chemokine 2;CCNA2:周期蛋白A2 cyclin-A2;CDKN1A:细胞周期蛋白依赖性激酶抑制剂1A cyclin-dependent kinase inhibitor 1A;CDKN2A:细胞周期蛋白依赖性激酶抑制剂2A cyclin-dependent kinase inhibitor 2A;CYCS:细胞色素C cytochrome C;CYP19A1:细胞色素P450家族19亚族A多肽1 cytochrome P450 19A1;CYP1A1:细胞色素P450家族1亚族A多肽1 cytochrome P450 1A1;CYP1A2:细胞色素P450家族1亚族A多肽2 cytochrome P450 1A2;CYP1B1:细胞色素P450家族1亚族B多肽1 cytochrome P450 1B1;CYP3A4:细胞色素P450家族3亚族A多肽4 cytochrome P450 3A4;DCAF5:DDB1与CUL4蛋白链接因子5 DDB1- and CUL4-associated factor 5;DPP4:二肽激肽酶-4 dipeptidyl peptidase IV;TOP1:DNA拓扑异构酶1 DNA topoisomerase 1;TOP2A:DNA拓扑异构酶2α DNA topoisomerase 2-alpha;ALG5:多萜基磷酸β-葡糖基转移酶5 dolichyl-phosphate beta-glucosyltransferase 5;DUOX2:双氧化酶2 dual oxidase 2;EGFR:表皮生长因子受体 epidermal growth factor receptor;SELE:选择素E E-selectin;ESR1:雌激素受体1 estrogen receptor 1;ESR2:雌激素受体β2 estrogen receptor beta 2;SULT1E1:类固醇硫酸酯酶 estrogen sulfotransferase;ELK1:转录激活因子ETS样蛋白Elk-1 ETS domain-containing protein Elk-1;EEF1E1:真核翻译延伸因子1 eukaryotic translation elongation factor 1 epsilon-1;EIF6:真核翻译起始因子6 eukaryotic translation initiation factor 6;CCND1:周期蛋白D1 G1/S-specific cyclin-D1;CCND2:周期蛋白D2 G1/S-specific cyclin-D2;CCNB1:周期蛋白B1 G2/mitotic-specific cyclin-B1;GABRA1:γ氨基丁酸亚基受体α1 gamma-aminobutyric acid receptor subunit alpha-1;GABRA2:γ氨基丁酸亚基受体α2 gamma-aminobutyric-acid receptor alpha-2 subunit;NR3C1:糖皮质激素受体 glucocorticoid receptor;G6PC2:葡萄糖-6-磷酸酶 glucose-6-phosphatase;GSTM1:谷胱甘肽S转移酶Mu 1 glutathione S-transferase Mu 1;GSTM2:谷胱甘肽S转移酶Mu 2 glutathione S-transferase Mu 2;GSTP1:谷胱甘肽S转移酶P glutathione S-transferase P;PYGM:肌肉糖原磷酸化酶 glycogen phosphorylase muscle form;GSK3B:糖原合酶激酶3β glycogen synthase kinase 3 beta;HSF1:热休克因子蛋白1 heat shock factor protein 1;HSPB1:热休克蛋白β1 heat shock protein beta 1;HMOX1:血红素氧合酶1 heme oxygenase 1;HPSE:乙酰肝素酶 heparanase;MET:肝细胞生长因子受体 hepatocyte growth factor receptor;HK2:己糖激酶2 hexokinase 2;MS4A2:高亲和力免疫球蛋白ε受体亚基β high affinity immunoglobulin epsilon receptor subunit beta;HAS2:透明质酸合成酶2 hyaluronan synthase 2;HIF1A:缺氧诱导因子1α hypoxia-inducible factor 1 alpha;IER3IP1:即早反应蛋白3相互作用蛋白1 immediate early response 3-interacting protein 1;MCL1:诱导骨髓白血病细胞分化蛋白1 induced myeloid leukemia cell differentiation protein 1;CHUK:核因子抑制剂kappa-B激酶亚基α inhibitor of nuclear factor kappa-B kinase subunit alpha;INS:胰岛素 insulin;INSR:胰岛素受体 insulin receptor;IGF1R:胰岛素样生长因子受体1 insulin-like growth factor 1 receptor;IGF2:胰岛素样生长因子2 insulin-like growth factor 2;IGFBP3:胰岛素样生长因子结合蛋白3 insulin-like growth factor-binding protein 3;ICAM1:细胞间黏附分子1 intercellular adhesion molecule 1;IFNG:干扰素γ interferon gamma;IRF1:干扰素调节因子1 interferon regulatory factor 1;IL-1α:白细胞介素-1α interleukin-1 alpha;IL-1β:白细胞介素-1β interleukin-1 beta;IL-10:白细胞介素-10 interleukin-10;IL-13:白细胞介素-13 interleukin-13;IL-2:白细胞介素-2 interleukin-2;IL-4:白细胞介素-4 interleukin-4;IL-6:白细胞介素-6 interleukin-6;IL-8:白细胞介素-8 interleukin-8;MMP1:间质胶原酶1 interstitial collagenase 1;ALPI:肠道碱性磷酸酶 intestinal-type alkaline phosphatase;FCER2:低亲和力免疫球蛋白εFc受体2 low affinity immunoglobulin epsilon Fc receptor 2;MMP9:基质金属蛋白酶9 matrix metalloproteinase 9;NR3C2:盐皮质激素受体 mineralocorticoid receptor;MAPK1:丝裂原活化蛋白激酶1 mitogen-activated protein kinase 1;MAPK14:丝裂原活化蛋白激酶14 mitogen-activated protein kinase 14;CHRM1:毒蕈碱型乙酰胆碱受体M1 muscarinic acetylcholine receptor M1;CHRM2:毒蕈碱型乙酰胆碱受体M2 muscarinic acetylcholine receptor M2;CHRM3:毒蕈碱型乙酰胆碱受体M3 muscarinic acetylcholine receptor M3;MPO:过氧化酶 myeloperoxidase;NQO1:NAD(P)H醌脱氢酶1 NAD(P)H dehydrogenase [quinone] 1;MT-ND6:NADH复合体6 NADH-ubiquinone oxidoreductase chain 6;POR:NADPH细胞色素P450还原酶 NADPH-cytochrome P450 reductase;NCF1:中性粒细胞胞质因子1 neutrophil cytosol factor 1;IKBKG:NF-kappa-B基本调节剂 NF-kappa-B essential modulator;NFKBIA:NF-kappa-B抑制剂α NF-kappa-B inhibitor alpha;NOS3:一氧化氮合酶3 nitric oxide synthase 3;NOS2:一氧化氮合酶2 nitric oxide synthase 2;NFE2L2:核因子相关因子2 nuclear factor erythroid 2-related factor 2;NCOA1:核受体共激活因子1 nuclear receptor coactivator 1;NCOA2:核受体共激活因子2 nuclear receptor coactivator 2;ODC1:鸟氨酸脱羧酶1 ornithine decarboxylase 1;SPP1:骨桥蛋白1 osteopontin 1;PPARG:过氧化物酶体增值物活化受体γ peroxisome proliferator activated receptor gamma;PPARA:过氧化物酶体增值物活化受体α peroxisome proliferator-activated receptor alpha;PPARD:过氧化物酶体增值物活化受体δ peroxisome proliferator-activated receptor delta;SERPINE1:纤溶酶原激活物抑制剂1 plasminogen activator inhibitor 1;PARP1:多聚ADP核糖聚合酶1 poly [ADP-ribose] polymerase 1;PCOLCE1:前胶原C内肽酶增强子1 procollagen C-endopeptidase enhancer 1;EGF:表皮生长因子 pro-epidermal growth factor;PGR:孕激素受体 progesterone receptor;PCNA:增殖细胞核抗原 proliferating cell nuclear antigen;PTGES:前列腺素E合成酶 prostaglandin E synthase;PTGER3:前列腺素E2受体EP3 prostaglandin E2 receptor EP3 subtype;PTGS1:前列腺素内过氧化物合酶1 prostaglandin G/H synthase 1;PTGS2:前列腺素内过氧化物合酶2 prostaglandin G/H synthase 2;ACP3:前列腺酸性磷酸酶3 prostatic acid phosphatase 3;PSME3:蛋白酶体激活因子复合体亚基3 proteasome activator complex subunit 3;PRKCA:蛋白激酶C-α protein kinase C alpha type;PRKCB:蛋白激酶C-β protein kinase C beta type;FOS:转录因子c-Fos proto-oncogene c-Fos;NPEPPS:嘌呤霉素敏感性氨肽酶 puromycin-sensitive aminopeptidase;AKT1:丝氨酸/苏氨酸蛋白激酶1 serine/threonine-protein kinase 1;RAF1:原癌基因丝氨酸/苏氨酸蛋白激酶 RAF proto-oncogene serine/threonine-protein kinase;RASSF1:包含Ras关联域的蛋白1 Ras association domain-containing protein 1;RASA1:GTP酶活化蛋白1 Ras GTPase-activating protein 1;ERBB2:受体酪氨酸蛋白激酶2 receptor tyrosine-protein kinase 2;ERBB3:受体酪氨酸蛋白激酶3 receptor tyrosine-protein kinase 3;RB1:视网膜母细胞瘤相关蛋白1 retinoblastoma-associated protein 1;RXRA:维甲酸X受体α retinoic acid receptor RXR-alpha;RXRB:维甲酸X受体β retinoic acid receptor RXR-beta;RUNX2:Runt相关转录因子2 Runt-related transcription factor 2;CHEK1:丝氨酸/苏氨酸蛋白激酶1 serine/threonine-protein kinase 1;CHEK2:丝氨酸/苏氨酸蛋白激酶2 serine/threonine-protein kinase 2;PON1:对氧磷酸1 serum paraoxonase/arylesterase 1;STAT1:信号转导子与转录激活子1 signal transducer and activator of transcription 1;SCN10A:钠通道蛋白10亚基α sodium channel protein type 10 subunit alpha;SCN5A:钠通道蛋白5亚基α sodium channel protein type 5 subunit alpha;SLC5A5:钠/碘同向转运蛋白5 sodium/iodide cotransporter 5;FXYD2:钠/钾转运ATP酶亚基γ 2 sodium/potassium-transporting ATPase subunit gamma 2;SLC6A3:钠依赖性多巴胺转运蛋白 sodium-dependent dopamine transporter;SLC6A2:钠依赖性去甲肾上腺素转运蛋白 sodium-dependent noradrenaline transporter;SLC6A4:钠依赖性5-羟色胺转运体 sodium-dependent serotonin transporter;SOD1:超氧化物歧化酶1 superoxide dismutase 1;THBD:血栓调节蛋白 thrombomodulin;F3:组织因子 tissue factor;PLAT:组织型纤溶酶原激活剂 tissue-type plasminogen activator;JUN:转录因子AP-1 transcription factor AP-1;E2F1:转录因子E2F1 transcription factor E2F1;E2F2:转录因子E2F2 transcription factor E2F2;RELA:转录因子p65 transcription factor p65;TGFB1:转化生长因子β1 transforming growth factor beta-1;TRPM2:瞬时受体电位阳离子通道亚家族M成员2 transient receptor potential cation channel subfamily M member 2;PRSS1:胰蛋白酶1 trypsin-1;TNF:肿瘤坏死因子 tumor necrosis factor;DIO1:Ⅰ型碘甲状腺原氨酸脱碘酶 type I iodothyronine deiodinase;TYR:酪氨酸酶 tyrosinase;PLAU:尿激酶型纤溶酶原激活物 urokinase-type plasminogen activator;VCAM1:血管细胞黏附蛋白1 vascular cell adhesion protein 1;VEGFA:血管内皮生长因子A vascular endothelial growth factor A;KDR:血管内皮生长因子受体 vascular endothelial growth factor receptor;XDH:黄嘌呤脱氢酶 xanthine dehydrogenase/oxidase。下同 the same as below。

绿色V型形代表中药,蓝色菱形代表苦参的药效成分,橙色圆形代表苦参潜在作用靶点。

Fig.1 Medicinal ingredients-target network of Sophorae flavescentis Radix

The green shapes represent Chinese medicine, the blue diamonds represent the medicinal ingredients of Sophorae flavescentis Radix, and the orange circles represent the potential targets of Sophorae flavescentis Radix.

2.3 苦参药物-成分-疾病-靶点网络图的构建

利用GeneCards和OMIM数据库筛选抑菌相关靶点去除重复靶点后得到907个靶点。苦参潜在作用靶点与抑菌相关靶点取交集后得到75个交集靶点,如图2所示。通过Cytoscape 3.7.0软件构建药物-成分-疾病-靶点的网络图(图3)。作用靶点越多的药效成分,其在生理生化作用中越关键(表3)。筛选出排名前5位的关键药效成分,分别为槲皮素、木犀草素、刺芒柄花素、8-异戊烯基山奈酚、菜豆素。
图2 苦参与抑菌靶点韦恩图

Fig.2 Venn diagram of Sophorae flavescentis Radix and antibacterial targets

图3 药物-成分-疾病-靶点网络图

苦参药效成分为蓝色菱形,苦参为绿色的V形,交集靶点为橙色圆形,抑菌为紫色三角形。

Fig.3 Drugs-ingredients-diseases-targets network diagram

The blue diamonds represent the medicinal ingredients of Sophorae flavescentis Radix, the green V shape represents Sophorae flavescentis Radix, the orange circles represent the intersection targets, and the purple triangle represents bacteriostasis.

表3 苦参药效成分对应的抑菌靶点

Table 3 Antibacterial targets corresponding to medicinal ingredients of Sophorae flavescentis Radix

药效成分 Medicinal ingredients 抑菌靶点基因 Antibacterial target genes
槲皮素 Quercetin MMP2、BAX、BCL2、ALOX5、AHR、BCL2L1、CASP3、CD40LG、TP53、CRP、CXCL10、CXCL11、
CXCL2、CYP3A4、DPP4、TOP1、TOP2A、DUOX2、EGFR、SELE、SULT1E1、CCND1、GSTM1、
CHUK、ICAM1、IFNG、IRF1、IL-1α、IL-1β、IL-10、IL-2、IL-6、MMP1、MMP9、MAPK1、MYC、
MPO、NCF1、NFKBIA、NFE2L2、NR1I2、ODC1、SPP1、PPARG、PTEN、SERPINE1、
PARP1、KCNH2、PTGS1、PTGS2、PRKCA、PRKCB、AKT1、
STAT1、SOD1、JUN、TGFB1、TNF、XDH
木犀草素 Luteolin MMP2、XIAP、BCL2L1、CASP3、CD40LG、CDK4、TP53、DPP4、TOP1、TOP2A、EGFR、
CCND1、MCL1、ICAM1、IFNG、IL-10、IL-2、IL-4、IL-6、MMP1、MMP9、MAPK1、NFKBIA、
PPARG、PTGS1、PTGS2、AKT1、JUN、TNF、XDH
刺芒柄花素 Formononetin DPEP1、DPP4、ESR1、ESR2、IL-4、MAPK14、SIRT1、NOS2、PPARG、
PTGS1、PTGS2、SLC6A4、JUN
8-异戊烯基山奈酚
8-isopentenyl-kaempferol		 KCNMA1、DPP4、ESR1、MAPK14、PPARG、KCNH2、PTGS1、PTGS2
菜豆素 Phaseolin ADRA1D、ESR1、ESR2、MAPK14、CHRM3、NOS2、PPARG、PTGS1、PTGS2
Wighteone DPP4、ESR1、ESR2、MAPK14、NOS2、PPARG、PTGS2
高丽槐素 Inermin HTR3A、ADRA1D、CHRM3、PTGS1、PTGS2、SLC6A4
大豆抗毒素 Glyceollin KCNMA1、ESR1、ESR2、NOS2、PTGS1、PTGS2
高丽槐素 Inermine HTR3A、ADRA1D、CHRM3、PTGS1、PTGS2
甘草素
(2R)-7-hydroxy-2-(4-hydroxyphenyl)chroman-4-one		 DPEP1、ESR1、PTGS1、PTGS2、SLC6A4
苦参素 Kushenin DPP4、ESR1、ESR2、NOS2、PTGS1、PTGS2
异黄腐酚
(2S)-7-hydroxy-2-(4-hydroxyphenyl)-5-methoxy-8-(3-methylbut-2-enyl)chroman-4-one		 ESR1、NOS2、KCNH2、PTGS2
苦参碱 Matrine MMP2,CASP3、ICAM1、IL-6、MYC、TNF
柚皮素
(2R)-5,7-dihydroxy-2-(4-
hydroxyphenyl)chroman-4-one		 DPEP1、ESR1、PGR、PTGS1、PTGS2
二氢黄酮甙
5,7-dihydroxy-2-(3-hydroxy-4-
methoxyphenyl)chroman-4-one		 PTGS1、PTGS2
里查酮G Leachianone G ESR1、NOS2、PTGS2
花旗松素 Cis-dihydroquercetin PTGS1、PTGS2
苦参醇J Kushenol J PTGS1、PTGS2
贯叶金丝桃素 Hyperforin CYP3A4、ICAM1、NR1I2
苦参醇T Kushenol T PTGS2
桑叶活性黄酮 Norartocarpetin PTGS1、PTGS2
槐果碱 Sophocarpine IL-6、TNF
槐定碱 Sophoridine IL-6、TNF
(-)-maackiain-3-O-glucosyl-6'-
O-malonate		 TOP2A

2.4 苦参抑菌的PPI网络图的构建

利用STRING数据库将75个交集靶点进行PPI分析,其中1个靶点蛋白上的连线越多,说明与其相互作用的蛋白也越多,该蛋白的等级越高。将构建的PPI网络图导入Cytoscape 3.7.0软件中并进行拓扑分析。如图4所示,此网络图包括30个节点、1 070条相互关系,等级越高的靶点颜色越深,节点越大。根据靶点的degree值排名(表4)可知,筛选排名前5的关键靶点用于后续分子对接。
图4 苦参关键抑菌靶点PPI网络图

通过拓扑分析,确定苦参抑菌相关靶点的PPI网络,颜色越深,圆圈越大,其度值越高。

Fig.4 PPI network of key antibacterial targets of Sophorae flavescentis Radix

Through topological selection, the PPI network of targets related to the antibacterial effect of Sophorae flavescentis was determined, and the darker color, the bigger circle, the higher degree value.

表4 苦参抑菌STRING网络关键抑菌靶点

Table 4 Key antibacterial targets of STRING network of Sophorae flavescentis Radix

序号
No.		 基因名称
Gene name		 度
Degree		 序号
No.		 基因名称
Gene name		 度
Degree
1 AKT1 62 39 CD40LG 28
2 IL-6 62 40 SELE 28
3 TNF 60 41 SPP1 27
4 IL-1β 58 42 CHUK 25
5 TP53 58 43 PGR 25
6 JUN 56 44 CDK4 24
7 PTGS2 54 45 CXCL2 24
8 CASP3 53 46 IRF1 24
9 MMP9 51 47 XIAP 24
10 MYC 49 48 NCF1 23
11 EGFR 46 49 PRKCA 23
12 NFKBIA 46 50 AHR 22
13 PPARG 46 51 DPP4 20
14 ESR1 45 52 BAX 19
15 CCND1 42 53 ESR2 19
16 ICAM1 42 54 CXCL11 18
17 IFNG 40 55 SOD1 18
18 IL-10 40 56 ALOX5 15
19 IL-2 40 57 CYP3A4 14
20 STAT1 40 58 TOP2A 14
21 IL-4 39 59 BCL2 13
22 MMP2 39 60 NR1I2 12
23 PTEN 39 61 PRKCB 12
24 BCL2L1 37 62 TOP1 12
25 SIRT1 35 63 PTGS1 10
26 IL-1α 33 64 DUOX2 9
27 TGFB1 33 65 GSTM1 9
28 CXCL10 32 66 ODC1 7
29 MAPK1 32 67 XDH 7
30 MAPK14 32 68 SLC6A4 6
31 MPO 32 69 SULT1E1 4
32 CRP 31 70 HTR3A 3
33 NFE2L2 31 71 ADRA1D 2
34 NOS2 31 72 KCNH2 2
35 SERPINE1 31 73 KCNMA1 2
36 MCL1 30 74 CHRM3 1
37 MMP1 29 75 DPEP1 1
38 PARP1 29

2.5 苦参GO功能富集分析

75个苦参关键抑菌靶点利用DAVID数据库进行GO功能富集分析,共获取616条GO条目,分别选取前10个分析条目绘制柱状图,结果如图5所示。苦参抑菌靶点的功能通过生物过程(biological process)、分子功能(molecular function)和细胞组分(cellular component)3个部分呈现。生物过程主要涉及细胞因子介导的信号通路(cytokine-mediated signaling pathway)、炎症反应(inflammatory response)、对药物的反应(response to drug)、凋亡过程的负调控(negative regulation of apoptotic process)、对异物刺激的反应(response to xenobiotic stimulus)、缺乏配体的外源性凋亡信号通路(extrinsic apoptotic signaling pathway in absence of ligand)、基因表达的正向调控(positive regulation of gene expression)、凋亡过程的正向调控(positive regulation of apoptotic process)等496个;细胞组分主要分布于细胞外空间(extracellular space)、核质(nucleoplasm)、大分子复合物(macromolecular complex)、细胞外区(extracellular region)、细胞质(cytoplasm)、胞质溶胶(cytosol)、染色质(chromatin)、线粒体(mitochondrion)等40个;分子功能主要体现在酶结合(enzyme binding)、蛋白结合(protein binding)、细胞因子活性(cytokine activity)、转录因子结合(transcription factor binding)、蛋白质同源二聚化活性(protein homodimerization activity)、BH3结构域结合(BH3 domain binding)、序列特异性 DNA 结合(sequence-specific DNA binding)等80个。
图5 苦参抑菌靶点的GO功能富集分析

Biological process:生物过程;cytokine-mediated signaling pathway:细胞因子介导的信号通路;inflammatory response:炎症反应;response to drug:对药物的反应;negative regulation of apoptotic process:凋亡过程的负调控;response to xenobiotic stimulus:对异物刺激的反应;extrinsic apoptotic signaling pathway in absence of ligand:缺乏配体的外源性凋亡信号通路;positive regulation of gene expression:基因表达的正向调控;positive regulation of apoptotic process:凋亡过程的正调控; positive regulation of transcription from RNA polymerase Ⅱ promoter:RNA聚合酶Ⅱ启动子对pri-miRNA转录的正调控;positive regulation of transcription, DNA-template:转录的正调控,DNA模板;cellular component:细胞组分;extracellular space:细胞外空间;nucleoplasm:核质;macromolecular complex:大分子复合物;extracellular region:细胞外区;cytoplasm:细胞质;cytosol:胞质溶胶;chromatin:染色质;mitochondrion:线粒体;transcription factor complex:转录因子复合物;nuclear membrane:核膜;molecular function:分子功能;enzyme binding:酶结合;protein binding:蛋白结合;cytokine activity:细胞因子活性;transcription factor binding:转录因子结合;protein homodimerization activity:蛋白质同源二聚化活性;BH3 domain binding: BH3结构域结合;sequence-specific DNA binding:序列特异性DNA结合;identical protein binding:相同蛋白结合;RNA polymerase Ⅱ transcription factor activity, ligand-activated sequence-specific DNA binding:RNA聚合酶Ⅱ转录因子活性,配体激活特殊DNA结合序列;steroid binding:胆固醇结合。

Fig.5 GO functional enrichment analysis of antibacterial targets of Sophorae flavescentis Radix

2.6 KEGG通路富集分析

为了深入探讨苦参抑菌的潜在机制,75个苦参抑菌靶点的KEGG通路富集分析利用DAVID数据库进行,获得156条通路抑菌相关通路,选取前20条通路绘制气泡图(图6)。结果显示,苦参抑菌靶点涉及多条与抑菌密切相关的信号通路,主要涉及肿瘤坏死因子信号通路(TNF signaling pathway)、白细胞介素-17信号通路(IL-17 signaling pathway)、C型凝集素受体信号通路(C-type lectin receptor signaling pathway)、晚期糖基化终末化产物信号通路在糖尿病并发症中的作用(AGE-RAGE signaling pathway in diabetic complications)等。
图6 苦参关键抑菌靶点KEGG通路富集分析

TNF signaling pathway:肿瘤坏死因子信号通路;IL-17 signaling pathway:白细胞介素-17信号通路;C-type lectin receptor signaling pathway:C型凝集素受体信号通路;AGE-RAGE signaling pathway in diabetic complications:晚期糖基化终末化产物信号通路在糖尿病并发症中的作用;Pathways in cancer:癌症途径;Lipid and atherosclerosis:血脂和动脉粥样硬化;Leishmaniasis:利什曼病;Toxoplasmosis:弓形虫病;Hepatitis B:乙型肝炎;Small cell lung cancer:肺小细胞癌;Fluid shear stress and atherosclerosis:流体剪切应力与动脉粥样硬化;Chagas disease:美洲锥虫病;Measles:麻疹;Influenza A:甲型流感;Endocrine resistance:抵抗内分泌;Human T-cell leukemia virus 1 infection:人类T细胞白血病;Human cytomegalovirus infection:人类巨细胞病毒感染;Kaposi sarcoma-associated herpesvirus infection:卡波西肉瘤相关疱疹病毒;Epstein-Barr virus infection:EB病毒感染;Hepatitis C:丙型肝炎。

Fig.6 KEGG pathway enrichment analysis of key antibacterial targets of Sophorae flavescentis Radix

2.7 分子对接

将关键苦参药效成分槲皮素、木犀草素、刺芒柄花素、8-异戊烯基山奈酚、菜豆素与关键抑菌靶点AKT1、IL-6、TNF、IL-1β、TP53编码的蛋白分子进行对接,结果见表5,所有组合对接结合能均小于0 kcal/mol,表明蛋白与小分子能够自发结合。其中结合能≤-5.0 kcal/mol的蛋白-小分子对接结果3D示意图见图7~图11,AKT1、TNF、TP53与槲皮素(quercetin)、木犀草素(luteolin)、刺芒柄花素(formononetin)、8-异戊烯基山奈酚(8-isopentenyl-kaempferol)、菜豆素(phaseolin)对接结合能较低,可形成稳定的复合物。因此,AKT1、TNF、TP53为苦参较好的结合靶点。
表5 苦参核心药效成分与关键抑菌靶点的结合能

Table 5 Binding energy of core medicinal ingredients of Sophorae flavescentis Radix and key antibacterial targetskcal/mol

分子名称
Molecular
names		 化学式
Chemical
formula		 PubChem
编号
PubChem
ID		 与AKT1
结合能
AKT1
binding
energy		 与IL-6
结合能
IL-6
binding
energy		 与TNF
结合能
TNF
binding
energy		 与IL-1β
结合能
IL-1β
binding
energy		 与TP53
结合能
TP53
binding
energy
槲皮素 Quercetin C15H10O7 5280343 -11.05 -7.38 -12.37 -9.57 -11.00
木犀草素 Luteolin C15H10O6 5280445 -8.15 -5.86 -9.61 -7.99 -9.24
刺芒柄花素 Formononetin C16H12O4 5280378 -8.54 -6.25 -8.05 -7.50 -9.88
8-异戊烯基山奈酚
8-isopentenyl-kaempferol		 C20H18O6 129716399 -7.86 -6.12 -7.79 -7.27 -8.53
菜豆素 Phaseolin C20H18O4 4063834 -7.37 -6.14 -8.01 -7.57 -8.24
图7 AKT1与苦参核心药效成分分子对接3D图(结合能≤-5.0 kcal/mol)

Fig.7 Molecular docking 3D diagram of AKT1 and core medicinal ingredients of Sophorae flavescentis Radix (binding energy≤-5.0 kcal/mol)

图8 IL-6与苦参核心药效成分分子对接3D图(结合能≤-5.0 kcal/mol)

Fig.8 Molecular docking 3D diagram of IL-6 and core medicinal ingredients of Sophorae flavescentis Radix (binding energy≤-5.0 kcal/mol)

图9 TNF与苦参核心药效成分分子对接3D图(结合能≤-5.0 kcal/mol)

Fig.9 Molecular docking 3D diagram of TNF and core medicinal ingredients of Sophorae flavescentis Radix (binding energy≤-5.0 kcal/mol)

图10 IL-1β与苦参核心药效成分分子对接3D图(结合能≤-5.0 kcal/mol)

Fig.10 Molecular docking 3D diagram of IL-1β and core medicinal ingredients of Sophorae flavescentis Radix (binding energy≤-5.0 kcal/mol)

图11 TP53与苦参核心药效成分分子对接3D图(结合能≤-5.0 kcal/mol)

Fig.11 Molecular docking 3D diagram of TP53 and core medicinal ingredients of Sophorae flavescentis Radix (binding energy≤-5.0 kcal/mol)

3 讨论

苦参是清热燥湿代表药物之一,具有治疗湿热泻痢、黄疸、尿闭、疥癣等功效。有研究发现,苦参对金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌等有抑制作用[7-8]。但苦参具体的抗菌药效成分及其作用机制尚未知。目前,有许多药物通过网络药理学来预测其作用靶点及机制,并取得良好成效。Li等[9]在研究维生素C对治疗肺炎作用机制中,运用TCMSP数据库、GeneCard数据库、STRING数据库等预测关键蛋白,发现维生素C对治疗肺炎作用的靶点主要有丝裂原活化蛋白激酶1(MAPK1)、丝裂原活化蛋白激酶3(MAPK3)、血管紧张素-2(AT-2)、促醛固酮激素2(AGTR2)、信号转导和转录激活因子3(STAT3),其抗肺炎作用与抑制炎症作用和增强免疫调节有关。Pan等[10]通过网络药理学方法研究桂枝少瑶对治疗慢性疼痛相关有效成分和潜在靶点进行了预测,发现桂枝少瑶对治疗慢性疼痛的潜在机制主要与炎症反应、免疫调节和神经递质调节有关。本研究利用网络药理学和分子对接技术,预测苦参药效成分及其抑菌的潜在作用机制和靶点。
通过网络药理学技术发现筛选出的苦参药效成分主要是生物碱类化合物和黄酮类化合物。文献中也报道了黄酮类化合物、生物碱类化合物是苦参的主要药效成分[11-12]。其中槲皮素、木犀草素、刺芒柄花素、菜豆素、高丽槐素等黄酮类化合物有抑菌多种病原微生物生长的倾向[13]。槲皮素对金黄色葡萄球菌、大肠杆菌有良好的抑制作用,槲皮素的抗菌机制主要包括破坏细菌细胞壁,改变细胞通透性,影响蛋白质合成和表达,降低酶活性,抑制核酸合成[14]。有研究发现,木犀草素对幽门螺旋杆菌有抑制作用[15]。苦参碱、槐定碱等生物碱类化合物对细菌也有抑制作用[16],其中苦参碱灌胃给药的生物利用度仅为(17.1±5.4)%[17],因此其适用于调控肠道菌群。实验室前期结果显示,苦参碱显著增加了拟杆菌门(Bacteroidetes)、拟杆菌纲(Bacteroidia)、拟杆菌目(Bacteroiidales)、嗜酸乳杆菌(Lactobacillus acidophilus)的丰度,而显著减少了厚壁菌门(Firmicutes)、梭菌纲(Clostridia)、梭菌目(Clostridiales)、瘤胃菌科(Ruminococcaceae)和脱硫弧菌属(Desulfovibrio)的丰度[18],其中嗜酸乳杆菌添加到肉鸡饲粮中,能提高肉鸡生长性能和免疫性能,降低大肠杆菌感染组肉鸡死亡率[19]。基于此,推测苦参可通过这24个药效成分发挥抑菌作用。
PPI蛋白互作网络分析结果表明,苦参抑菌作用核心靶点AKT1、IL-6、TNF、、IL-1β、TP53等互作频率较大,为苦参关键抑菌靶点。AKT1与TP53均参与细胞凋亡与葡萄糖代谢作用,影响细胞的增殖和生长[20]。AKT1可以通过抑制肌动蛋白动力学过程、吞噬小体与溶酶体融合,来抑制细菌的杀灭和促进细菌在细胞内生长[21]。研究表明,TP53具有控制细菌感染的作用,抑制TP53可能赋予细菌一定的生长优势[22]。IL-1β与IL-6是多功能的细胞因子,能够引起T细胞、B细胞、中性粒细胞等免疫细胞在炎症局部聚集和促进抗体的产生[23-24]。TNF是一种多效应细胞因子,在免疫系统中能上调炎症因子IL-8的表达,刺激炎症的发生[25]。有研究表明,感染病原体的宿主细胞产生一系列促炎细胞因子,包括IL-1β、IL-6和TNF[26],在促进和调节细菌病原体的免疫反应中起关键作用[27]。
本研究中,GO富集分析表明苦参活性成分可在细胞质、核质、线粒体、染色质等细胞的不同部位,通过发挥酶结合、蛋白结合、细胞因子活性、转录因子活性等分子功能,在细胞因子介导的信号通路、炎症反应、凋亡过程的正负调控、对药物的反应等不同的生物过程中发挥作用,从而在抑菌中起作用。同时KEGG通路富集分析显示与苦参作用于抑菌作用相关性较大发代谢通路有IL-17信号通路、TNF信号通路、C型凝集素受体信号通路、AGE-RAGE信号通路、在糖尿病并发症中的作用等,这说明苦参可能通过多靶点多通路来抑制细菌。TNF广泛存在于抗病毒、抗细菌及免疫调节等生物学过程[28-29]。IL-17主要由免疫细胞产生,具有强大的促炎特性[30]。它通常通过结合它们的特定受体(IL-17受体)发挥作用,并在保护宿主免受病原体侵袭方面发挥重要作用[31]。C型凝集素是一个含有钙离子(Ca2+)依赖性碳水化合物识别结构域(CRD)的蛋白质家族[32]。有研究表明,细菌可以通过Toll样受体(TLR)、NOD样受体(NLR)和C型凝集素受体(CLR)刺激Th17反应。当激活时,Th17淋巴细胞会产生几种细胞因子,主要是IL-17和趋化因子,它们进一步吸引和激活吞噬细胞以介导细菌清除[33]。
分子对接结果表明,苦参核心药效成分与关键靶点的所有组合均能自发对接,并且结合能均<-5.0 kcal/mol。这表明了苦参药效成分能够通过AKT1、IL-6、TNF、IL-1β、TP53靶点蛋白起到抑菌作用,验证了上述网络药理学预测结果的可靠性。其中AKT1、TNF、TP53与苦参药效成分结合能较其他靶点低,因此,AKT1、TNF、TP53可能是苦参发挥抑菌作用的主要靶点。为接下来将验证苦参是否通过这些重要靶点来抑制有害菌,改变肠道菌群多样性以及提高免疫力,从而开发为饲料添加剂提供理论基础。

4 结论

本研究基于网络药理学和生物信息学预测和分析苦参发挥抑菌作用的核心靶点和通路结果表明,苦参抑菌的分子机制可能通过槲皮素、木犀草素、刺芒柄花素等调控AKT1、TNF、TP53等靶点,参与IL-17信号通路、TNF信号通路等通路来达到,通过分子对接初步验证了苦参关键药效成分与抑菌靶点的相互作用模式,为进一步研究苦参抑菌的作用机制和将其研发为新型饲料添加剂提供一定的理论基础。

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