Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 8: 5560 - 5572

DOI: https://doi.org/10.12418/CJAN2025.451

RESEARCH PAPER

Study on Mechanism of Red Clover Enhancing Anti-Immune Stress in Rabbits Based on Network Pharmacology Method and Molecular Docking Technology

  • LIU Jinyao , 1 ,
  • BAO Huyang 1 ,
  • SUN Xiuzhu 2 ,
  • REN Zhanjun 1 ,
  • WANG Shuhui , 1, *
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  • 1 College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
  • 2 College of Grassland Agriculture, Northwest A&F University, Yangling 712100, China
*associate professor, E-mail:

Received date: 2024-12-30

  Online published: 2025-08-14

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Abstract

This experiment was conducted to study the mechanism of red clover enhancing anti-immune stress in rabbits based on network pharmacology method and molecular docking technology. All the components of red clover were obtained through the HERB database, the active ingredients and corresponding related targets of red clover were obtained according to the information calculated by the SwissADME database and SwissTarget Preliminary platform, and the potential targets of anti-immune stress in rabbits were mined by combining the disease information platforms GeneCard and OMIM, and the protein-protein interaction (PPI) network was constructed by using the target data, and the gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out, finally, the Autodock software and PyMOL software were used to molecularly dock and visualize the core targets with the corresponding components to verify the docking activity. The results showed that there were 29 active ingredients corresponding to 344 potential targets for anti-immune stress in rabbits, including 49 key targets, and the top 10 degree value were caspase 3 (CASP3), epidermal receptor growth factor (EGFR), estrogen receptor 1 (ESR1), proto-oncotyrosine protein kinase (SRC), matrix metalloproteinase 9 (MMP9), prostaglandin G/H synthase 2 (PTGS2), peroxisome proliferator-activated receptor γ (PPARG), Toll-like receptor 4 (TLR4), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α isoform (PIK3CA), tyrosine protein kinase Nyn (FYN). GO analysis showed that the target functions included 92 biological processes, 21 cellular components and 32 molecular functions, and 123 pathways were enriched by KEGG pathway (P<0.05), and the core targets were mainly involved in cancer pathway, prostate cancer, endocrine therapy resistance, estrogen signaling pathway, phosphatidylinositol 3-kinase-protein kinase pathway, breast cancer, lipid and atherosclerosis, glioma, chemocarcinogenic-receptor activation, interleukin-17 signaling pathway and other pathways. Finally, through molecular docking verification, the calculated binding chemical energy was less than 0 kJ/mol, indicating that the active ingredient of red clover and the corresponding core target could be spontaneously and stably combined. In conclusion, this study reveal that the red clover has multi-component, multi-target and multi-channel anti immune stress effects in rabbits through network pharmacological methods and molecular docking technology, which provide a theoretical basis for the development and application of red clover.

Key words： network pharmacology; rabbits; immune stress; molecular docking; red clover

Cite this article

LIU Jinyao , BAO Huyang , SUN Xiuzhu , REN Zhanjun , WANG Shuhui . Study on Mechanism of Red Clover Enhancing Anti-Immune Stress in Rabbits Based on Network Pharmacology Method and Molecular Docking Technology[J]. Chinese Journal of Animal Nutrition, 2025 , 37(8) : 5560 -5572 . DOI: 10.12418/CJAN2025.451

家兔具有繁殖力强、养殖周期短、饲料利用率高等优点,在肉类生产中具有巨大潜力,在确保全球粮食安全和发展我国经济振兴中发挥重要作用[1]。我国是世界上第一养兔大国,兔相关产品总产量和出口量已连续多年位居世界第一[2]。家兔产业已经成为我国畜牧业的特色优势产业。但家兔天性敏感胆小,且消化系统较为脆弱,易受病原体攻击造成免疫应激(immune stress,IS)[3],出现采食量减少、生长缓慢和抗病能力降低等症状[4-5]。在当前家兔养殖集约化、产业化的模式下,预防和控制免疫应激对提高家兔生产性能具有重要意义。在我国实行全面禁抗政策的背景下,植物提取物以其低毒性、高安全性和改善健康效果明显等特点,已成为饲料添加剂领域的研究热点。因此,开发抗免疫应激的植物提取物存在广阔的发展前景[6]。
红三叶草(Trifolium pratense L.)是豆科多年生草本植物[7],具有重要的饲用和药用价值,国外民间将其入药可治感冒和肺结核、利尿消炎,国内药典载其有“清热、凉血、宁心功效”[8]。红三叶草含有多种多糖、黄酮类和异黄酮类物质,具有调节血脂、抗氧化、抗肿瘤以及雌激素样活性等生理功能[9-10],同时还可以抗球虫病、抗菌、预防心血管疾病,以及治疗更年期综合征、抗衰老、防止骨质流失、预防癌症等[11-13]。目前关于红三叶草的研究主要集中在异黄酮[14-15],但有学者指出,红三叶草中发挥的作用不仅取决于异黄酮,还可能与其他活性成分有关[16]。Zhang等[17]提取出的红三叶草多糖RCP-1.1(3.0 mg/mL)对1,1-二苯基-2-吡啶肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)和2,2'-联氮基-双(3-乙基苯并噻唑啉-6-磺酸)[(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid),ARTS]自由基的清除率分别达到抗坏血酸(3.0 mg/mL)的91.82%和98.95%,而且对α-葡萄糖苷酶的抑制能力可达到阿卡波糖的86.72%,降血糖与抗氧化作用显著。研究表明,饲喂红三叶草可以显著降低高脂血症兔C反应蛋白(C-reactive protein,CRP)、甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)和低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)含量,显著提高高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)含量[18]。Chen等[19]研究发现,红三叶草异黄酮(red clover isoflavones)作为佐剂通过调节Th1/Th2细胞因子和转录因子的基因表达,增加了小鼠对抗原的特异性抗体和细胞应答。
网络药理学是基于系统生物学理论、生物系统网络分析、多靶点药物分子设计特异性信号节点选择的新学科,而中草药多成分、多途径、多靶点的特点与网络药理学策略的整体性、网络性原理基本吻合[20]。目前,分子对接技术已经成为药物开发领域较为成熟的技术,有助于预测分子的相互作用,特别是小分子配体和生物大分子之间的相互作用,也有助于估计结合自由能[21]。红三叶草存在多种有效成分,对多种疾病均有较好作用。因此,本研究通过网络药理学方法构建网络富集分析,探索红三叶草抗家兔免疫应激的物质基础与潜在机制,并通过分子对接技术进行验证,为红三叶草在家兔生产进一步利用提供理论依据。

1 材料与方法

1.1 红三叶草的主要活性成分筛选与潜在靶点预测

本研究通过HERB数据库(http://herb.ac.cn/)以“红车轴草”为关键词检索,筛选出了红三叶草主要成分,将所获得成分的分子名称输入PubChem数据库(https://pubchem.ncbi.nlm.nih.gov/)中获取主要成分的Canonical SMILES格式的分子表达式,把所得Canonical SMILES格式的分子表达式导入SwissADME数据库(http://www.swissadme.ch/index.php)中获得主要成分的药理学性质,根据lipinsk五原则(满足Lipinski、Ghose、Veber、Egan和Muegge指标中任意3个Yes,且生物利用度评分≥0.55)[22]对成分进行筛选后获得活性成分,将活性成分的Canonical SMILES格式的分子表达式导入SwissTargetPrediction数据库(http://www.swisstargetprediction.ch/),以P值>0为阈值收集活性成分的靶点,将所有活性成分的靶点合并去除重复值,即可获得红三叶草有效成分全部潜在靶点。将红三叶草活性成分及其对应的靶点导入软件Cytoscape 3.10.0构建活性成分-潜在靶点网络。

1.2 家兔免疫应激靶点收集

在GeneCards数据库(https://www.genecards.org/)中以“immune stress”为关键词检索相关靶点,以同样的方式在OMIM数据库(https://www.omim.org/)与TTD数据库(https://db.idrblab.net/ttd/)检索靶点,将获得的靶点取交集并去除重复值,获得免疫应激靶点。

1.3 蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络构建及关键靶点筛选

利用Venny 2.1.0(https://bioinfogp.cnb.csic.es/tools/venny/index.html)获取红三叶草潜在靶点与家兔免疫应激靶点的交集作为有效靶点,利用STRING数据库(https://cn.string-db.org/)计算上述有效靶点之间的相互作用,物种选择“Oryctolagus cuniculus”,置信区间>0.400,并隐藏不存在相互作用的节点,所得数据导入Cytoscape 3.10.0进行可视化操作,在Cytoscape 3.10.0中利用Centis-cape 2.2插件对所得的PPI网络进行拓扑分析,以介数中心性(betweenness centrality)、紧密中心性(closeness centrality)及度值(degree value)作为筛选条件,筛选出均大于上述指标平均值的作为核心靶点。

1.4 基因本体功能(GO)与京都基因与基因组百科全书(KEGG)通路富集分析

将上述红三叶草作用家兔免疫应激的核心靶点导入DAVID数据库(https://davidbioinformatics.nih.gov/)后,设置物种为“Oryctolagus cuniculus”进行分析,以P<0.05为条件,保存所得GO-生物过程(biological process,BP)、GO-细胞组分(cellular component,CC)、GO-分子功能(molecular function,MF)、KEGG通路进一步分析。

1.5 构建活性成分-靶点-免疫应激网络

将所得红三叶草活性成分与对应的有效靶点、核心靶点导入Cyctscape 3.10.0构建网络。

1.6 分子对接验证

将1.3获得核心靶点中度值排名前3位的靶点与其对应的红三叶草活性成分进行分子对接验证试验。在PubChem数据库中获得红三叶草活性成分的化学结构,在PDB数据库(https://www.rcsb.org/)中输入靶点名称获得相应的蛋白质晶体结构,将蛋白质晶体结构导入Pymol 2.6中去除水分子与配体分子后,将其与对应的红三叶草活性成分分子结构导入Autodock 1.5.7计算结合能,并用Pymol对结果进行可视化。

2 结果

2.1 红三叶草的主要活性成分筛选与潜在靶点预测结果

如表1所示,在HERB数据库中共获得成分54个,在SwissADME数据库以lipinsk五原则筛选后得到29个活性成分。
表1 红三叶草中29个活性成分信息

Table 1 Information of 29 active components in red clover

编号
No.		 HERB数据库编号
HERB database No.		 化合物名称
Chemical compound name
1 HBIN003962 2,3-二甲基苯酚2,3-dicresol
2 HBIN005632 糠醛2-furaldehyde
3 HBIN005640 2-呋喃甲酸2-furancarboxylicacid
4 HBIN007430 3,4-二甲氧基苯甲酸3,4-dimethoxybenzoicacid
5 HBIN011862 5-O-甲基染料木素5-O-methylgenistein
6 HBIN015566 α-铁(顺式) Alpha-iron (cis)
7 HBIN018523 鸡豆黄素Abiochanina
8 HBIN022577 大豆黄酮Daidzein
9 HBIN023694 双香豆素Dicoumarin
10 HBIN023881 黄颜木素Dihydrofisetin
11 HBIN026630 单叶芸香定Foliosidine
12 HBIN026662 刺芒柄花素Formononetin
13 HBIN026663 芒柄花素-7-葡萄糖苷Formononetin-7-glucoside
14 HBIN027456 染料木黄酮Genistein
15 HBIN030303 鸢尾蛋白Birilinb
16 HBIN030305 德鸢尾苷元Irilone
17 HBIN031114 异鼠李素Isorhamnetin
18 HBIN033803 木犀草素luteolin
19 HBIN034625 美迪紫檀素Medicarpin
20 HBIN034628 苜蓿紫檀酚-3-O-葡萄糖苷Medicarpin-3-O-glucoside
21 HBIN036774 假荆芥内酯Nepetalactone
22 HBIN038577 二氢樱黄素Padmakastein
23 HBIN038995 对香豆酸p-coumaricacid
24 HBIN039031 柳穿鱼素Pectolinarigenin
25 HBIN040658 红车轴草素Pratensein
26 HBIN040662 车轴草醇Pratol
27 HBIN041013 野靛黄素Pseudobaptigenin
28 HBIN041495 槲皮素Quercetin
29 HBIN047035 红车轴草根苷Trifolirhizin

2.2 红三叶草抑制家兔免疫应激靶点分析

将红三叶草活性成分导入SwissTargetPredicion数据库,获得红三叶草活性成分靶点1 384个,其中可获得概率>0靶点的成分有26个,将全部活性成分靶点合并去除重复值之后获得靶点344个。将活性成分及其对应的靶点导入到Cytoscape 3.10.0中构建活性成分-潜在靶点网络(图1),通过拓扑分析可得度值最高的成分分别是异鼠李素、木犀草素、美迪紫檀素、柳穿鱼素、车轴草醇、槲皮素,度值均为100,可能为红三叶草中主要的活性物质。
图1 红三叶草活性成分-潜在靶点网络图

仅列出靶点中度值排名前10的靶点名称 List only the top 10 target names in degree values。CYP19A1:芳香酶 aromatase;CA12:碳酸酐酶12 carbonic anhydrase 12;CA1:碳酸酐酶 1 carbonic anhydrase 1;CA2:碳酸酐酶2 carbonic anhydrase 2;ESR2:雌激素β受体 estrogen receptor beta;ADORA1:腺苷受体A1 adenosine receptor A1;CA4:碳酸酐酶4 carbonic anhydrase 4;CA7:碳酸酐酶7 carbonic anhydrase 7;TYR:酪氨酸酶 tyrosinase;MAOA:胺氧化酶[含黄素]A amine oxidase [flavin-containing] A。

Fig.1 Network diagram of active ingredient of red clover-potential target

将在Gene Cards、OMIM等数据库所获得免疫应激相关靶点取并集后获得16 075个靶点,将免疫应激相关靶点与红三叶草活性成分靶点导入Venny 2.1.0获得交集靶点321个,绘制韦恩图(图2)。
图2 红三叶草活性成分靶点与家兔免疫应激靶点交集

Fig.2 Intersection of target of active ingredient of red clover and target of immune stress in rabbits

2.3 构建PPI网络

将红三叶草活性成分靶点与家兔免疫应激靶点交集的321个靶点导入STRING数据库,得到交集靶点PPI网络,隐藏不存在相互作用的节点后获得276个节点与1 798条边,平均节点度值为13.029,可证明靶点编码蛋白质间存在较强的相互作用。将蛋白质相互作用相关数据导入到Cytoscape 3.10.0进行可视化处理与分析,结果见图3。由分析可知,度值排名前10位的靶点有半胱天冬酶3(CASP3)、表皮生长因子受体(EGFR)、雌激素受体1(ESR1)、原癌酪氨酸蛋白激酶(SRC)、基质金属蛋白酶9(MMP9)、前列腺素G/H合酶2(PTGS2)、过氧化物酶体增殖物激活受体γ(PPARG)、Toll样受体4(TLR4)、磷脂酰肌醇4,5-二磷酸3-激酶催化亚基α亚型(PIK3CA)、酪氨酸蛋白激酶Fyn(FYN),推测这些靶点可能在家兔免疫应激中起到关键作用,使用Cytoscape 3.10.0中利用Centis-cape 2.2插件对所得的PPI网络进行拓扑分析,以介数中心性平均值506.73、紧密中心性平均值0.001 3及度值平均值13.02为阈值,筛选出49个满足均大于上述指标阈值的核心靶点,这些靶点之间的相互作用更复杂,核心靶点网络图见图4。
图3 红三叶草作用于家兔免疫应激靶点的PPI网络图

A:PPI网络图;B:PPI网络图可视化。

Fig.3 PPI network diagram of red clover acting on immune stress targets in rabbits

A: PPI network diagram;B: PPI network diagram visualization.

图4 红三叶草作用家兔免疫应激核心靶点图

仅列出靶点中度值排名前10的靶点名称 List only the top 10 target names in degree values。CASP3:半胱天冬酶3 caspase 3;EGFR:表皮生长因子受体 epidermal receptor growth factor;ESR1:雌激素受体1 strogen receptor 1;SRC:原癌酪氨酸蛋白激酶 proto-oncotyrosine protein kinase;MMP9:基质金属蛋白酶9 matrix metalloproteinase 9;PTGS2:前列腺素G/H合酶2 prostaglandin G/H synthase 2;PPARG:过氧化物酶体增殖物激活受体γ peroxisome proliferator-activated receptor γ;TLR4:Toll样受体4 Toll-like receptor 4;PIK3CA:磷脂酰肌醇4,5-二磷酸3-激酶催化亚基α亚型 phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α isoform;FYN:酪氨酸蛋白激酶Fyn tyrosine protein kinase Fyn。

Fig.4 Diagram of key targets of red clover on immune stress in rabbits

2.4 构建活性成分-靶点-免疫应激网络

将上述交集基因、关键基因与红三叶草活性成分导入Cytoscape 3.10.0,绘制活性成分-靶点-免疫应激靶点网络,包括370个节点(344个红三叶草活性成分靶点与26个活性成分),活性成分-靶点-免疫应激网络图见图5。经计算可得异鼠李素、木犀草素、美迪紫檀素、柳穿鱼素、车轴草醇、槲皮素6种成分度值最高,是红三叶草中作用免疫应激的主要有效成分。
图5 活性成分-靶点-免疫应激网络图

圆圈代表对应靶点;正方形对应红三叶草活性成分。

Fig.5 Network diagram of active ingredient-targets-immune stress

Circles represent the corresponding targets; square represent the active ingredients of red clover.

2.5 GO功能注释分析与KEGG通路富集分析

将49个关键靶点导入DAVID数据库中,分别进行功能注释分析与通路富集分析,以P<0.05为阈值进行筛选,获得生物过程相关108条,细胞组分相关29条,分子功能相关35条,通路富集分析得到134条信号,P值从小到大排列,分别选择前10条进行可视化,结果见图6和图7。
图6 核心靶点的GO功能分析

Biological process:生物过程;phosphorylation:磷酸化;protein phosphorylation:蛋白质磷酸化;insulin-like growth factor receptor signaling pathway:胰岛素样生长因子受体信号通路;cellular response to cadmium ion:细胞对镉离子的反应;cellular response to amyloid-beta:细胞对淀粉样蛋白的反应;negative regulation of protein catabolic process:负向调节蛋白质分解代谢过程;protein destabilization:蛋白质去稳定化;cellular response to reactive oxygen species:细胞对活性氧的反应;positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction:磷脂酰肌醇3-激酶/蛋白激酶B信号转导的正调控;positive regulation of non-canonical NF-kappaB signal transduction:非经典核转录因子-κB信号转导的正向调控;Cellular component:细胞组分;perinuclear region of cytoplasm:细胞质核周区;cytosol:胞质;nucleus:细胞核;axon:轴突;cytoplasm:细胞质;early endosome:早期内体;melanosome:黑色素颗粒;protein-containing complex:含蛋白质的复合物;insulin receptor complex:胰岛素受体复合物;late endosome:晚期内体;Molecular function:分子功能;identical protein binding:相同的蛋白质结合;ATP binding:ATP结合;insulin receptor substrate binding:胰岛素受体底物结合;estrogen response element binding:雌激素反应元件结合;nuclear receptor activity:核受体活性;protein serine kinase activity:蛋白质丝氨酸激酶活性;disordered domain specific binding:无序结构域特异性结合;steroid binding:甾体结合;signaling receptor binding:信号受体结合;cyclin binding:细胞周期蛋白结合。

Fig.6 GO functional analysis of core targets

图7 核心靶点的KEGG通路富集分析

Pathways in cancer:癌症通路;Prostate cancer:前列腺癌;Endocrine resistance:内分泌治疗耐药;Estrogen signaling pathway:雌激素信号通路;PI3K-Akt signaling pathway:磷脂酰肌醇3-激酶-蛋白激酶B通路;Breast cancer:乳腺癌;Lipid and atherosclerosis:脂质和动脉粥样硬化;Glioma:胶质瘤;Chemical carcinogenesis-receptor activation:化学致癌-受体激活;IL-17 signaling pathway:白细胞介素-17信号通路。

Fig.7 KEGG pathway enrichment analysis of core targets

2.6 分子对接验证

将核心靶点网络中度值排名前3位的靶点与其对应的红三叶草成分进行分子对接验证试验,共33组,结合能均<-25 kJ/mol,表示受体配体之间均较易自发结合[23],对接结果见表2。其中,靶点与化合物结合能较小的有ESR1-美迪紫檀素、ESR1-木犀草素、EGFR-野靛黄素。选择结合能最小的前10组受体配体可视化,结果见图8。该结果进一步验证了网络药理学预测分析结果的可靠性。
表2 核心靶点与相应成分分子对接结果

Table 2 Results of molecular docking of core targets and corresponding components

靶点
Targets		 PDB数据库蛋白质
PDB database protein		 编号
No.		 结合能
Binding energy/(kJ/mol)

半胱天冬酶3
CASP3
1NME		 19 -26.36
20 -30.12

表皮生长因子受体
EGFR
2EB2		 5 -28.45
7 -29.29
8 -29.29
12 -28.87
14 -29.71
15 -30.12
16 -30.96
17 -30.96
18 -32.64
20 -33.05
22 -29.29
24 -29.29
25 -29.29
27 -34.31
28 -30.96

雌激素受体1
ESR1
7RS8		 1 -23.43
5 -34.31
7 -32.22
8 -34.31
12 -32.22
14 -33.89
15 -30.96
16 -32.64
18 -34.73
19 -35.15
22 -29.29
23 -24.69
24 -29.71
25 -33.89
26 -34.31
27 -32.64
图8 靶点与化合物分子对接模式图

a:EGFR-苜蓿紫檀酚-3-O-葡萄糖苷 EGFR-medicarpin-3-o-glucoside;b:EGFR-野靛黄素 EGFR-pseudobaptigenin;c:ESR1-5-O-甲基染料木素 ESR1-5-O-methylgenistein;d:ESR1-大豆黄酮 ESR1-daidzein;e:ESR1-染料木黄酮 ESR1-genistein;f:ESR1-德鸢尾苷元 ESR1-irilone;g:ESR1-木犀草素 ESR1-luteolin;h:ESR1-美迪紫檀素 ESR1-medicarpin;i:ESR1-红车轴草素 ESR1-pratensein;j:ESR1-车轴草醇 ESR1-pratol。

Fig.8 Docking pattern diagram of target and compound molecules

3 讨论

本文运用网络药理学方法构建网络分析,得到异鼠李素、木犀草素、美迪紫檀素、柳穿鱼素、车轴草醇、槲皮素等26个红三叶草活性成分,通过构建红三叶草活性成分作用于家兔免疫应激PPI网络图,经相关参数计算筛选得到关键靶点49个,最后本试验把度值排名前3的靶点CASP3、EGFR、ESR1与其对应的活性成分进行分子对接,结果显示分子可与靶点稳定结合,经计算得结合能最小的组合有ESR1-美迪紫檀素、ESR1-木犀草素、EGFR-野靛黄素等复合物,表明红三叶草活性成分抗家兔免疫应激作用可能与这些靶点密切相关。
根据活性成分-靶点-免疫应激网络图筛选可知,异鼠李素、木犀草素、美迪紫檀素、柳穿鱼素、车轴草醇、槲皮素可能是红三叶草中抗家兔免疫应激主要有效物质。Shi等[24]研究了异鼠李素对骨髓来源的树突状细胞(BMDCs)的作用和机制,发现异鼠李素可有效抑制脂多糖(LPS)处理的BMDCs的成熟,表明异鼠李素是一种有效的免疫抑制剂,可能对免疫应激的缓解有一定作用。现有研究表明,异鼠李素也可作用于SRC/胞外信号调节激酶(ERK)/环磷腺苷反应元件结合蛋白(CREB)通路和磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路[25]。木犀草素是具有抗炎、抗氧化、抗肿瘤、抗变应性和免疫调节作用的类黄酮物质,Verbeek等[26]研究发现,木犀草素可抑制小鼠和人自身免疫性T细胞体外抗原特异性增殖和干扰素-γ的产生,提示木犀草素是效果明显的T细胞抑制剂,证实木犀草素在免疫调节存在作用。柳穿鱼素在细胞试验中被证实可以通过作用于PI3K/AKT/哺乳动物雷帕霉素靶蛋白(mTOR)/ERK信号通路和含有Src同源结构域的蛋白酪氨酸磷酸酶-1(SHP-1)/Janus激酶2(JAK2)/信号转导因子和转录激活因子3(STAT3)信号通路作用于多种肿瘤细胞[27]。车轴草醇相关研究比较少,有报道指出其可促进细胞内黑色素形成与抗炎作用[28]。槲皮素是一种生物活性较高的黄酮类物质,有抗氧化、免疫调节、抗炎症、抗肿瘤等药理作用[29],是天然的免疫抑制剂,抑制在免疫过程中起到关键作用的肥大细胞类胰蛋白酶和白细胞介素-6(IL-6)的释放以及HMC-1细胞系的组氨酸脱羧酶(HDC)mRNA转录[30]。此外,有研究指出槲皮素可促进胰岛素分泌有效改善高血糖[31],与GO功能富集分析所得结果中最显著的途径相关,证明槲皮素可能在红三叶草抗家兔免疫应激中具有重要意义。红三叶草中富含异黄酮类物质,而本研究筛选出的主要有效物质均为黄酮类,表明红三叶草抗家兔免疫应激发挥主要作用的并非异黄酮,为红三叶草非异黄酮类活性物质的研究提供了新思路。
本研究构建PPI网络后分析得到49个关键靶点,其中CASP3、EGFR、ESR1等靶点在红三叶草作用家兔免疫应激中起到重要作用。CASP3是一种半胱氨酸-天冬氨酸蛋白酶,是介导细胞凋亡的关键介质,位于半胱氨酸-天冬氨酸蛋白酶级联反应的末端,可被内源性和外源性死亡途径激活,在集落刺激因子-1(CSF-1)诱导单核细胞分化为巨噬细胞的过程中扮演重要角色,也在组织分化、再生和神经发育中的发挥重要作用[32]。在受体酪氨酸激酶(receptor tyrosine kinase,RTK)/ErbB家族中EGFR扮演着重要角色,广泛分布于细胞表面并作为信号接收受体传导信息,主要激活的信号通路包括丝裂原活化蛋白激酶(MAPK)通路、PI3K通路、c-Src通路,各个通路之间并非独立而是存在交互作用的,最终影响细胞的凋亡、增殖、分化、迁移等生物过程,并且与肿瘤的发生与形成息息相关[33]。基质金属蛋白酶能够水解细胞外信号蛋白,调节免疫细胞运输[34]。有研究发现,活化的MMP9可以提高细胞内超氧化物歧化酶3(superoxide dismutase 3,SOD3)的活性[35],而SOD3可以通过抑制T细胞分化而起到免疫调节作用,这种酶可以抑制受体蛋白的表达、通路激活和细胞因子的产生,还可通过干扰核因子-κB(NF-κB)信号通路来调节对病毒感染的炎症反应,在一定程度上抑制了病毒诱导的细胞凋亡[36]。
通过GO功能富集分析发现,在生物功能、细胞组分、分子功能中最显著的分别是胰岛素样生长因子受体信号通路、胰岛素受体复合物、胰岛素受体底物结合,胰岛素样生长因子受体信号通路在许多正常组织的生长和发育中起着关键作用,并调节生物体的整体生长。该信号通路包括配体、胰岛素样生长因子受体(IGFR)和胰岛素样生长因子结合蛋白(IGFBP),配体包括胰岛素样生长因子-Ⅰ(IGF-Ⅰ)、胰岛素样生长因子-Ⅱ(IGF-Ⅱ)和胰岛素本身,而可以与配体发生作用的有胰岛素样生长因子受体1(IGF-ⅠR)、胰岛素样生长因子受体2(IGF-ⅡR)、胰岛素受体(IR)以及胰岛素样生长因子和胰岛素的杂交受体,此外该系统还包括6种可以调节配体受体结合活性的结合蛋白[37-38]。GO功能富集分析结果中胰岛素受体复合物与胰岛素受体底物结合均发生在IGFR通路,提示红三叶草活性成分主要通过影响家兔IGFR通路中IR结合活性来发挥免疫应激作用。研究人员在小鼠和人肾培养物上进行试验,发现IR信号被抑制的小鼠更容易感染尿路致病性大肠埃希菌,发现IR信号转导失调的小鼠集合管插层细胞和人肾细胞无法整合应激反应的核心成分,包括激活转录因子4(ATF4)。沉默ATF4会损害NF-κB活化并促进感染,表明IR是细胞代谢和基本生物过程(包括免疫反应)的重要调节剂[39]。
目前关注中草药抑制免疫应激的研究比较少,并且免疫应激的研究在猪和家禽上较多,而网络药理学分析需借助前沿文献与研究热点获取信息的数据库来探究作用机理,故可供分析的家兔免疫应激靶点、PPI网络及富集数据不足,结果存在一定局限性,所得红三叶草作用家兔免疫应激的靶点与通路待进一步试验验证。

4 结论

本研究通过网络药理学分析及分子对接的方法分析了红三叶草抗家兔抗免疫应激的物质基础和作用机制,经分析得到了红三叶草发挥抗家兔免疫应激的异鼠李素、木犀草素、美迪紫檀素等29个活性成分和CASP3、EGFR、ESR1等49个关键靶点,并得到了相关通路,为红三叶草缓解家兔免疫应激奠定理论基础。

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