Fatigue first appeared in Synopsis of the Golden Chamber^[1]. Fatigue is a common and complex symptom. Physiological fatigue is caused by the accumulation of metabolites in the body caused by a large amount of exercise. If it cannot be eliminated in time, with the increase of exercise intensity and amount, there will beNow mood changes, physical discomfort, spleen and stomach symptoms and body fluid loss show sub-health fatigue. Further development of this state will inevitably lead to dysfunction of the five organs, depletion of essence and blood, and chronic fatigue syndrome^[2]。There are about10%Of people suffer from chronic fatigue^[3]However, its pathogenesis is not completely clear. Studies have shown that fatigue may be related to the inflammatory response and dysfunction of energy metabolism, immune and endocrine systems, and antioxidant defense systems^[4-6]。

Astragalus membranaceus, first published in the Shennong materia medica classic, is listed as the top grade^[7], Astragalus membranaceus, a leguminous plantAstragalusmembranaceus (Fisch.)Bge. Or Astragalus mongholicusA. membranaceus (Fisch.) Bge. var. mongholicusBge. HsiaoIt has the effects of Invigorating Qi and Yang, strengthening the surface and stopping sweating, promoting water and swelling, generating fluid and stopping bleeding, and is used for Qi deficiency and fatigue, eating less and loose stools, sinking of middle Qi, Qi deficiency and edema, etc^[8]The main components of Astragalus membranaceus include flavonoids, saponins, sugars, organic acids, amino acids, etc^[9]Traditional Chinese medicine believes that Astragalus membranaceus is a good medicine for tonifying Qi, known as "ten drugs and eight Qi". Literature research shows that Astragalus membranaceus has significant anti fatigue^[10], anti-inflammatory^[11], anti immunity^[12]However, the molecular mechanism of its action is not very clear. Therefore, it is of great significance to explore the targets and pathways of anti fatigue effect of Astragalus membranaceus by using the method of network pharmacology, and scientifically demonstrate the mechanism of anti fatigue effect of Astragalus membranaceus from a micro perspective.

Network pharmacology integrates the technology and content of systems biology, multidirectional pharmacology, computational biology, network analysis and other disciplines to carry out "disease-phenotype-gene-The construction of multi-level network of "drugs" explores the correlation between drugs and diseases from the perspective of the whole, which has the characteristics of integrity and systematicness, and is especially suitable for multi-component, multi-targetStudy on the pharmacodynamic mechanism and material basis of traditional Chinese medicine in multiple ways^[13-15]。The previous research of our research group reproduced the fatigue rat model through exhaustive swimming and restricted diet. The results of exhaustive swimming time, serum biochemical indicators, pathological sections, muscle metabolomics and other results of the fatigue rats after giving Astragalus showed that Astragalus had obvious anti fatigue effect. Therefore, the main primary and secondary metabolites of Astragalus membranaceus discovered by the research group, including sugars, flavonoids and saponins, were taken as the research objects in this study. The network pharmacology method was used to explain the mechanism of Astragalus membranaceus' anti fatigue effect, providing a theoretical basis for subsequent experiments.

1  method

1.1  Construction of chemical composition database of Astragalus membranaceus

Our research group recognized from the middle finger of Astragalus membranaceus in the early stage12Four secondary metabolic components as well as amino acids, organic acids, sugars and other primary metabolic components, combined with the pharmacology database and analysis platform of traditional Chinese medicine system（TCMSP，http://lsp.nwu.edu.cn/，Version 2.3）The chemical components of Astragalus membranaceus were obtained according to the method established in the laboratory^[16]Determine the content of the components.

1.2  Construction of compound molecular structure

The above active ingredients of Astragalus membranaceus inChembiodraw Ultra 12.0The structure diagram is drawn in the software, and themol2. utilizePubChem（https://pubchem.ncbi.nlm.nih.gov/）、TCMSP、ChemSpider（http://www.chemspider.com）、Chemical Book（http://www.chemicalbook.com/）Its molecular structure was confirmed.

1.3  Prediction and screening of targets of active ingredients of Astragalus membranaceus

Will“1.2”Obtained under11Molecular structure of compoundsmol2Format file import toPharmMapperThe potential targets of Astragalus membranaceus were selected. utilizeUniProtDatabaseUniProt KB（https://www.uniprot.org/uniprot/）Search function, introducing Astragalus protein targetPDB ID. Download the reverse docking prediction results of each component, and score the docking（Z）Values are arranged in descending order, and the top of each component is selected300Targets were used for subsequent studies.

1.4  Anti fatigue target screening

By using theGeneCardsDatabase（http://www. genecards.org/，Version 4.5.0）AndOMIMDatabase（http://www.ncbi.nlm.nih.gov/omim）Enter keywords infatigue、anti-fatigue, searching for reported fatigue related bases.

1.5  active ingredient -Construction of anti fatigue target network

. Import dataCytoscape Version 3.6.0Software to construct the active ingredients of Astragalus membranaceus-Anti fatigue action target network.CytoscapeThe core architecture of the software is the network, and each node（node）（edge）（degree）Represents the number of nodes connected to each other in the network. The greater the degree, the more likely this target will become the key target of the compound.

1.6  Construction and analysis of protein interaction network

StringDatabase（https://string-db.org/，Version 10.5）Is a protein containing known and predicted-Database of protein interactions. Introducing the protein target of Astragalus membranaceus intoStringDatabase, defining species as human, obtaining protein interaction relationships, resultsGuaranteed depositTSVFormat, keep in filenode1、node2andCombinedscoreInformation and importCytoscape（Cytoscape→Tools→ Networkanalyzer→Networkanalysis→Analyzenetwork），Save the network analysis results and useCytoscapeMediumGenerate style from statisticsTools（Cytoscape→Tools→Networkanalyzer→Networkanalysis→Generate style from statistics）The node size and color settings are used to reflect the size of the degree value, and the edge thickness settings are used to reflect the binding rate score（Combine score）To obtain the final protein interaction network.

1.7  Target type attribution

DisGeNET Database（http://www.disgenet.org/ web/DisGeNET/menu，Version 5.0）It contains human diseasesOne of the platforms for disease associated genes and variants. stayDisGeNETGenes were selected for searching in the database, and the above targets of Astragalus membranaceus were successively imported into the database to obtain target type information（Protein Class）。

1.8  Biological process and pathway analysis

Biological information annotation database（DAVID，https:// david.ncifcrf.gov/summary.jsp，Version 6.8）It can provide systematic and comprehensive biological function annotation information for large-scale genes or proteins, and can find the most significantly enriched biological annotations. Introducing the target of Astragalus membranaceus intoDAVIDDatabase,SelectIdentifierset upbyOFFICIAL GENE SYMBOL，List TypeSet toGeneList, define the species as human, and conduct gene ontology on the action targets of Astragalus membranaceus（GO）Analysis andKEGGPathway analysis and save the results. Set thresholdP＜0.05And sort according to the number of targets involved, and screen the top biological processes or pathways.

1.9  Drawing of access map

utilizeUniProtDatabaseUniProt KBSearch function to get Astragalus respectively76Of anti fatigue targetsUniprotID. utilizationKEGGDatabase（http://www.kegg.jp）KEGG mappingMediumKEGG MapperTool to integrate the above key targets of Astragalus membranaceusUniProt IDImport, define the species as human, obtain the pathway map of anti fatigue effect of Astragalus membranaceus, screen the top several pathways and integrate them to draw the final pathway map.

2  result

2.1  Screening of active ingredients from Astragalus membranaceus

In this study, a total of11Active ingredients of Astragalus membranaceus (table1）, including6,4Astragaloside, a saponin componentI、AstragalosideII、AstragalosideIII、AstragalosideIVAnd sucrose.

2.2  Active ingredients of Astragalus membranaceus-Prediction of anti fatigue targets

takePharmMapper、ChemmapperAll the targets obtained from the database are obtained after removing duplicates and false positives11Active ingredient targets231ByGeneCards、OMINFatigue of database retrieval integration/Anti fatigue target861The active ingredients were obtained by taking the intersection of the corresponding target of Astragalus active ingredients and the fatigue related target-Disease targets76See table for details2。

2.3  active ingredient -Construction of action target network

applyCytoscapeSoftware construction active ingredient-The network diagram of anti fatigue targets is shown in Fig1As shown, there are87Nodes,166Bar edges, where yellow indicates6Flavonoids, indicated in green4species. Edges represent interactions of active ingredients with anti fatigue targets. The analysis results showed that11The active ingredients correspond to11、14、17、10、19、14、19、15、22、16、9Targets. 2）As can be seen in, flavonoidsAction target of class components40Target of saponins35Targets of carbohydrate components9Among them, flavonoids and saponins act as targets together16Flavonoids and sucrose act together as targets4Saponins and sucrose act together as targets3Nos,3The common targets of class components are only1PCs. 2（MMP2）, vitaminsDReceptors（VDR）、E3（TRIM21）（CAT）, myoglobin（MB）, choline kinaseβ（CHKB）8、7、6、6、5、5The active ingredients are related. Each active ingredient can act on multiple targets, and each target is also associated with multiple compounds, which shows that different compounds have common effects when Astragalus membranaceus plays its pharmacodynamic role.

2.4  Construction and analysis of protein interaction network

As shown in Fig3As shown in, nodes in the figure represent proteins, and edges represent the associations between proteins, involving71Nodes,367. The larger the degree value is, the larger the node is, and the larger the position it occupies in the whole network. By calculating the topological properties of each node, the key targets of Astragalus membranaceus's anti fatigue effect are found. The target proteins with the highest degree value screened in the protein interaction network play an important role in the anti fatigue process of Astragalus membranaceusWith. Last choice21Individual degree value ≥15，Betweenness centrality≥0.020479，Center proximity（closeness）≥0.555555As the main node, and the key targets are imported in turnDisGeNETDatabase to obtain the corresponding types of targets. See table for main information3The results showed that there were insulin, signal transduction factors and coagulation factors in the anti fatigue process of Astragalus membranaceusII, neural cell adhesion molecule, receptor (peroxisome proliferator activated receptor, estrogen receptor), protein (annexin, tumor proteinp53, microglobulin, heat shock eggBai et al.)Enzymes (catalase, tyrosine kinase, nitric oxide synthase, polymerase, protease, heme oxygenaseI, thrombin) and other substances.

2.5  Gene function and pathway analysis

For the selected21Key targetsGOAnalysis andKEGG.GOEnrichment analysis included biological processes（biological process，BP）, molecular function（molecular function，MF）And cellular components（cellularcomponent，CC）3Branches. Set thresholdP＜0.05, common enrichment134Biological processes or pathways, screening the top biological processes or pathways, usingGraphPadPrism 5.0Drawing, see Fig4～7。

As shown in Fig4As shown, these targets mainly involve nitric oxide（NO）The biosynthetic process of（positiveregulation of nitric oxide biosynthetic process）Hydrogen peroxide reaction（response to hydrogen peroxide）, positive regulation of brown adipocyte differentiation（positive regulation of brown fat cell differentiation）Metabolic process of reactive oxygen species（positive regulation ofreactive oxygen species metabolic process）. Part of the reason for fatigue is that the body's energy supply is insufficient. Mitochondria produce almost all the energy and heat in the body,NOAs a vasodilator, it can stimulate the synthesis of new mitochondria and can directlyRegulate the binding and release of oxygen and heme, and control the supply of oxygen to mitochondria in this way, so as to play an anti fatigue role^[17]。The anti exercise fatigue effect of total saponins of Astragalus may be related to its inhibition of exercise oxidationThe increase of free radicals induced by stress is related to the improvement of hippocampal neuronal damage^[18]。

MFAnalysis (Fig5）As can be seen in, the targets are mainly involved in protein binding（protein binding）, enzyme binding（enzymebinding）, heme binding（heme binding）（protein phosphatase binding）. HemeOxygenase-1（HO-1）Is an inducible rate limiting enzyme that helps inhibit inflammatory cytokines, such as tumor necrosis factor-α（TNF-α）, interleukin-1β（IL-1β）AndIL-6, and has become a key molecule in regulating inflammatory responses and oxidative stress in various diseases^[19]。

CCAnalysis (Fig6）It can be seen that the target mainly involves cellsQualitative（cytoplasm）, nuclear（nucleus）, membrane（membrane）, extracellular secretion（extracellular exosome）. Nuclear transcription factorE2Correlation factor2（Nrf2）Usually present in the cytoplasm,Wangetc.^[20]It is pointed out that drug anti fatigue may be associated withNrf2Mediated oxidative stress.

KEGGPathway analysis results (Fig7）Showed that the anti fatigue targets of Astragalus membranaceus mainly involvedFoxOsignaling pathway（5Targets/23%）Cancer related pathways（7Targets/33%）, hypoxia inducible factor（HIF-1）signaling pathway（4Targets/19%）（3Targets/14%）And other signaling pathways. .

2.6  Target pathway analysis

utilizeKEGG MapperTool to obtain the anti fatigue pathway map of Astragalus membranaceusKEGGDerived before10The pathways were integrated to draw a pathway map (Fig8）. The arrows in the figure indicate the promoting effect,T-Arrows indicate inhibitory effects, and different colored arrows indicate different pathways. Pathway targets are marked in blue, and anti fatigue targets of Astragalus membranaceus are marked in pink. The figure shows that the anti fatigue effect of Astragalus membranaceus mainly involves cancer pathwaysFoxO, phosphatidylinositol3-Kinases（PI3K）-protein kinaseB（Akt）、HIF-1, vascular endothelial growth factor（VEGF）, mitogen activated protein kinase（MAPK）、Ca²⁺signaling pathway、 Oxidative stress responseDNARepair process, etc., involving10Astragalus anti fatigue targets (accounting for49%）. Suggested that Astragalus membranaceus anti fatigue targetPoints are located in these pathways and play a role by regulating several links. The vast majority of targets play a role in multiple pathways, such asPI3K、AktEt al.

3  discuss

. Network pharmacology and the whole of traditional Chinese Medicine

It is helpful to systematically and comprehensively explain the anti fatigue mechanism of Astragalus membranaceus. In this study, the main primary and secondary metabolic components identified by the research group in the early stage were selected as active components, and a total of calycosin glucoside, calycosin, formononetin, formononetin, isoflavones, pterostane, astragaloside were obtainedI、II、III、IVAnd sucrose Co11Active ingredients, including6Flavonoids4Saponins and sucrose. Studies have shown that astragaloside significantly increases the peroxidase of skeletal muscle in Exercise-induced Fatigue Rats（POD）（T-AOC）AndCa²⁺-Mg²⁺-ATPEnzyme activity, reducing lactate dehydrogenase（LDH）It can protect the muscle tissue of exercise-induced fatigue rats^[21]The chromatographic peaks of flavonoids and some other components (including saponins) in Astragalus membranaceus showed an enhanced effect on anti fatigue^[22]Astragalus polysaccharide can prolong the swimming time of weight-bearing exhaustion in mice, reduce the accumulation of blood lactic acid, increase the storage of liver glycogen, and delay the occurrence of fatigue/The effect of improving exercise ability can improve the superoxide dismutase in mouse liver tissue（SOD）, GuCystatin peroxidase（GSH-Px）It can protect against exercise-induced oxidative stress^[23]。

active ingredient -. The results of this study showed that11All active ingredients have anti fatigue effects, among which astragalosideI、III. Insulin（INS）、IL-10、TP53、CATIt is predicted that it will play a major role in the anti fatigue activity of Astragalus membranaceus.INS、CAT,INSIt is a multifunctional protein hormone that plays an important role in regulating the metabolism of sugar, fat and protein^[24]. higherCATThe activity has a high antioxidant function, which can timely remove free radicals in the body, reduce the damage of peroxidation to tissues and cells of the body and promote the recovery of fatigue^[25]。IL-10、TP53Is the main target of sucrose,IL-10Is due toTh2A cytokine produced by cells that inhibitsThCell clone a pleiotropic immune regulator characterized by cytokine synthesis, which plays an important role in the recovery of exercise-induced fatigue^[26]。P53It is a tumor suppressor gene, and the cell signal transduction pathway it mediates plays an important role in regulating the normal life activities of cells. Some studies have shown that by inhibiting rat skeletal musclep53Protein expression, can reduce the occurrence of muscle apoptosis caused by exercise^[27]。

The results of enrichment analysis and target attribution analysis showed that the anti fatigue of Astragalus membranaceus involved biological processes such as cellular processes, metabolic processes and responses to stress, cellular components such as organelles, cell membranes and cytoplasm, molecules such as small molecules, cations, metal ions, and a variety of substances such as signaling molecules, transcription factors, receptors, proteins and enzymes, which was a complex process.

It can be seen from the pathways that Astragalus membranaceus anti fatigue mainly involves cancer pathwaysFOXO、Nrf2、HIF-1、MAPK、PI3K/Akt、VEGF、Ca²⁺, adenylate activated protein kinase（AMPK）/p53andinsulinsignaling pathway， Involving oxidative stressDNARepair, cell proliferation, reactive oxygen species（ROS）Detoxification and other reactions. Studies have shown thatFoxOTranscription factors asFoxThe main member of the family is insulin/Insulin like growth factor（INS/IGF-1）Key factors in signaling pathway play important roles in physiological regulation, metabolism and cell cycle of animals through transcriptional regulation and signal transduction pathways^[28]。Hribaletc.^[29]Found that in myoblastsc2c12Medium, viasiRNAinhibitionFoxolTo increase the expression of sarcoplasmic globulin is conducive to myocyte differentiation; In dependent onAktDuring sarcoplasmic protein production,FoxO1Protein plays a key role, suggesting that Astragalus membranaceus canFoxOPathway affects muscle differentiation in fatigue rats to alleviate fatigue.Nrf2Is a key regulator of cellular oxidation at the transcriptional level, which directly controlsSOD、HO-1andCATConcentration of.HO-1It helps to convert heme into bilirubin, which is an effective antioxidant. WhenROSIn case of excessive accumulation,Nrf2Is activated and accumulates in the cytoplasm. By improving the in vivoNrf2, which can effectively prevent oxidative stress damage and thus alleviate fatigue^[20]。HIF-1It is a nuclear protein produced by tissue cells under hypoxic conditions, which is used to maintain the key substance of oxygen homeostasis. It combines with its downstream target genes to enhance the body's ability to tolerate hypoxia. Exercise will lead to exercise-induced hypoxia in body tissues and cells, and in skeletal muscleHIF-1αProtein content and gene expression will rise^[30]。MAPKsignalThe system plays an important role in the body's adaptation to environmental stress and maintaining the dynamic balance of oxidation and antioxidation in the body. Studies have shown that^[31]，Wolfberry fruit can effectively alleviate the imbalance of the body's internal environment caused by excessive training, improve the body's oxygen free radical scavenging ability, especially skeletal muscle, and inhibitMAPKThe protein expression of signaling pathway can prevent and delay the occurrence and development of exercise fatigue.PI3KIt plays an important role in the phosphokinase family. It can regulate cell growth and apoptosis,AktIt's serine/Threonine kinase, yesPI3KOne of the most important downstream effector molecules. Renzenghui^[32]The study found that high-intensity incremental load training would make the hippocampus of ratsPI3K、AktProtein andmRNAThe increase of expression level can indicate that high-intensity incremental load exercise can activatePI3K/AktSignal transduction pathway to exert its anti apoptotic effect.AMPKIs a key regulatory protein in cellular energy metabolism,AMPKActivation can regulate apoptosis^[33-34]。AMPKActivation leads top53The accumulation of apoptosis in muscle tissue is thus induced by inhibition ofAMPK/p53Signaling pathway can inhibit the apoptosis of muscle tissue cells, reduce tissue cell damage, and relieve muscle fatigue.INSIt is the main regulatory hormone in the process of sugar and fat energy metabolism.INSFirst with the cell surface insulin receptor（INSR）Binding, activating itsβSubunit of protein tyrosine kinase（PTK）. Insulin receptor substrate protein（IRS）As an anchoring protein can activate at least2Known signaling pathways: one is throughIRSactivationPI3K; The other is through growth factor receptor binding protein2（Grb2）/SOSandRASProtein activationMAPKchannel^[35]。

In conclusion, the results of this study showed that the11Active ingredients act on76Targets, involving a variety of processes, molecules and pathways, reflecting the multi-component of Astragalus membranaceus-Multitarget-The function characteristics of multiple pathways. The anti fatigue effect of Astragalus membranaceus was predicted by protein interaction network21Three important target proteins, and the roles of key targets were assigned, the pathway map was drawn, and the pathways were verified through literature research, but the relevant targets should be further verified by experiments.

References (omitted)  

Come   Source: Zhang   ; Ke, qinxuemei Study on the anti fatigue mechanism of Astragalus based on network pharmacology [j]. Chinese herbal medicine, 2019, 50 (8): 1880-1889