Steamed Panax notoginseng Attenuates Anemia in Mice With ...

Panax notoginseng (Burk.) F. H. Chen is a medicinal herb used to treat blood disorders since ancient times, of which the steamed form ... DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Supplementaldata totalviews ViewArticleImpact SHAREON VincentKamWaiWong MacauUniversityofScienceandTechnology,Macao,SARChina LeiChen GuangdongOceanUniversity,China SongxiaoXu ZhejiangCancerHospital,UniversityofChineseAcademyofSciences,China CharlesJ.Malemud CaseWesternReserveUniversity,UnitedStates Theeditorandreviewer'saffiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. AbstractIntroductionMaterialsandMethodsResultsDiscussionConclusionsDataAvailabilityStatementEthicsStatementAuthorContributionsFundingConflictofInterestAbbreviationsSupplementaryMaterialReferences Opensupplementaldata Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex Checkforupdates Peoplealsolookedat ORIGINALRESEARCHarticle Front.Pharmacol.,21January2020Sec.Ethnopharmacology https://doi.org/10.3389/fphar.2019.01578 SteamedPanaxnotoginsengAttenuatesAnemiainMiceWithBloodDeficiencySyndromeviaRegulatingHematopoieticFactorsandJAK-STATPathwayZejunZhang1,YimingZhang1,MinGao1,XiumingCui1,YangYang1,BertvanDuijn2,3,MeiWang2,4,5,YupiaoHu1,ChengxiaoWang1andYinXiong1,2,3,4*1FacultyofLifeScienceandTechnology,KunmingUniversityofScienceandTechnology,Kunming,China2InstituteofBiologyLeiden,LeidenUniversity,Leiden,Netherlands3FytagorasBV,Leiden,Netherlands4LU-EuropeanCenterforChineseMedicine,LeidenUniversity,Leiden,Netherlands5SUBioMedicineBV,Leiden,NetherlandsPanaxnotoginseng(Burk.)F.H.Chenisamedicinalherbusedtotreatblooddisorderssinceancienttimes,ofwhichthesteamedformexhibitstheanti-anemiaeffectandactswitha“blood-tonifying”functionaccordingtotraditionaluse.Thepresentstudyaimedtoinvestigatetheanti-anemiaeffectandunderlyingmechanismofsteamedP.notoginseng(SPN)onmicewithblooddeficiencysyndromeinducedbychemotherapy.Blooddeficiencysyndromewasinducedinmicebycyclophosphamideandacetylphenylhydrazine.Anumberofperipheralbloodcellsandorgans(liver,kidney,andspleen)coefficientsweremeasured.ThemRNAexpressionofhematopoieticfunction-relatedcytokinesinthebonemarrowofmicewasdetectedbyRT-qPCR.Thejanuskinase-signaltransducerandactivatoroftranscription(JAK-STAT)signalingpathwaywasscreenedbasedonourpreviousanalysisbynetworkpharmacology.TheexpressionofrelatedproteinsandcellcyclefactorspredictedinthepathwaywasdeterminedbyWesternblotandRT-qPCR.SPNcouldsignificantlyincreasethenumbersofperipheralbloodcellsandreversetheenlargementofspleeninadose-dependentmanner.ThequantitiesofrelatedhematopoieticfactorsinbonemarrowwerealsoincreasedsignificantlyafterSPNadministration.SPNwasinvolvedinthecellcyclereactionandactivationofimmunecellsthroughtheJAK-STATpathway,whichcouldpromotethehematopoiesis.IntroductionBlooddeficiency,acommonsyndromeseenintheclinic,isapathologicalstateofblooddysfunctionandorgansdystrophyaccordingtothetheoryoftraditionalChinesemedicine(Liet al.,2015a).Thediagnosticindicatorofblooddeficiencysyndrome(BDS)mainlyreferstothereductionofbloodcellsorhemoglobin,whichissimilarasanemiainmodernmedicine.PatientsoranimalswithBDSoftensufferfromimpairedhemopoieticfunction,peripheralbloodpancytopenia,hypofunctionofinternalorgans,malnutrition,orevenmyelosuppressionduringseverediseases(Zhanget al.,2014a;Jiet al.,2017).TofindtherapiestoalleviateBDSandtreatanemiahasattractedtremendousattentioninrecentyears.Hematopoieticcytokines,suchasgranulocyte-macrophagecolony-stimulatingfactor(GM-CSF),interleukin-2(IL-2),erythropoietin(EPO),andthrombopoietin(TPO),aresometimesusedtoacceleratethehemopoieticrecoveryduringcancertherapy.However,duetotheunstableefficacy,highcost,andadverseeffectslikeosteomuscularpain,arthralgia,andallergicreactions(Dygaiet al.,2012)causedbythosetreatments,theapplicationsofabovecompoundsregimensarelimited.ItwasreportedthatmanyChineseherbalmedicineswithefficaciesofenrichingandregulatingblood,couldpreventandtreatBDSaswellasanemia(Liet al.,2012;ChenJ.etal.,2019;Liet al.,2015b).Panaxnotoginseng(PN)Burk.,aplantingenusPanax(Araliaceae),isahighlyvaluedChineseherbalmedicineusedtotreatblooddisordersinAsiaforthousandsofyears(Wanget al.,2006).Itwasrecordedin“SupplementsforCompendiumofMateriaMedica”(1786)byXueMinZhaoassuchthatPNrootcouldbeusedasahematinicdrug(Guoet al.,2010).Asteamingprocessisoftenusedtoenhancethe“blood-tonifying”functionofPN(Lauet al.,2003).Accordingtoourpreviousstudy,alongwiththedurationofsteaming,thelevelsofsomemajoractivesaponinsinrawPNweredecreasedandsomeothernewsaponinswereproducedorincreased(Xionget al.,2017a;Xionget al.,2017b).SuchtransformationofactiveconstituentsinrawandsteamedPN(SPN)contributestothedifferenceintheirefficacies,suchasthatSPNisbetteratnourishingthebloodandsupplementingqi(vitalenergy)(TheStatePharmacopoeiaCommissionofPeople'sRepublic,2005;Xionget al.,2017b).AlthoughmultiplepharmaceuticstudieshavebeencarriedouttoconfirmthetherapeuticeffectofSPNonBDS(TheStatePharmacopoeiaCommissionofPeople'sRepublic,2005;Zhouet al.,2014;Xionget al.,2017a;Xionget al.,2017b),theunderlyingactionmechanismisstillunclear,whichhindersthedevelopmentofanti-anemiadrugsfromthisherbalmedicineofwhichsideeffectsareonlyrarelyreported.TobetterelucidatethemechanismofSPNtreatingBDS,theclassicalBDSmodelinducedbyacetylphenylhydrazine(APH)andcyclophosphamide(CY)wasusedinthisstudy(Zhanget al.,2014a).BasedonourpreviousanalysisofthepossiblesignalingpathwaysrelatedtothehematiniceffectofSPNusingthenetworkpharmacology,thejanuskinase-signaltransducerandactivatoroftranscription(JAK-STAT)signalingpathwaywaschosentofurtherelucidatesinceitwaspredictedtobeoneofthepathwayswithalargenumberofpotentialtargetsinvolved(FigureS1)(Xionget al.,2019).Thepathwayinvolvesmanymembersofthecytokinereceptorsuperfamily,includingGM-CSF,EPO,TPO,interferons,andnumerousinterleukins,whichmakesitcentraltohematopoieticcellbiologyandhematologictherapyalike(Darnellet al.,1994;Wardet al.,2000).Inthisstudy,basedontheclassicalBDSmicemodel,thehematopoieticeffectandworkingmechanismofSPNwereinvestigatedbyevaluatingtheroutinebloodparameters,organcoefficients,andhematopoiesis-relatedfactorsinvolvedintheJAK-STATpathway.ThesedataprovideatheoreticalbasisfortheuseofSPNanditsproductsinhumanhealthandprovidescluesfordevelopingnewdrugstotreatBDSaswellasanemia.MaterialsandMethodsPreparationandChemicalAnalysisofSteamedPanaxnotoginsengSampleswerecollectedfromWenshan,YunnaninChina(104°077ˊE,23°188ˊN),whichwereidentifiedtobethedriedrootandrhizomeofP.notoginseng(Burkill)F.H.ChenbyProf.XiumingCuifromKunmingUniversityofScienceandTechnology.Thespecimen(No.WSPN15101)isdepositedinYunnanKeyLaboratoryofP.notoginseng,KunmingUniversityofScienceandTechnology(FigureS2),whichcanbefullyvalidatedusinghttp://mpns.kew.org/mpns-portal/?_ga=1.111763972.1427522246.1459077346.AndthequalityofsampleswasconsistentwiththerequirementsofChinesePharmacopoeiaof2015edition(ChinesePharmacopoeiaCommission,2015).ThepreparationofSPNwasdescribedinourpreviousstudy(Xionget al.,2017b).Inshort,SPNsampleswerepreparedbysteamingthecrushedrawmaterialsinanautoclave(ShanghaiBoxunIndustryandCommerceCo.,Ltd,China)for6hat120°C.Thesteamedpowderwasthendriedinaheating-airdryingovenatabout45°Ctoconstantweight,thenpowderedandsievedthrougha40-meshsieve.QualitativeandquantitativeanalysesofSPNwereperformedaspreviousreport(Xionget al.,2017a).Inbrief,notoginsenosideR1andginsenosidesRg1,Re,20(R)-Rh1,Rb1,Rd,Rk3,Rh4,20(S)-Rg3,20(R)-Rg3(SichuanWeikeqiBiologicalTechnologyCo.,Ltd.Sichuan,China)withpurity≧98%wereusedasstandardcompounds.TheanalysiswasperformedonanAgilent1260HPLCsystem(AgilentTechnologies)equippedwithaG1311BPump,aG4212BDADdetector,andaG1329Bautosampler.Chromatographicseparationwascarriedoutat30°ConaVisionHTC18column(250×4.6mm,5μm).ThemobilephasewascomprisedofA(ultrapurewater)andB(methylcyanide).Thegradientmodewasasfollows:0–20min,80%A;20–45min,54%A;45–55min,45%A;55–60min,45%A;60–65min,100%B;65–70min,80%A;70–90min,80%A.Theflowratewassetat1.0ml/min.Thedetectionwavelengthwassetat203nmandsamplevolumewassetat10μl.TheHPLCchromatogramofSPNsampleandchemicalstructuresofdominatingcompoundscharacterizedwereshowninFigureS3.AnimalAnimalexperimentalproceduresinthestudywerestrictlyconformtheGuidefortheCareandUseofLaboratoryAnimalsandrelatedethicsregulationsofKunmingUniversityofScienceandTechnology.TheprotocolwasapprovedbytheExperimentalAnimalWelfareandEthicsCommittee,KunmingUniversityofScienceandTechnology.SixtyKunming(KM)mice,halfmaleandhalffemale,weighing18–22g,werepurchasedfromTianqinBiotechnologyCo.Ltd.,Changsha,China[SCXK(Xiang)2014-0011].Micewererandomlydividedintosixgroups,namelythecontrolgroup,modelgroup,FufangE'jiaoJiang(FEJ)group,high-doseSPN(H-SPN)group,moderate-doseSPN(M-SPN)group,andlow-doseSPN(L-SPN)group,10miceineachgroup(thepoweranalysistodeterminethesamplesizewasgiveninTableS1).Theexperimentalmethodwasdescribedinourpreviousstudy(Xionget al.,2017a).“TheBDSmodelwasestablishedbyintraperitonealinjectionof0.07g·kg-1ofCYforthefirst3daysandahypodermicinjectionof0.02g·kg-1ofAPHonthefourthday.Afterthat,miceinthecontrolgroupwereadministeredwith0.9%normalsaline,whereasothergroupswereadministeredwithFEJ(8ml/kg-1),orSPNpowder(1.8,0.90,and0.45g·kg-1,respectively),respectively,bygavagefor12days.”BloodRoutineTestHalfanhourafterthelastexperimentaladministration(saline,FEJ,orSPN),themicewereslightlyanesthetizedwithasmallamountofdiethylether,andbloodwastakenfromtheorbitandcollectedintoasterilecentrifugetubecontainingsodiumcitrate.BloodwassubjectedtoaperipheralhemogramanalysisbyaHEMAVET950full-automaticbloodcellanalyzer(DrewScientificGroup,Dallas,TX,USA),forthequantificationofredbloodcells(RBCs),whitebloodcells(WBCs),hemoglobin(Hb)andplatelets(PLTs).OrganCoefficientAftertheanesthetizationandbloodcollectionasdescribedin2.3,themiceweresacrificedbycervicaldislocation.Theliver,kidney,andspleenswerewashedinnormalsalinesolution,driedwithfilterpaper,andweighedimmediately.ThespleenswerestoredinliquidnitrogenforWesternblotanalysis.Organcoefficientwascalculatedbythefollowingequation:organcoefficient=[organwetweight(g)/bodyweight(g)]×100%(Zhanget al.,2014b).AnalysisofEPO,EPOReceptor(EPOR),TPO,TPOReceptor(C-Mpl),GM-CSF,andGATA-1inMiceBoneMarrowNucleatedCellsOnefemurwasisolatedafterthesacrificeofeachmouse.Theattachedmuscleswereremovedbygauzepolishing.Thefemurwasbrieflysoakedin75%ethanolbeforewashingwithphosphatebufferedsolutionforthreetimesunderasepticconditions.Thebonemarrowcellswerewashedoutofthefemurwithphosphatebufferedsolutionseveraltimesandfilteredwitha0.4mmdiameterneedle.Thebonemarrowcellsuspensionswereseparatedwithmicelymphocyteseparationsolutiontopreparethebonemarrownucleatedcells,afterwhich800μlofRNAisoPluskit(TaKaRa,Kusatsu,Japan)wasaddedbeforestorageat−80°Cuntilreal-timequantitativepolymerasechainreaction(RT-qPCR)analysiswasdone.TotalRNAfromthebonemarrownucleatedcellswasextractedusingtheRNAisoPluskit(TaKaRa,Kusatsu,Japan)inaccordancewiththemanufacturer'sprotocols.ThentotalRNA(microgram)wasreverse-transcribedintocDNAusingaPrimer-ScriptFirstStrandcDNAsynthesiskit(TaKaRa,Kusatsu,Japan)(Yanget al.,2018).Thedesignofgene-specificprimerswasdonewithPrimer5.0softwarebasedonpublishedmicesequencesfromtheGeneBank/NationalCenterforBiotechnologyInformationdatabaseandtheyweresynthesizedbyTsingKeBiologicalTechnology(Beijing,China).ThenucleotidesequencesprimersusedforPCRwereshowninTable1.TheRT-qPCRprogramwasconductedusingaLightCycler®96System(DBI®Bioscience,Ludwigshafen,Germany).Thermocyclingparametersincludedaninitialphaseof95°Cfor2minfollowedby40cyclesof95°Cfor10sand60°Cfor32s.Therelativegeneexpressionvalueswerecalculatedwiththe2−△△Ctmethod(Chenet al.,2017).TABLE1Table1Primerssequenceandparametersforareferencegeneandsixtargetgenes.PathwayandTargetsAnalysesPredictionBasedonpreviousresearch(Xionget al.,2019),20compoundsincludingginsenosidesofF2,Rb1,Rb2,Rb3,Rc,Rd,Re,Rg1,Rh2,Rh4,Rk3,20(R)-Rg2,20(S)-Rg2,20(R)-Rg3,20(S)-Rg3,20(R)-Rh1,and20(S)-Rh1;andnotoginsenosidesofC,R1,andR2reportedinSPNwereselectedtoconstructthenetwork.“ThechemicalstructuresofthecompositecompoundsinSPNwereobtainedfromtraditionalChinesemedicineDatabase@Taiwan(TDT)ordrawnwithChemDrawprofessional15.0(Chen,2011).ThetargetsofconstituentswerepredictedbytheonlinetargetpredictionsoftwareofPharmmapperwithacriterionof“fitscore”>4(http://lilab.ecust.edu.cn/pharmmapper/index.php)(Wanget al.,2017).GeneandproteintargetsassociatedwiththediseaseofanemiawerecollectedfromtheOnlineMendelianInheritanceinMan(OMIM)database(Ambergeret al.,2015).DatabaseofInteractingProteinsforprotein-proteininteractions(PPI)wasemployedtoidentifythepossibleinteractionsoftheaforementionedtargets.AllproteinIDcodeswereconvertedtoUniProtIDs(Weiet al.,2016).”WeusedtheDAVID(aDatabaseforAnnotation,Visualization,andIntegratedDiscovery)FunctionalAnnotationBioinformaticsMicroarrayAnalysis(https://david.ncifcrf.gov/)forpathwayenrichmentanalysisandtheKyotoEncyclopediaofGenesandGenomes(KEGG)toconstructthe“SPNcomponents-target-anemia”networkwithprotein-proteininteractionsinformation.Threetopologicalparametersof“degree,”“betweennesscentrality”and“closecentrality,”andCytoscape4.3wereappliedtoscreenpotentialtargetsforSPNtreatinganemia(Xionget al.,2019).TheserelevantproteinswereputintoDAVIDtoperformgeneontologytermsandKEGGpathwayanalysis.VerificationTotalProteinExtractionandWesternBlottingAnalysisSpleenproteinswereextractedusingRIPAlysisbuffer(Solarbio,Beijing,China)andcentrifugedat12,000 gfor10 min at4°C.ProteinconcentrationwasassessedusingtheBCAProteinAssayKit(Biosharp,Beijing,China).Samplescontainingequalamountsofprotein(80μg)wereseparatedby10%sodiumdodecylsulfate-polyacrylamidegelelectrophoresisandthentransferredto0.45μmpolyvinylidenedifluoridemembranes(Biosharp,Beijing,China).Thenon-specificbindingsitesonthemembranewerethenblockedwith5%freshnon-fatdrymilkinTrisbufferedsalineTBSwithTween(10mMTris,150mMNaCl,pH7.4)with0.1%Tween20)for2h.Subsequently,theywereincubatedwithprimaryantibodies(anti-IL-2,anti-STAT1,anti-SHP2,anti-JAK1,andanti-p-JAK1;1:1000dilution;Abcam,Cambridge,UK)andprimaryβ-actinantibody(1:1,000dilution;abmart,Shanghai,China)overnightat4°C,respectively.Afterthis,themembranewaswashedfollowedbythree10minwashesinTBST,andthenincubatedwithanti−mousesecondaryantibody(1:2000dilution;CellSignalingTechnology,Shanghai,China)for2hatroomtemperaturefollowedbythree10minwashesinTTBS.Finally,thechemiluminescentsignalswereobservedwithamultifunctionalgelimagingsystem(Bio-Rad,USA)anddensitometryforimmunoreactivebandswasperformedwithNationalInstitutesofHealthsoftware(ImageJ).AnalysisofIL-2,STAT1,SHP2,p-JAK1,Bcl-2,Bcl-XL,c-Myc,andp21inMiceSpleenbyRT-qPCRTheanalysismethodreferstoitem2.5.IL-2,STAT1,SHP2,p-JAK1,Bcl-2,Bcl-XL,c-Myc,andp21genessequencesynthesizedbyTsingKeBiologicalTechnology(Beijing,China),andprimerssequenceareshowninTable2.TABLE2Table2PrimerssequenceandparametersforIL-2,STAT1,SHP2,p-JAK1,Bcl-2,Bcl-XL,c-Myc,andp21genes.StatisticalAnalysesAlldataareexpressedasmeans±standarddeviation.SPSS21.0software(StatisticalProgramforSocialSciences,SPSSInc,USA)wasappliedtocarryoutthetwo-tailedunpairedt-test.AvalueofP<0.05wasconsideredtoindicateasignificantdifference.AvalueofP<0.01wasconsideredtoindicateahighlysignificantdifference.QuantitativegeneexpressionsweredeterminedbyRT-qPCRandhistogramsweredrawnbyGraphPadPrism7software(GraphPadSoftware,USA).ResultsGeneralBehaviorofMiceThechangeofgeneralbehaviorofmicecanreflecttheoccurrenceandrecoveryofBDSofmicetocertaindegree(Liet al.,2012).AftertheinductionofBDS,miceinthemodelgroupbecamesluggish,exhaustedandsomnolent,alongwithweightloss,sparsehair,paleearsandtail,andlossofappetite.ThosesymptomswereconsistentwiththedescriptionofBDSinChinesemedicine(Jiet al.,2017).Incontrast,miceinthecontrolgroupandBDS-inducedmicetreatedwithFEJorSPNwererelativelyvigorousandstrong,presentingthickandlustroushair,pinkandmoistnoseandlips,roundandpinktail,gainedweightandbetterappetite.BloodRoutineTestAftertheadministrationofsaline,FEJorSPNfor12days,thequantitiesofWBC,RBC,Hb,andPLTfromtheperipheralbloodofmiceweredetermined(Figure1).Comparedtothecontrolgroup,thelevelsofWBC,RBC,Hb,andPLTinthemodelgroupweredecreasedsignificantly(p<0.01),indicatingtheanemiamodelwasestablishedsuccessfully.ComparisonwithinthemodelgroupshowsthatWBC,RBC,HbandPLTlevelsinmicetreatedwithFEJandH-SPNwereincreasedsignificantly(p<0.01orp<0.05).Inparticular,allthreedoses(H,M,andL)ofSPNsignificantlyimprovedthelevelofWBC(p<0.01).RBCandHblevelsinmicetreatedwithM-SPNandL-SPNwerenotsignificantlyincreased.FIGURE1Figure1Thebloodparametersofthecontrolandmodelmiceaftertreatmentwithsalinesolution(model),FEJanddifferentamountsdosesofSPN.(A)ThenumberofWBC.(B)ThenumberofRBC.(C)ThecontentofHb.(D)ThenumberofPLT.Eachvaluerepresentsthemean±SD(n=10);*p<0.05and**p<0.01,comparedtothecontrolgroup;▲p<0.05and▲▲p<0.01,comparedtothemodelgroup.OrganCoefficientsTheresultsfororgancoefficientsareshowninFigure2.Comparedtothecontrolgroup,theliverandkidneyofthemodelgroupwererelativelysmall,butthespleenwasabnormallyenlarged.Exceptforthespleencoefficient,therewasnosignificantdifferenceinotherorgancoefficientsbetweentheexperimentalgroups(model,FEJ,H-SPN,M-SPN,andL-SPNgroup)andthecontrolgroup.Comparedtothecontrolgroup,thespleencoefficientofthemodelgroupwasincreasedsignificantly(p<0.01).ComparisonwithinthemodelgroupshowsthatthespleencoefficientsoftheFEJandH-SPNgroupsweredecreasedsignificantly(p<0.01),theM-SPNgroupwasdecreasedsignificantly(p<0.05),whereastherewasnosignificantdifferencefortheL-SPNgroup.Accordingtheresults,SPNinducesareversingeffectontheenlargementofspleeninadose-dependentway.FIGURE2Figure2Theorgancoefficientsofcontrolandmodelmiceaftertreatmentwithsalinesolution(model),FEJanddifferentdosesofSPN.(A)Thelivercoefficient.(B)Thespleencoefficient.(C)Thekidneycoefficient.Eachvaluerepresentsthemean±SD(n=10);**p<0.01,comparedtothecontrolgroup;▲p<0.05and▲▲p<0.01,comparedtothemodelgroup.EffectofSPNonEPO,EPOR,TPO,c-Mpl,GM-CSF,andGATA-1inMiceBoneMarrowTheeffectsofFEJandSPNonthemRNAexpressionofhematopoieticfunction-relatedcytokinesinthebonemarrowofmiceisshowninFigure3.Comparedtothecontrolgroup,thelevelsofEPO,EPOR,TPOandc-Mplinthemodelgroupwereincreasedsignificantly(p<0.01orp<0.05).ComparisonwithinmodelgroupshowsthatthelevelsoftheabovecytokinesinmicetreatedwithFEJandH-SPNwereincreasedsignificantly(p<0.01).IncreasestodifferentdegreeswerealsoseenintheM-SPNandL-SPNgroups.Comparedtothecontrolgroup,thelevelsofGM-CSFandGATA1inthemodelgroupweredecreasedsignificantly(p<0.01orp<0.05).ComparisonwithinthemodelgroupshowsthatbothcytokinesweresignificantlyincreasedinmicetreatedwithFEJ,H-SPN,M-SPNandL-SPN(p<0.01orp<0.05).ThemRNAexpressionlevelsofthesehematopoieticrelatedcytokineswerechangedinaSPNdose-dependentmanner.FIGURE3Figure3ThemRNAexpressionof(A)EPO,(B)EPOR,(C)TPO,(D)c-Mpl,(E)GM-CSF,and(F)GATA1incontrolandmodelmiceaftertreatmentwithsalinesolution(model),FEJanddifferentdosesofSPN.Eachvaluerepresentsthemean±SD(n=10);*p<0.05and**p<0.01,comparedtothecontrolgroup;▲p<0.05and▲▲p<0.01,comparedtothemodelgroup.PathwayandTargetsAnalysesPredictionAccordingtothepathwayenrichmentanalysis,theJAK-STATsignalingpathwaywaspredictedtobeoneofthemostenrichedpathways(FigureS1).BasedontheKEGGanalysis,threeanemia-relatedtargetsareinvolvedinthepathwayandcorrespondingactiveconstituentsinPNarepredictedandshowninTable3(Xionget al.,2019).Anotherkinaseprotein,JAK1,betweenIL-2andSTAT1orSHP2,alsoplaysasignificantpivotalroleintheJAK-STATpathway(Figure4).Therefore,theexpressionofIL-2,JAK1,p-JAK1,andSTAT1proteinsinBDSmicewasinvestigatedinthefollowinganalysis.Inaddition,themRNAexpressionofdownstreamcytokinesincludingBcl-2,Bcl-XL,c-Myc,andp21relatedtothecellcyclewasinvestigatedbasedonthepredictionandreferencestudies(Fröjmarket al.,2010;Karimianet al.,2016).TABLE3Table3TheinformationofthreetargetsandtheircorrespondingactiveconstituentsinvolvedinJAK-STATsignalpathwaypredictedbyKEGG.FIGURE4Figure4Targetproteins(IL-2,SHP2,andSTAT1)predictedtobeinvolvedintheJAK-STATpathwayandthedownstreamcytokines(Bcl-2,Bcl-XL,c-Myc,andp21).IL-2,interleukin-2;JAK1,januskinase-1;SHP2,tyrosine-proteinphosphatasenon-receptortype11;GRB,growthfactorreceptor-boundprotein;SOS,guaninenucleotideexchangefactor;STAT1,signaltransducerandactivatoroftranscription;Bcl-2,B-celllymphoma-2;Bcl-XL,Bcl-2like1;c-Myc,v-mycavianmyelocytomatosisviraloncogenehomolog;andp21,cki.WesternBlottingAnalysisTheproteinexpressionofJAK1,p-JAK1,STAT1,SHP2,andIL-2inthespleenofmiceineachgroupisshowninFigure5.TheresultsofthegrayscaleanalysisareshowninTable4.Accordingtotheresults,therewasnosignificantdifferenceinJAK1expressionamongthegroups(p>0.05).Theexpressionlevelofp-JAK1inthemodelgroupwassignificantlyhigherthaninthecontrolgroup(p<0.01).BothFEJ,H-,andM-SPNresultinsignificantlylowerproteinexpressionlevelsthaninthemodelgroup(p<0.01).Comparedtothecontrolgroup,theexpressionlevelsofproteinSTAT1,SHP2andIL-2inthemodelgroupweresignificantlydecreased(p<0.01orp<0.05).ComparisonwithinthemodelgroupshowsthattheexpressionlevelsoftheproteinIL-2aftertreatmentwithFEJ,H-SPN,M-SPNorL-SPNweresignificantlyincreased(p<0.01orp<0.05).TheexpressionlevelsoftheproteinSTAT1andSHP2inFEJandH-SPNgroupsweresignificantlyhigherthanthoseinthemodelgroup,andthedifferenceswerestatisticallysignificant(p<0.01orp<0.05),whilethoseforinM-SPNandL-SPNgroupswerenotstatisticallysignificant(p>0.05).FIGURE5Figure5TheexpressionofpredictedtargetproteinbyWesternblot.Lanes1–6representcontrol,model,FEJ,H-SPN,M-SPN,andL-SPNgroup,respectively.TABLE4Table4Grayvalueratioofpredictedtargetproteins/β-actinbyWesternblot.QuantificationofIL-2,STAT1,SHP2andp-JAK1byRT-qPCRinMiceSpleenTheexpressionofIL-2,STAT1,SHP2andp-JAK1mRNAinthespleenofmiceisshowninFigure6.Comparedtothecontrolgroup,theexpressionofIL-2andSHP2mRNAinthemodelgroupwassignificantlydown-regulated(p<0.01).ComparisonwithinthemodelgroupshowsthataftertreatmentwithFEJordifferentdoseofSPN,theexpressionofIL-2andSHP2mRNAintheadministeredgroupswasincreasedtovaryingdegreesinadose-dependentmanner.TherewasnosignificantdifferenceintheexpressionofIL-2andSHP2betweentheL-SPNgroupandthemodelgroup(p>0.05),othersarestatisticallysignificant(p 0.05).Therewasnosignificantdifferenceintheexpressionofp-JAK1betweentheM-SPNgroupandthemodelgroup(p>0.05).Andtherewassignificantdifferencebetweentheothergroups(p<0.01).FIGURE6Figure6ThemRNAexpressionof(A)IL-2,(B)STAT1,(C)SHP2,and(D)p-JAK1incontrolandmodelmiceaftertreatmentwithsalinesolution(model),FEJanddifferentdosesofSPN.Eachvaluerepresentsthemean±SD(n=10);*p<0.05and**p<0.01,comparedtothecontrolgroup;▲p<0.05and▲▲p<0.01,comparedtothemodelgroup.QuantificationofJAK1-STAT1DownstreamCytokinesinMiceSpleenbyRT-qPCRTheexpressionofBcl-2,Bcl-XL,c-Myc,andp21mRNAinthespleenofmiceisshowninFigure7.Comparedtothecontrolgroup,theexpressionofBcl-2andBcl-XLmRNAinthemodelgroupwashighlysignificantlydown-regulated(P<0.01).ComparisonwithinthemodelgroupshowsthataftertreatmentwithFEJordifferentdoseofSPN,theexpressionofBcl-2andBcl-XLmRNAintheadministeredgroupwasincreasedtovaryingdegreesinadose-dependentmanner.ExceptthattherewasnosignificantdifferencebetweentheBcl-2mRNAofL-SPNandthemodelgroup(p>0.05),andtheothergroupswerestatisticallysignificant(p<0.01).Comparedtothecontrolgroup,theexpressionofc-Mycandp21mRNAinthemodelgroupwashighlysignificantlyup-regulated(p<0.01).ComparisonwithinthemodelgroupshowsthataftertreatmentwithFEJordifferentdoseofSPN,theexpressionofc-Mycandp21mRNAintheadministeredgroupdecreasedandwasnegativelyrelatedtothedrugconcentration.ExceptthattherewasnosignificantdifferencebetweentheL-SPNandthemodelgroup(p>0.05),andtheothergroupswerestatisticallysignificant(p<0.01).FIGURE7Figure7ThemRNAexpressionof(A)Bcl-2,(B)Bcl-XL,(C)c-Myc,and(D)p21incontrolandmodelmiceaftertreatmentwithsalinesolution(model),FEJanddifferentdosesofSPN.Eachvaluerepresentsthemean±SD(n=10);*P<0.05and**P<0.01,comparedtothecontrolgroup;▲P<0.05and▲▲P<0.01,comparedtothemodelgroup.DiscussionAccordingtotraditionalChinesemedicinetheory,thepathogenesisofblooddeficiencycouldbethedisharmonyofYin-Yang,qideficiency,bloodstasis,andthedysfunctionofinternalorgans.Therefore,herbalmedicineswhichcannourishqiandblood,aswellasinvigoratethespleenandkidneyareoftenusedintheclinictotreatblooddeficiencyandanemia(Hijikataet al.,2009;Wuet al.,2013;Sunet al.,2016).Forexample,FEJ,thepositivecontrolinthestudy,isawidelyusedimmune-boostingtraditionalChineseformulawhichhasbeenclearlyconfirmedtopromotetherecoveryofbonemarrowhemopoieticactivityandenhancetheimmunefunction(Liuet al.,2014;Shenet al.,2016).WhileforSPN,anothertonicandhemostaticdrugusedformorethan400years,thepossiblemechanismofthe“blood-tonifying”effectisunclearyet.Afterthemodeling,therewasanobviouschangeintheappearanceofmice,includinghairloss,movementretardation,andweightloss,withthesignificantdecreaseintheperipheralWBC,RBC,Hb,andPLT,whicharecharacteristicclinicalmanifestationsofblooddeficiencyaswellasanemia(Sunet al.,2016).WiththeH-SPNandFEJtreatments,thedecreaseofperipheralbloodcellswasreversedsignificantly(Figure1),suggestingthatH-SPNandFEJcouldenhancethehematopoieticeffectofmicewithBDS,consistentwiththetraditionaluseofSPNandFEJtotonifytheblood(Guet al.,2015).Meanwhile,M-SPNandL-SPNcouldnotincreasethenumbersofRBC,HbandPLTsignificantly,suggestingthatahigherdosageofSPNwasrequiredtoachieveamoreobvioustherapeuticeffect,whichwasconsistentwithourpreviousstudy(Xionget al.,2017a).Anotherdiagnosticconsiderationforanemiaistheenlargedsizeofspleen,whichisoneofthemostcommonandearlyorganstobeaffectedindifferenttypesofanemia(Dhaliwalet al.,2004;Alsalem,2011).Itisgenerallyacceptedthatthehematopoieticdeficiencyisassociatedtoamassivemigrationofbonemarrowprogenitorcellsoutofthemarrow,viatheblood,leadingtoanaccumulationinthespleen(Queirozet al.,2004).AndthepoolingofRBCsintheenlargedspleencanexacerbatethesyndromeofanemiainturn(Gasparet al.,2015).Therefore,thedeclineofRBCsincirculationandanemiacanbebothcorrelatedwiththeextentoftheenlargementofthespleen(Wagneret al.,2000).AccordingtoFigure3,theweightandsizeofspleeninthemodelgroupwereincreased.WhilethespleenofmicetreatedwithFEJ,H-SPN,andM-SPNwaskeptfromenlargingcomparedtothemodelgroup(Figure2C).ItindicatesthatFEJ,H-SPN,andM-SPNcouldalleviatethesideeffectsofchemotherapyonthespleenandprotecttheimmuneorgan.Theprocessofbloodcellformation,bywhichasmallnumberofself-renewingstemcellsgiverisetolineagecommittedprogenitorcellsthatsubsequentlyproliferateanddifferentiatetoproducethecirculatingmaturebloodcells,isregulatedbyaseriesofrelatedhematopoieticrelatedcytokines(Sieff,1987).Hematopoieticgrowthfactors(HGFs)includingEPO,TPO,GM-CSF,andIL-3arecytokinesinvolvedintheregulationofhematopoiesis(Ferrettiet al.,2006;Genovaet al.,2016).Amongthem,EPOisaglycoproteinhormoneandamajoragonistfortheproductionofWBC(Frankeet al.,2013).EPObindstoitsreceptorEPOR,activatesthedownstreamJAK-STATsignalingpathway,promotesthegrowthanddifferentiationoferythroidprogenitorcells,inhibitstheirapoptosis,andmobilizeshematopoieticstemcells(HSCs)(Madonnaet al.,2009;Elliottet al.,2014).TheresultsinFigures3A,BshowthattheexpressionsofEPOandEPORmRNAinthemodelgroupwashigherthanthoseinthecontrolgroup,suggestingthatthechemotherapy-inducedanemiacausedafeedbackincreaseinEPOcontent,andtheninducedtheexpressionofEPOR.Thisisacompensatorymechanism.Duetothedamageofchemotherapydrugstobonemarrowcells,thereactivityofreactivecellstoHGFsisreduced.SoahigherconcentrationofHGFsisrequiredtopromotetherecoveryofhematopoieticfunction,whichisconsistentwiththeaboveliteraturereports.Whileaftertreatingwiththedrugs,theexpressionofEPOandEPORmRNAintheadministrationgroups(FEJ,H-SPN,M-SPN,andL-SPNgroups)washigherthanthatinmodelgroup,indicatingthatFEJandSPNcouldpromotetherecoveryoferythropoiesisbypromotingthesynthesisandsecretionofEPOinmicebonemarrow.AsimilartendencywasalsoobservedinthelevelchangeofTPOanditsreceptorc-Mplinthebonemarrowofmice(Figures3C, D),theformerofwhichisthemostimportantregulatorofplateletproduction,aswellasamajorcytokinerequiredforthedevelopmentofHSCs,monocytes,granulocytes,mastcellsanddendriticcells(Chen,2004;Kirshenbaumet al.,2004).AfteradministrationofFEJandSPN,theexpressionofTPOandc-MplmRNAinbonemarrowwassignificantlyincreased,whichwouldbebeneficialtopromotetherecoveryofmegakaryocyteshematopoiesisandincreasethenumberofperipheralPLTinmicewithBDS.AnotherimportantHGFisGM-CSF,whichmainlyactsonHSCs/HPCsandbonemarrowstromalcellsorfibroblasts,andplaysanimportantroleinregulatinghematopoiesisandleukocyteformation(Kimuraet al.,1997;Waller,2007).TheresultsinFigure3EshowedthatAPHandCYsignificantlyinhibitedtheexpressionofGM-CSFmRNA,whichwasincreasedsignificantlyaftertheadministrationofFEJandSPN.ItindicatedthatFEJandSPNcouldinhibitthecytotoxicityofCYandAPHtosomeextent.GM-CSFlevelwasinducedtoincrease,promotingtherecoveryofgranulocytichematopoieticfunction.WealsoinvestigatedthechangeofGATA1,anecessarytranscriptionfactorforthedevelopmentandmaturationofnormalerythrocytes.TheincreasedexpressionofGATA1mRNAaftertreatingFEJandSPNwasconsistentwiththeabovementionedcytokines(Figure3F).Fromtheresultsofresponsesofthehematinicfunction-relatedcytokines,wenotedthattheexpressionofthemwerecloselyrelatedtothesignalpathwayofJAK-STAT,whichplaysacriticalroleintransductionofextracellularsignalsfromcytokinesandgrowthfactorsinvolvedinhematopoiesis,immuneregulationandinflammation(VainchenkerandConstantinescu,2013;Chenet al.,2016).Accordingtoourpreviousanalysisofnetworkpharmacology,JAK-STATwasalsopredictedtobeoneofthemajorpathwaysrelatedtothetreatmentofanemiawithSPN(FigureS1).IL-2,STAT1,andSHP2inFigure4,werepredictedtobethemajorthreetargetsinvolvedintheJAK-STATpathwaytotreatanemia(Xionget al.,2019).Toverifytheprediction,weinvestigatedtheeffectofSPNontheexpressionofthethreetargetsandalsothedownstreamcytokinesofJAK-STATincludingBcl-2,Bcl-XL,c-Myc,andp21.Accordingtotheresults,thetreatmentbyAPHandCYcouldsignificantlyinhibittheexpressionofIL-2andSHP2proteins(Figure5)andthecorrespondedmRNA's(Figures6A,C).SinceIL-2isaTh1typecytokinesecretedbyTcells(Vafadariet al.,2012)andSHP2playsanup-regulationroleinthedevelopmentandfunctionofhematopoieticcells(Kratzet al.,2007;Zhuet al.,2011),thechemotherapy-inducedanemiacouldberelatedtothedevelopmentofimmunosuppressionandcytotoxicityonhematopoieticcells.Afterthedrugadministration,theexpressionofIL-2andSHP2wasincreased,suggestingthatFEJandSPNcouldmakeapositiveeffectonenhancingtheTcellimmunityandreducingthecytotoxicity.Meanwhile,itwasreportedthatSHP2directlymediatesmanycytokinesandgrowthfactorreceptorslikeGM-CSFandEPOreceptors(Myerset al.,1998).TheincreasedmRNAexpressionofGM-CSFandEPORshowninFigure3couldbepartlyattributedtotheup-regulationroleofSHP2intervenedbySPN.Theroleofdrugsonanotherimportantsignaltransducerandactivatoroftranscription,STAT1,wasinvestigatedbyWesternblotandRT-qPCR.AsshowninFigure6B,thechemotherapy-inducedtheexpressionofSTAT1mRNA,whichwasdecreasedinadose-dependentwaywhentreatedwithSPN.However,theactivationofSTAT1proteinlevel(Figure5)wasinconsistentwithitsmRNAexpression.Thismightbeduetothatthepost-transcriptionalRNAandthebindingsitesofregulatoryproteinschangedthemodeoftranslationandthenaffectedtheexpressionofproteins.Suchphenomenonwasalsoinvestigatedinpreviousstudies(Saunderset al.,2007;Nicolosoet al.,2016).Anotherpossibleexplanationmaybethatthepost-translationalmodificationledtoadecreaseinthestabilityorhalf-lifeoftheprotein.Also,theactivityofJAK1anditsphosphorylatedform,theenzymesessentialforthereceptorsignalinglocatedintheupstreamofSTAT1,wasinvestigated.Accordingtotheresults,thetreatmentofdrugsmadenosignificanteffectontheexpressionofJAK1,whereastheexpressionofp-JAK1wasincreasedafterthechemotherapyandthendecreasedaftertreatingwithFEJandH-SPN.ItsuggestedthatFEJandH-SPNmightregulatethehematinicfunctionthroughphosphorylatingJAK1.Besidestheabovetargetsinvolvedinthepathway(Figure8),networkpharmacologyalsoprovidesthechemicalinformationrelatedtotheefficacyofSPN,whichisthereforebeneficialtouncoveringtheinteractionsbetweenactiveconstituentsinSPNandthecomplexsyndromesystems(Taoet al.,2013;Shenget al.,2014).Bytheanalysisofnetworkpharmacology,sixcompoundsinSPNwerepredictedtobetheanti-anemiaonescorrespondingtothethreetargetsinvolvedintheJAK-STATsignalpathway,whichwereginsenosidesRh4,Rk3,Rb2,20(R)-Rg3,Rd,andnotoginsenosideC(Figure9).Differencesinginsenosidestructurewhichincludethetype,position,andnumberofsugarmoietiesattachedbyaglycosidicbondcancharacteristicallyinfluencethebiologicalresponses(Tenget al.,2017;Chenetal.,2019).Amongthem,20(R)-Rg3,Rh4,andRk3showedincreasedlevelsalongwiththedurationofsteamingandhadakeyroleintheactivitiesofSPNinourpreviousstudy(Xionget al.,2017b).Zhouet al.(2016)reportedthatRg3helpedrepairblooddisorderssuchasanemia,leukopenia,andthrombocytopenia.20(R)-Rg3couldalsostimulatedtheproductionofIL-2andenhancethecellularimmunityintumor-bearingmice(Wuet al.,2014),whichwasconsistentwithourresultofIL-2expression.GinsenosidesRh4andRk3wereconsideredtobenewtherapeuticdrugsfortreatinganemiasinceRk3promotedbonemarrowandextramedullaryhematopoiesiswhileRh4promotedextramedullaryhematopoiesis(Weiet al.,2018).DifferentfromRg3,Rb2andRdhaveanadditionofoneglucosemoietyandtwoglucosemoietiesattachedatC-20position.Rb2couldprotectthehematopoieticsystemfromtheirradiation-causedinjuryandpromotethesynthesisofserumproteinandDNAofmarrowcells(Leeet al.,2011;Zhanget al.,2017).GinsenosidesRdcouldregulatetheexpressionofBcl-2(Wanget al.,2013).NotoginsensideCwasonlymentionedinafewreports(Yoshikawaet al.,2001;Chenet al.,2010)whichcouldhavetheanti-tumorandimmunologicaladjuvantactivity.Infurtherstudies,theeffectsofthoseconstituentsandrelatedmechanismwillbeinvestigatedandconfirmed.FIGURE8Figure8(A)ChangesofbodyparametersandcytokinesinBDSmicecomparedtothecontrolgroup.(B)ChangesofbodyparametersandcytokinesinSPNadministrationgroupsmicecomparedtothemodelgroup.IL-2,interleukin-2;JAK1,januskinase-1;SHP2,tyrosine-proteinphosphatasenon-receptortype11;GRB,growthfactorreceptor-boundprotein;SOS,guaninenucleotideexchangefactor;STAT,signaltransducerandactivatoroftranscription;Bcl-2,B-celllymphoma-2;Bcl-XL,Bcl-2like1;c-Myc,v-mycavianmyelocytomatosisviraloncogenehomolog;p21,cki.FIGURE9Figure9ThesixcompoundscorrespondingtothethreetargetsinvolvedintheJAK-STATsignalpathwayrelatedtothetreatmentofBDSwithSPN.(A)GinsenosidesRh4,(B)ginsenosidesRk3,(C)ginsenosidesRb2,(D)ginsenosides20(R)-Rg3,(E)ginsenosidesRd,and(F)notoginsenosideC.Bcl-2,Bcl-XL,c-Myc,andp21arecellcycle-relatedfactorsofJAK1-STAT1.SPNcouldactontheJAK1-STAT1signalingpathwayandsimultaneouslyaffectthedownstreamcytokines,improvingthecellapoptosisofmicewithBDS.AccordingtoFigure7A,theexpressionofBcl-2wasup-regulatedalongwiththedoseincreaseofSPN,whichcouldmaintainthesurvivalandproliferationoferythroidprogenitorcellswhenGATA-1wasreducedinthemodelofanemia(Figure3F)(KapurandZhang,2001).Anotheranti-apoptoticproteinofBcl-XLwasalsoup-regulatedwhentreatingwithSPN(Figure7B),whichmightbeduetothemediationofGATA1,EPOandTPOthroughtheJAK/STATspathway,supportingthesurvivalofhematopoieticcells(Chibaet al.,1991;Kitajima,2006).c-Mycandp21,theexpressionofwhichwereoftenincreasedinhematologicalmalignanciescausedbyBDSorDNAdamage,werebothdown-regulatedsignificantlyaftergivingSPN(Figure7C),suggestingthatSPNmightattenuateanemiabyinhibitingthearrestofcellcyclebecauseofDNAdamage.Therefore,theexpressionofvarioushematopoieticfactorscouldbecloselyrelatedtothecellapoptosis.ThisisconsistentwiththereportofĆmielováandŘezáčová,(2011).ConclusionsThehematopoieticeffectandmechanismofSPNonmicewithBDSwasstudiedatthreelevels.Firstly,SPNrevertedthedecreaseinthenumberofperipheralbloodcellsandpreventedtheenlargementoftheextramedullaryhematopoieticorganofspleen.Secondly,SPNpromotedtheexpressionofrelatedhematopoieticcytokinesincludingEPO,EPOR,TPO,c-Mpl,GM-CSF,andGATA-1inbonemarrownucleatedcellsofmice.Thirdly,SPNwasinvolvedintheactivationofJAK-STATsignalingpathway,throughregulatingthetargetsexpressionofIL-2,p-JAK,STAT1,andSHP,andalsothedownstreamcellcyclefactorsofBcl-2,Bcl-XL,c-Myc,andp21(Figure8).SPNcouldinfluencethecellcycleandpossiblytheapoptosisprocess,enablingthedamagedcellstoobtainafavorablesurvivalconditioninablooddeficiencyenvironment.ThepresentstudyprovidesfurtherguidancefordevelopmentsintheclinicaluseandaswellasprovidescluesfordrugdevelopmentofSPN.DataAvailabilityStatementAlldatasetsgeneratedforthisstudyareincludedinthearticle/SupplementaryMaterial.EthicsStatementTheanimalstudywasreviewedandapprovedbytheExperimentalAnimalWelfareandEthicsCommittee,KunmingUniversityofScienceandTechnology.AuthorContributionsYXsupervisedtheprojectandparticipatedinthedesignofthestudy.ZZperformedtheexperimentsandstatisticalanalysisaswellasthewritingofthepaper.YYgaveexperimentalguidance.MGprovidedtherevisionandcorrectionworks.YZ,YH,CW,andXCcollectedandprocessedsamples.BDandMWguidedthedesignandmodifiedthemanuscript.FundingThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(81660661),theYunnanAppliedBasicResearchProject(2016FD040),andtheNationalResearchProgramofChina(2017YFC02503).ConflictofInterestAuthorBDwasemployedbycompanyFytagorasBV.Theremainingauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest.AbbreviationsAPH,acetylphenylhydrazine;Bcl-2,B-celllymphoma-2;BDS,blooddeficiencysyndrome;c-Mpl,thrombopoietinreceptor;c-Myc,v-mycavianmyelocytomatosisviraloncogenehomolog;CY,cyclophosphamide;EPO,erythropoietin;EPOR,erythropoietinreceptor;FEJ,FufangE'jiaoJiang;GM-CSF,granulocyte-macrophagecolony-stimulatingfactor;Hb,hemoglobin;IL-2,interleukin-2;JAK-STAT,januskinase-signaltransducerandactivatoroftranscription;KEGG,KyotoEncyclopediaofGenesandGenomes;PN,Panaxnotoginseng;PLT,platelet;RBC,redbloodcell;PT-qPCR,real-timequantitativepolymerasechainreaction;SHP-2,tyrosine-proteinphosphatasenon-receptortype11;SPN,steamedPanaxnotoginseng;TPO,thrombopoietin;WBC,whitebloodcell.SupplementaryMaterialTheSupplementaryMaterialforthisarticlecanbefoundonlineat:https://www.frontiersin.org/articles/10.3389/fphar.2019.01578/full#supplementary-materialReferencesAlsalem,A.H.(2011).Spleniccomplicationsofsicklecellanemiaandtheroleofsplenectomy.Isrn.Hematol.2011,1–7.doi:10.5402/2011/864257CrossRefFullText|GoogleScholarAmberger,J.S.,Bocchini,C.A.,Schiettecatte,F.,Scott,A.F.,Hamosh,A.(2015).OMIM.org:OnlineMendelianInheritanceinMan(OMIM®),anonlinecatalogofhumangenesandgeneticdisorders.NucleicAcidsRes.43(1),789–798.doi:10.1093/nar/gku1205CrossRefFullText|GoogleScholarChen,G.,Yi,J.Y.,Wang,L.,Liu,J.H.(2010).DeterminationmethodfornotoginsenosideCinmouseplasma.J.JilinUniv.(EarthSci.Edition).48(3),516–519.doi:10.1016/S1872-2067(10)60116-7CrossRefFullText|GoogleScholarChen,L.,Teng,H.,Fang,T.,Xiao,J.B.(2016).AgrimonolidefromAgrimoniapilosasuppressesinflammatoryresponsesthroughdown-regulationofCOX-2/iNOSandinactivationofNF-κBinlipopolysaccharide-stimulatedmacrophages.Phytomedicine23(8),846–855.doi:10.1016/j.phymed.2016.03.016PubMedAbstract|CrossRefFullText|GoogleScholarChen,Y.L.,Wang,H.M.,Hu,W.,Wang,S.S.,Wang,Y.H.,Snider,J.L.,etal.(2017).Combinedelevatedtemperatureandsoilwaterloggingstressesinhibitcellelongationbyalteringosmolytecompositionofthedevelopingcotton(GossypiumhirsutumL.)fiber.PlantSci.256,196–207.doi:10.1016/j.plantsci.2017.01.001PubMedAbstract|CrossRefFullText|GoogleScholarChen,J.,Wang,F.,Huang,S.,Liu,X.,Li,Z.,Qi,A.,etal.(2019).Jian-Pi-Yi-Shendecoctionrelievesrenalanemiain5/6nephrectomizedrats:productionoferythropoietinviahypoxiainduciblefactorsignaling.Evid-Based.Compl.Alt.2019,1–8.doi:10.1155/2019/2807926CrossRefFullText|GoogleScholarChen,W.(2004).ThrombopoietincooperateswithFLT3-ligandinthegenerationofplasmacytoiddendriticcellprecursorsfromhumanhematopoieticprogenitors.Blood103(7),2547–2553.doi:10.1182/blood-2003-09-3058PubMedAbstract|CrossRefFullText|GoogleScholarChen,C.Y.(2011).TCMdatabaseatTaiwantheworld'slargesttraditionalChinesemedicinedatabasefordrugscreeninginsilico.Plos.One6(1),e15939.doi:10.1371/journal.pone.0015939PubMedAbstract|CrossRefFullText|GoogleScholarChiba,T.,Ikawa,Y.,Todokoro,K.(1991).GATA-1transactivateserythropoietinreceptorgene,anderythropoietinreceptor-mediatedsignalsenhanceGATA-1geneexpression.NucleicAcidsRes.19(14),3843–3848.doi:10.1093/nar/19.14.3843PubMedAbstract|CrossRefFullText|GoogleScholarChinesePharmacopoeiaCommission.(2015).PharmacopoeiaofthePeople"sRepublicofChina(Beijing,China:ChineseMedicalScienceandTechnologyPress).GoogleScholarĆmielová,J.,Řezáčová,M.(2011).Proteinanditsfunctionbasedonasubcellularlocalization.J.CellBiochem.112(12),3502–3506.doi:10.1002/jcb.23296PubMedAbstract|CrossRefFullText|GoogleScholarDarnell,J.,Kerr,I.,Stark,G.(1994).Jak-STATpathwaysandtranscriptionalactivationinresponsetoIFNsandotherextracellularsignalingproteins.Science264(5164),1415–1421.doi:10.1126/science.8197455PubMedAbstract|CrossRefFullText|GoogleScholarDhaliwal,G.,Cornett,P.A.,Tierney,L.M.(2004).Hemolyticanemia.Am.Fam.Physician69(11),2599–2606.doi:10.1016/j.pop.2004.04.007PubMedAbstract|CrossRefFullText|GoogleScholarDygai,A.M.,Goldberg,V.E.,Artamonov,A.V.,Bekarev,A.A.,Vereschagin,E.I.,Madonov,P.G.,etal.(2012).Effectsandmechanismsofhemopoiesis-stimulatingactivityofimmobilizedoligonucleotidesunderconditionsofcytostaticmyelosuppression.B.Exp.Biol.Med.152(4),451–455.doi:10.10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norreproductioninotherforumsispermitted,providedtheoriginalauthor(s)andthecopyrightowner(s)arecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms.*Correspondence:YinXiong,[email protected] 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