黃芪多糖對人外周血單個核細(xì)胞與宮頸癌細(xì)胞共培養(yǎng)的調(diào)節(jié)作用,SCI醫(yī)學(xué)論文

黃芪多糖對人外周血單個核細(xì)胞與宮頸癌細(xì)胞共培養(yǎng)的調(diào)節(jié)作用,SCI醫(yī)學(xué)論文Abstract：OBJECTIVE:ToinvestigateimmunomodulatoryeffectsofAstragaluspolysaccharides(APS)ontheco-cultureofperipheralbloodmononuclearcells(PBMCs)withHeLacervicalcancercellline.METHODS:ToassesstheproliferationofPBMCs,carboxyfluoresceinsuccinimidylester(CFSE)-labeledPBMCswereco-culturedwithHeLacellsandtreatedwithdifferentconcentrationsofAPS.Supernatantsofcellculturewerecollectedforcytokinesassayviaenzyme-linkedimmunosorbentassay(ELISA).TheimpactofAPSontheproliferationofPBMCs,inductionofregulatoryTcells(Tregs),andmyeloid-derivedsuppressorcells(MDSCs)wascarriedoutbyflowcytometry.RESULTS:ItwasobservedthatAPScouldincreasetheproliferationofPBMCsco-culturedwithHeLacells(P0.05).However,APShadnosignificanteffectsontheinductionofTregsandMDSCsintheco-cultureassay(P0.05).Furthermore,ELISAresultsdemonstratedthatAPScoulddecreaseIL-10andTGF-βconcentration(P0.05).CONCLUSION:Theabove-mentionedcharacteristicsshowedthatAPSmightbeabletomodulateimmuneresponsesandimproveanti-tumoreffectsthroughincreasingtheproliferationofPBMCsanddecreasinginhibitorycytokinessecretionascriticalmediatorsofimmunesuppressioninthetumormicroenvironment.Keyword：Astragalus;polysaccharides;uterinecervicalneoplasms;HeLacells;coculturetechniques;cytokines;T-lymphocytes,regulatory;Cervicalcancer(CC)isknownasthefourthfrequentcancertypeinwomenwithanapproximatelyestimated570000newcasesin2021.[1,2]However,traditionaltherapies,suchaschemotherapy,radiotherapy,andsurgicalresectionshavemostlybeenappliedtoimproveclinicalsymptomsofcancerpatientsandtumorremissions.Giventheadversecomplicationsofsuchtherapies,immunomodulatorycomponentsextractedfromtraditionalmedicinalplantsarerecentlybeinginvestigatedaseffectivetherapeuticapproachesformodulationofsuppressiveimmuneresponsesinthetumormicroenvironment(TME).[3]TMEhasbeenacceptedasanaidingfactortocancerprogression.Inthisregard,theTMEcomponentsincludefibroblasts,bloodandlymphaticvascularnetworks,immuneandinflammatorycells,aswellasadipose-relatedcells.Accordingly,regulatoryTcells(Tregs)andmyeloid-derivedsuppressorcells(MDSCs)aretwotypesofimmunesuppressorcellsthatmayplayleadingrolesinTMEthatwerealsostudiedinthepresentstudy.[4,5,6])RadixAstragaliMongolici(Huangqiinchinese)isknownasamedicinalherbusedinTraditionalChineseMedicineforcenturies.[7]Themainconstituentsofthisherbarepolysaccharides,saponins,flavonoids,aminoacidsincludinggamma-aminobutyricacid(GABA)andL-canavanine,andtraceelements.Therearealso14polysaccharidesinthedriedrootofAstragalusmembranaceus(Fisch.)whose13typespossessβ-D-(1→3)-galactanmoietiessplitwithβ-D-(1→6)-galactooligosaccharideside-chains.[8]Asthemainactiveextractfromastragalusmembranaceus,APShasbeenassociatedwithavarietyofproprietieslikeimmune-stimulant,anti-inflammatory,anti-oxidative,anti-diabetic,hepato-protective,anti-viral,andanti-canceractivities[9]aswellasseveralbiologicaleffectsbasedontheregulationofcellularandhumoralimmunity.[10,11]Additionally,numerousstudieshavereportedthatAPShasstrongimmunomodulatoryeffectsinvitro/invivothatmaybeinvolvedininhibitingtumorgrowth,facilitatingcytokinesecretion,andpromotingimmuneorganindicesinEhrlichascitescarcinoma(EAC)-bearingmice.[6,12]However,theimmunomodulatoryeffectsofAPSonPBMCsco-culturedwithCCcellshavenotbeensofarelucidated.Herein,thepurposeofinvitroco-cultureofPBMCsandHeLacellswastoinvestigatetheimmunomodulatoryeffectsofAPSontheproliferationofPBMCs,inductionofTregcells,andMDSCs,aswellascytokinesecretionbyPBMCsco-culturedwithHeLacells.ReagentsAPSwaspurchasedfromNOWFoods(Bloomingdale,IL,USA).Itwassubsequentlydissolvedinnormalsalinetodevelopafinalstocksolutionof5mg/mL.Thegivensolutionwassubsequentlyfilteredutilizinga0.22?porefilter.FurtherdilutionwasalsopreparedinthecompleteRoswellParkMemorialInstituteMedium1640(RPMI1640)medium.RPMI1640medium,penicillin/streptomycin,fetalbovineserum(FBS),andtrypsinwerepurchasedfromGibco(Amarillo,Texas,USA).Additionally,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)reagentwasobtainedfromSigma-Aldrich,Germany.Theinterferon(IFN)-γ,TGF-β,IL-10,IL-6,andVascularendothelialgrowthfactor(VEGF)levelsweredeterminedusingacommercialELISAkit(BDBiosciences,SanDiego,CA,USA).CFSE(Waltham,Massachusetts,USA)wasusedforproliferationassayandthenstoredat-20℃indarkbottleswithafinalconcentrationof5mM.ThefrequencyofMDSCsandTregcellswasdeterminedonafluorescence-activatedcellsorter(FACS)caliber(BDBiosciences)withthefollowingmarkers:FITC-labeledanti-humanCD4(cloneRPA-T4),APC-labeledCD25(M-A251),PE-labeledCD127(HIL-7R-M21),PE-labeledHLA-DR(G46-6),APC-CY7-labelledCD11b(ICRF44),andAPC-labeledanti-CD33(WM53)(allfromBDBiosciences)basedonmanufacturersinstructionsfollowingisotype-matchedcontrols.MTTcytotoxicityassayTheeffectofAPSextractontheviabilityofPBMCsandHeLacellswasdeterminedusingMTTassay.PBMCsandHeLacells(15×10[4]and1×10[4]cells/well,respectively)wereculturedinseparatewellsusing96-wellplates.ThesecellsweretreatedwithvariousAPSconcentrations(1000,500,250,125,60,30,15?g/mL)andincubatedfor24,48,72,and96hat37℃and5%CO2.Thecellswerealsoculturedalone(anegativecontrol)andtreatedwith5?g/mLofPolyhydroxyalkanoates(PHA),asapositivecontrol.Theexperimentswereperformedintriplicate.Inthisrespect,followingeachdifferenttimegroup,20?LofanMTTsolutionat5mg/mLwasaddedintoeachwellandthenceforwardincubatedfor4hindarknessat37℃and5%CO2.Afterward,themediumalongwiththeMTTsolutionwasremovedfromeverywelland100?LDMSO(FisherScientific,LE,UK)wasthenaddedintoeachone.Ultimately,theabsorbancewascalculatedat570nmwavelengththroughELISAReader(LabtechLT-4000MicroplateReader)anditwasthendirectlycorrelatedwithviableculturedcells.Dividingtheabsorbanceoftreatedcellsbythatofthecontrolgroupineachexperiment,therelativepercentageofcellsurvivalwasalsocalculated.TheextractsofAPSthatdemonstratednotoxicityonPBMCsandHeLacellswerefurthertestedforproliferativeeffectsofPBMCsbyusingCFSEdyedilutionassay.ProliferationAssayofPBMCsTheproliferativeeffectsoftheextractsofAPSonPBMCswereperformedusingCFSEstaining.HumanPBMCswereseparatedbygradientcentrifugationusingFicoll(Lymphodex,Germany)at400×gfor20minat20℃.ThePBMCswerethencollectedandwashedwithphosphatebufferedsaline(PBS).Briefly,PBMCs(10×10[6]cells/well)werelabeledwith5?MofCFSEfor5minat37℃indarkness.ThereactionwascompletedbyaddingPBSsupplementedwith5%FBS.TheCFSE-labeledPBMCswerethenplatedin24-wellplates(5×10[5]cells/well)andculturedfor4dwithorwithoutAPSconcentrations(1000,500,250,125,60,30,15?g/mL).Theculturesupernatantswerecollectedandimmediatelyfrozenat-80℃forsubsequentcytokineassays.InvitrointeractionsofPBMCsandHeLacellsHeLaCCcellline(humancervicaladenocarcinoma,ATCC#HTB-26)waspurchasedfromPasteurInstituteofIran,Tehran,Iran.ThecellswereculturedinRP-MI-1640andthensupplementedwith10%(v/v)FBSandantibiotics(100U/mLpenicillinand100?g/mLstreptomycin)at37℃inahumidenvironmentwith5%CO2.Afterward,CFS-labeledPBMCs(1×10[6]cells/well)wereco-culturedwithHeLacellsatdifferentratios(1∶1,1∶5,1∶10)andtreatedwithorwithoutvariousconcentrationsofAPSbyat37℃with5%CO2.PHA-treatedPBMCswerethencomparedwithtests(apositivecontrol)anduntreatedPBMCs(negativecontrols).Followingtheco-culturefor3d,PBMCswereseparatedfromthesupernatants,andproliferationofCFSE-labeledcellswasmeasuredviaflowcytometrybasedondiversefluorescenceintensitiesofCFSE.Eachextractandcontrolwereassayedintriplicateorder.AnalysisoftregsinductionbyflowcytometryTheTregcellpopulationexpressesCD4aswellashighlevelsofCD25andlowCD127.Inthisrespect,PBMCs(1×10[6]cells/well)wereco-culturedwithHeLacellsusingdifferentratios(1∶10,1∶50,1∶100)in24-wellplatesandthentreatedwith(1000?g/mL)orwithoutAPSat37℃with5%CO2.After7d,PBMCswereharvestedandstainedforextracellularCD4,CD25,andCD127markersusinganti-humanCD4-FITC,anti-humanCD25-APC,andanti-humanCD127-PEmonoclonalantibodiesbasedonmanufacturersinstructions.Forflowcytometryanalysis,thecellswerethenwashedandre-suspendedinstainingbuffer.ThefrequencyofCD4+CD25+CD127-/lowcellswasalsodetectedusingFACScaliber(BDBioscience).Inthisrespect,50000totaleventswerecollectedanddataanalysiswasfulfilledviaFLOWJOsoftware.AnalysisofMDSCinductionbyflowcytometryInthepresentstudy,theMDSCswereimmunophenotypedasCD33+CD11b+HLA-DRcellsinPBMCsofhealthydonorsco-culturedwithHeLacellswithandwithoutbeingtreatedwithAPS.Inbrief,freshlyisolatedPBMCs(1×10[6]cells/well)wereco-culturedwithHeLacellsforoneweekatdifferentratios(1∶10,1∶50,1∶100)in24-wellplateswith(1000?g/mL)andwithoutAPSat37℃with5%CO2.PBMCs,culturedinmediumalone,wereequallyrunasinductionofnegativecontrol.Afteroneweek,PBMCswerecollectedfromHeLa/PBMCsco-culturemediumandwerestainedonicefor30minwithanti-humanCD33-APC,CD11b-APC-Cy7,andHLA-DR-PEmonoclonalantibodiesbasedonthemanufacturersinstructions.Sampleswerethenwashedtwotimes,andre-suspendedinFACSbufferforanalysis.ThefrequencyofCD33+CD11b+HLA-DR-cellswasdetectedusingFACScaliber.CytokineDeterminationPBMCs(5×10[5]cells/well)wereco-culturedwithHelacells(1×10[5]cells/well)byusinga24-welltissuecultureplateandthentreatedwith1000?g/mLAPSorPHA(1%v/v)andalsowithoutanytreatment.Moreover,PBMCsandHelacellswereculturedinaseparatewellandtreatedwith15and1000?g/mLAPS,forPBMCsandHelacells,respectively.Asadditionalcontrols,PBMCsandHelacellswereculturedinaseparatewellwithoutanytreatment.TheTGF-β,IL-10,IL-6,IFN-γ,andVEGF-AlevelsinculturesupernatantsweredetectedbyELISAafter24hand72h.Dilutedsupernatantswerethentestedinatriplicateorder.Theabsorbancewasmeasuredat450nmwavelengthusinganELISAreader.Inthetestsamples,cytokineconcentrationswerealsocalculatedaccordingtostandardcurves.StatisticalanalysisToassessdifferencesbetweendatagroups,one-wayanalysisofvariance(ANOVA)followedbytheMann-WhitneyUtestwithaP0.05levelofsignificancewasused.AllthestatisticalanalyseswerefulfilledviaGraphPadPrismsoftware(version8.0.2)(SanDiego,CA,USA).Theresultsarerepresentedasmean±standarderrorofmean.Pvalueslessthan0.05,0.01,and0.001wereconsideredstatisticallysignificant.RESULTSEffectofAPSonPBMCsandhelacellsviabilityInitially,theimpactsofAPSonPBMCsandHeLacellsviabilitywereexamined.Thesecellswereexposedto15,30,60,120,250,500,and1000?g/mLofAPSfor24,48,72,and96hinnormalconditions.AsillustratedinTable1,asignificantincreasewasobservedincellviabilityofPBMCsfollowingtreatmentwith15?g/mLofAPSfor96h(P0.05).Accordingly,PBMCstreatedwithAPSat15and60?g/mLfor24h,respectively,showedincrease(Table1)incellviabilitycomparedwiththatinthecontrolgroup(n=3,P0.01andP0.05;respectively).AccordingtoTable1,resultsdemonstratedthatthecellviabilityofHeLacellshadsignificantlydecreasedcomparedwiththecontrolgroupwhenthesecellsweretreatedwith250and1000?g/mLofAPSfor48hrs(n=3,P0.05andP0.01;respectively).APScouldalsoinhibitthegrowthofHeLacellsbasedonconcentrationandtime.NotoxicconcentrationsofAPSwerechosentoinvestigatetheabilityoftheseextractstostimulatetheproliferationofPBMCsusingCFSEdyedilutionassay.IncreasedproliferationofPBMCsbyAPSinvitroTodeterminetheeffectsofAPSontheproliferationofPBMCsco-culturedwithHeLacells,theproliferationofculturedCFSE-labeledPBMCstreatedwithandwithoutconcentrationsofAPS(15to1000?g/mL)wasinitiallyassessedintheabsenceofHeLacellsfor4d.AsshowninFigure1,theproliferationofPBMCswassignificantlyincreasedasthecellsweretreatedwith15?g/mLofAPSfor96hcompared(Figure1C)withuntreatedcontrol(Figure1B)(PBMCswereculturedincompletemediaalone)(n=4,P0.001).ProliferationeffectofAPS-treatedPBMCsco-culturedwithHeLacellsToexaminetheeffectsofAPSontheproliferationofPBMCsco-culturedwiththeHeLacellline,theimpactofHeLacellsontheproliferationofPBMCswasinitiallydetermined.CFSE-labeledPBMCswerecol-lectedafter4dofbeingco-culturedwithHeLacellsandassessedbyFACSanalysis.AsdepictedinFigure2,theresultsindicatedthatPBMCsproliferationwassignificantlyincreasedasthecellshadbeenco-culturedwithHeLacells(Figure2D)comparedwithnaturallyproliferatedPBMCs(Figure2C)(n=4,P0.01).ToexaminetheimpactofAPSonproliferatedPBMCsco-culturedwithHeLacells,theproliferationofAPS-treatedPBMCsafterbeingco-culturedwithHeLacellswasassessedviaflowcytometry.AspresentedinFigure2,APScouldsignificantlyenhancetheproliferationofPBMCsco-culturedwithHeLacellsandtreatedwith1000?g/mLofAPS(Figure2E)for4dcomparedtothatinthecontrolgroup(n=4,P0.05).Table1MTTassayNotes:theeffectsofdifferentconcentrationsofAPSontheviabilityofPBMCsandHeLacells.ResultsindicatedthatAPScouldincreasetheviabilityofPBMCsatconcentrationsof15and60?g/mL.APSat500?g/mLalsoshowedminortoxicityonHeLacellsandreducedcellviabilitybyaround50%at1000?g/mL.MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide;APS:Astragaluspoly-saccharides;PBMCs:peripheralbloodmononuclearcells.Dataarerepresentedasmean±standarderrorofmean.aP0.05,bP0.01vscontrolgroup.Figure1ProliferationofCFSE-labeledPBMCsculturedwithandwithoutAPSA:statisticalanalysisdemonstratedsignificantproliferationofPBMCsintreatmentswith15μg/mLofAPS(n=4,P0.001);B-D:representativehistogramsofCFSEdilutionshowedthepercentageofproliferatedPBMCs(C,D)andundividedcontrolcells(B).APS:Astragaluspolysaccharides;PBMCs:peripheralbloodmononuclearcells;CFSE:carboxyfluoresceinsuccinimidylester.Dataarerepresentedasmean±standarderrorofmean.aP0.001vscontrolgroup.EffectofAPSoninductionofTregcellsToassesswhetherco-culturedPBMCsandHelacellswithAPScouldaffecttheinductionofTregcells,inducingcapabilityofPBMCstreatedwith15?g/mLofAPSandculturedintheabsenceofHeLacellsfor7dwasdetermined.Following7dofculturing,inductionofCD4+CD25+CD127-/lowTregcellswasanalyzedusingflowcytometry.TheCD4+CD25+CD127-/lowTregcellfrequencyinthefreshlyisolatedPBMCsofhealthydonorswasalsoexamined(Day0)byflowcytometry.ThedatashowninFigure3indicatedasignificantfallintheinductionofTregcellsasPBMCstreatedwith15?g/mLofAPS(Figure3F,3G)comparedwiththecontrolgroup(Figure3E)(n=3,P0.05).ModulatoryeffectofAPSoninductionofTregcellsco-culturedwithHeLacellsAquestionwasaddressedwhetherco-culturedofPBMCsandHelacellswouldresultintheinductionofTregsornot.So,PBMCswereinitiallyco-culturedwithHelacellsinacontact-dependentmannerinaratioof1∶50(HeLa∶PBMCs)toassesstheeffectofHelacellsontheinductionofTregcells.Following7dofco-culture,inductionofTregcellswasanalyzedviaflowcytometry,asillustratedinFigure4.Co-cultureofPBMCswithHeLacellsindicatedasignificantincreaseinfrequencyofTregcells(Figure4A,4D)comparedwithnaturallyoccurringones(Figure4A,4C)(n=3,P0.05).Then,Helacellswereco-culturedwithPBMCsandtreatedwith1000?g/mLofAPS.Following7dofco-culture,inductionofTregcellswasanalyzedbyusingflowcytometry.ItwasobservedthattheexpansionofCD4+CD25+CD127-/lowTregcellswasnotsignificantlydifferentinAPS-treatedPBMCsco-culturedwithHelacells(Figure4E)incomparisonwithuntreatedcontrol(Figure4D)(n=3,P0.05).EffectofAPSonMDSCinductionTofindwhetherAPSwasinvolvedintheinductioncapabilityofMDSCs,PBMCswereculturedaloneorco-culturedwithHeLacells,withorwithoutAPSfor7d.Following7dofculture,inductionofCD33+CD11b+HLA-DR-cellswasanalyzedviaflowcytometry.TheCD33+CD11b+HLA-DR-cellsfrequencyinthepopulationofPBMCsinhealthydonorswasalsoexamined(Day0)(Figure5B)throughflowcytometry.Figure2EffectofAPSontheproliferationofPBMCsco-culturedwithHeLacellsA:statisticalanalysisoftheproliferatedcellsdemonstratedsignificantproliferationofPBMCsco-culturedwithHeLacells(n=4,P0.01);B:theproliferationofCFSE-labeledPBMCswasanalyzedbyusingflowcytometryafterbeingco-culturedwithHeLacellsandtreatedwithAPS(n=4,P0.05);C-F:representativehistogramsofCFSEdilutiondemonstratingthepercentageofproliferatedPBMCs;E:themostsignificantenhancingeffectofAPSwasdetectedafterbeingtreatedwith1000μg/mLofAPSfor4d.APS:Astragaluspolysaccharides;PBMCs:peripheralbloodmononuclearcells;CFSE:carboxyfluoresceinsuccinimidylester.Dataarerepresentedasmean±standarderrorofmean.aP0.01,bP0.05vscontrolgroup.Figure3APSdecreasedtheinductionofTregsinvitroA-C:gatingstrategytoassesstheTregspopulationthroughflowcytometry;F:expansionofCD4+CD25+CD127-/lowTregsfromPBMCsofhealthydonorsastreatedwithAPS;D-F:resultsdemonstratedthefrequencyofTregsaftertreatingwith15μg/mLofAPSfor7dcomparedtothecontrolgroupandday0;G:expansionofCD4+CD25+CD127-/lowTregswasassessedafterPBMCswereculturedinCMaloneandtreatedwith15μg/mLofAPS.APS:Astragaluspolysaccharides;PBMCs:peripheralbloodmononuclearcells.Dataarerepresentedasmean±standarderrorofmean.aP0.05vscontrolgroup.Figure4ProportionsofCD4+,CD25+,CD127-/lowTregsfromAPS-treatedPBMCsco-culturedwithHeLacellsA:thestatisticalgraphillustratedtheproportionofCD4+CD25+CD127-/lowTregsinducedfromPBMCsco-culturedwithHeLacellsandtreatedwithAPS;C-E:plotsofrepresentativeflowcytometryshowedtheproductionofCD4+CD25+CD127-/lowTregsfromPBMCsalone,co-culturedwithHeLacellsandwithoutanytreatmentandtreatedwithAPS.APS:Astragaluspolysaccharides;PBMCs:peripheralbloodmononuclearcells,b:non-sinificant.Dataarerepresentedasmean±standarderrorofmean.aP0.05vscontrolgroup.AsshowninFigure5,nosignificantdecreasewasobservedwhenPBMCswasculturedaloneandtreatedwith15?g/mLofAPS(Figure5E,5C)incomparisonwithuntreatedcontrol(Figure5C)(n=3,P0.05).Then,Helacellswereinitiallyco-culturedwithPBMCsinacontact-dependentmannertoevaluatetheimpactofHelacellsoninductionofMDSCsfromthepopulationofPBMCs.Accordingly,asignificantrisingtrendwasobservedintheMDSCsfrequency(Figure5E,5C)comparedwiththatinthecontrolgroup(Figure5C).Therefore,theco-cultureofPBMCsandHeLacellsrevealedasignificantincreaseininductionofMDSCscomparedwithnaturallyoccurringMDSCs(n=3,P0.05).Moreover,nosignificantdecreaseininductionofMDSCswasreportedwhenPBMCsandHeLacellsco-culturedandbeingtreatedwith1000?g/mLofAPS(Figure5E,5D),comparedtothecontrolgroup(Figure5D)(n=3,P0.05).Figure5CD33+CD11b+HLA-DR-cellsproportionsfromPBMCstreatedwithAPSandco-culturedwithHeLacellsA:gatingstrategytoassessthepopulationofMDSCsviaflowcytometry;C,D:plotsofrepresentativedensityfromtheexpansionofCD33+,CD11b+,HLA-DR-cellsfromPBMCsculturedalone,andtreatedwithAPS,co-culturedwithHeLacells,andtreatedwithAPS;E:AnalysisoftheMDSCspercentageamongthePBMCsculturedaloneandtreatedwithAPSaswellasafterco-culturingwithHeLacells.PBMCs:peripheralbloodmononuclearcells;APS:Astragaluspolysaccharides;MDSCs:myeloid-derivedsuppressorcells,a:non-significant.Dataarerepresentedasmean±standarderrorofmean.bP0.05vscontrolgroup.ImpactofAPSoncytokineproductionSinceAPSshowedproliferationeffectonPBMCsandsuppressingeffectsoninductionofTregcellsinvitro,thechangesofcytokineproductionbyPBMCS/HeLacellsthatwereculturedaloneorco-culturedtogether,wereinvestigatedwithorwithoutAPS.ThemeanlevelofIL-6wassignificantlyhigherintheAPS-treatedPBMCsmonoculturegroup(15?g/mL)incomparisonwiththeuntreatedgroup(P0.05).Besides,theconcentrationsofIL-6,IFN-γ,andVEGF-AweresignificantlyhigherinAPS-treatedPBMCs(1000?g/mL)co-culturedwithHeLacellsincomparisonwiththeuntreatedcontrolgroup(P0.05).Incontrast,theconcentrationofIL-10andIFN-γdidnotvarybetweentheAPS-treatedPBMCsmonoculturegroupandtheuntreatedcontrolone(P0.05).Inaddition,theproductionofIL-10andTGF-βwassignificantlylowerinAPS-treatedPBMCsco-culturewithHeLacellsincomparisonwiththeuntreatedgroup(P0.05).After72hrsexposuretoAPS,HeLacellshadsignificantlyreleasedlessTGF-βandVEGF-AlevelscomparedwithuntreatedHeLacells(P0.05andP0.001,respectively;Table2).TheresultsofthisstudyclearlyshowedthatAPSmighthaveimmunomodulatoryactivitybymodifyingthesecretionofcytokinesinTME(Table2).DISCUSSIONTraditionalChineseMedicine(TCM)hasbeenthemostusefultherapeuticmethodinchinaforthousandsofyears.Polysaccharidesasoneoftheactiveandimpor-tantcomponentsofTCMwithvariousbiologicalactivitiesarebecomingacommonremedyforcancerpatientssincetheycanenhanceimmunesystempotentialsagainsttumorcells.[13,14]AmongthedifferentoriginsofAPS,theactivecomponentobtainedfromChinesemedicinalherb(i.e.astragalusmembranaceus)hasexhibitedvariousbiologicalactivities,likeimmunomodulatoryeffects.[15,16,17]Atpresent,numerousstudieshaveindicatedthatAPScanincreasetherapeuticeffectsandreduceadverseones,andconsequentlycontributetoimprovedimmuneresponsesinvivo.[6,18]However,littlehasbeenreportedregardingAPSpotentialinboostingimmunesystemabilityagainsttumors.TheresultsofthisstudyindicatedthatAPScouldmodulateimmuneresponsesinvitro.Theproliferationoflymphocytesisthemostdirectindicatorofsignalingduringcellularimmuneresponses.[19]PreviousstudieshavedemonstratedthatAPScanhaveimmunomodulatoryeffects,includingenhancedproliferationactivitiesoflymphocytesandaugmentedTLR4-mediatedMyD88-dependentsignalingpathwayactivation.[6,20,21,22,23,24]Furthermore,numerousstudieshaveshownthatTLRscanregulatetheproliferationofcellstomultiplybeneficialimmuneresponsesagainsttumors.[15,25,26]Moreover,IL-6isconsideredasacytokinewithseveralimmunomodulatoryfunctionsthatcanstimulatetheproliferationofTcells,Bcells,andstemcells,consequentlypromotingimmunoglobulinproductionbyBcells.[16,27]Inthisrespect,Tianetal[28]reportedthatAPShadananti-tumoreffectcombinedwithAdriamycininvivoduetoitscapacitytoenhanceIL-6andtumornecrosisfactor(TNF)-αexpressioninH22tumor-bearingmice.Inotherinvestigations,IFN-γwasreportedtohaveasignificantanti-tumoreffectviamodulatingtumorcells,immunecells,and/ornon-immunestromalonesinTME.[29]AnotherreportsuggestedthatAPScouldup-regulateT-betmRNAexpressionandconcentrationsofIFN-γinindividualswithCCaswellassimilarlyinlungcancermicemodels.[16,30]AnotherreportalsoimpliedanexplicitroleofIFN-γonnon-stimulatedCD4+Tcellsthatcouldboostadaptiveimmunitythroughaugmentingsurvivalinthecourseofinitiationofimmuneresponses.[31]TheseexperimentalresultsimplicatedthatAPScouldboosttheproliferationofPBMCs,andsecretionofIL-6andIFN-γ,whichwasinlinewiththeseexperimentalresultsinaco-culturemodel.Moreover,theresultsofourstudysuggestedthattheimpactofAPSonproliferatedPBMCsmightberelatedtoitsabilitytoenhanceIL-6expression.Nonetheless,theprecisemolecularmechanismandroleofinnateimmunityreceptorsrequiremuchmoreexplanation.ItshouldbenotedthatTregcellscanalsosuppressanti-tumorimmuneresponsesandbecontributingtothedevelopmentofimmunosuppressiveTMEincancertypes.[32]ApreviousreportrevealedthatmalignantplasmacellsmightleadtoasignificantinductionofCD4+CD25+FoxP3+Tregcellsbasedonacontact-dependentmanner[33]whichwasconsistentwiththeexperimentalresultsinourstudy.OtherstudieshadalsosuggestedtheinhibitoryroleofAPSonthefrequencyofTregsandexpressionofForkheadboxP3bothinvivoandinvitro.[34,35,36]Furthermore,astragalosideⅣ(AS-Ⅳ)couldinhibittumorprogressioninamurinelungtumormodelviadown-regulatingthepercentageofTregcellsinvitroandinvivo.[37]Theresultsofthepresentstudywereinagreementwiththisreport.TGF-β1andIL-10,asinhibitorycytokines,arealsoinvolvedinthesuppressivefunctionsofTregcellsandmayinhibiteffectorTcellproliferation.[38]APShasbeenabletomodifyresponsesthroughimmunomodulatoryactivitiestorepresstheproductionofIL-10fromculturedmacrophagesinadose-dependentmannerinvitro.[39]Likewise,ithasbeenreportedthatTGF-βexpressionandTregcellfrequencyweredown-regulatedfollowingadministrationofAPSinvivo.[35]Inthepresentstudy,theproductionofTGF-βandIL-10fromPBMCsco-culturedwithHeLacellswasdecreasedandtheinductionofTregcellswasinhibitedthroughAPSinmonocultureofPBMCs.But,inductionofTregcellshadnosignificantchangewhenPBMCsandHeLacellswereco-culturedandtreatedwithAPS.ItisassumedthattheimmunomodulatoryeffectsofAPSinvariouscancertypesmightbedifferent.However,therewerenorelevantreportsinthiscase.Therefore,itissuggestedtoexaminethecompletemolecularmechanismofthisphenomenonaswell