Gua Sha attenuates the pulmonary inflammation in mice infected with PR8 virus by balancing the ratio of Treg/Th17

Abstract

Background: Gua Sha, an ancient Chinese treatment which produces the pressure on the skin, is used to prevent and treat cold for thousands of years. There’re evidences to approve that it can activate immune response and reduce the inflammation. However, how it has the effect on T helper 17 cells (Th17) and regulatory T cells (Treg) is poorly understood. Here, this study aims at the relationship between the pressure-stoke in the skin and pulmonary Th17 as well as Treg in PR8-infected mice.
Methods: ICR mice were randomly divided into five groups. The body weight and survival rates of all groups were monitored through the experiment. At the end of experiment, lung inflammation was detected by HE staining and the expression of Matrix metalloproteinase-9 (MMP-9) was measured by immunohistochemistry. Th17 and Treg from lung tissues was analyzed by flow cytometry.
esults: Our results indicated that the survival rates of prophylactic and therapeutic group respectively showed 20% and 10% though Gua Sha treatment didn’t restore the weight-loss of PR8-infected mice. What’s more important, Gua Sha remarkably inhibited inflammatory infiltration and the expression of MMP-9 of lung tissues in infected mice ( p ＜0.05). Finally, the ratio of Treg/Th17 from lung tissues in PR8-infected mice was significantly increased as compared with control mice while Gua Sha treatment remarkably inhibited this enhancement. All these results indicated that Gua Sha has the efficacy on reducing the pulmonary inflammation in PR8-infected mice possibly via restoring the Treg/Th17 balance.
Conclusions: Our findings for the first time suggest that Gua Sha exhibits a significant inhibition of inflammatory infiltration with down-regulation of MMP-9 in lung tissues from RR8-infected mice, which might be associated with the differentiation of Th17 and Treg. Further research will be carried toward how Gua Sha functions on maintaining the homeostasis of Th17 and Treg in the lungs.

Full text

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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.
GuaShaattenuatesthepulmonaryinflammationin
miceinfectedwithPR8virusbybalancingtheratioof
Treg/Th17
CURRENTSTATUS:POS TED
YalanLi
BeijingUniversityofChineseMedicine
YonganWang
BeijingUniversityofChineseMedicine
JingweiKong
BeijingUniversityofChineseMedicine
ZiruiLiu
BeijingUniversityofChineseMedicine
DongyuGe
BeijingUniversityofChineseMedicine
RuijuanDong
BeijingUniversityofChineseMedicine
GuiyingPeng
BeijingUniversityofChineseMedicine
penggy@bucm.edu.cnCorrespondingAuthor
DOI:
10.21203/rs.3.rs-20934/v1
SUBJECTAREAS
Virology InfectiousDiseases
KEYWORDS
GuaSha,Physicaltherapy,Influenza,Inflammation,Th17,Treg

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Abstract
Background:GuaSha,anancientChinesetreatmentwhichproducesthepressureontheskin,isused
topreventandtreatcoldforthousandsofyears.There’reevidencestoapprovethatitcanactivate
immuneresponseandreducetheinflammation.However,howithastheeffectonThelper17cells
(Th17)andregulatoryTcells(Treg)ispoorlyunderstood.Here,thisstudyaimsattherelationship
betweenthepressure-stokeintheskinandpulmonaryTh17aswellasTreginPR8-infectedmice.
Methods:ICRmicewererandomlydividedintofivegroups.Thebodyweightandsurvivalratesofall
groupsweremonitoredthroughtheexperiment.Attheendofexperiment,lunginflammationwas
detectedbyHEstainingandtheexpressionofMatrixmetalloproteinase-9(MMP-9)wasmeasuredby
immunohistochemistry.Th17andTregfromlungtissueswasanalyzedbyflowcytometry.
esults:Ourresultsindicatedthatthesurvivalratesofprophylacticandtherapeuticgrouprespectively
showed20%and10%thoughGuaShatreatmentdidn’trestoretheweight-lossofPR8-infectedmice.
What’smoreimportant,GuaSharemarkablyinhibitedinflammatoryinfiltrationandtheexpressionof
MMP-9oflungtissuesininfectedmice(p0.05).Finally,theratioofTreg/Th17fromlungtissuesin
PR8-infectedmicewassignificantlyincreasedascomparedwithcontrolmicewhileGuaShatreatment
remarkablyinhibitedthisenhancement.AlltheseresultsindicatedthatGuaShahastheefficacyon
reducingthepulmonaryinflammationinPR8-infectedmicepossiblyviarestoringtheTreg/Th17
balance.
Conclusions:OurfindingsforthefirsttimesuggestthatGuaShaexhibitsasignificantinhibitionof
inflammatoryinfiltrationwithdown-regulationofMMP-9inlungtissuesfromRR8-infectedmice,which
mightbeassociatedwiththedifferentiationofTh17andTreg.Furtherresearchwillbecarriedtoward
howGuaShafunctionsonmaintainingthehomeostasisofTh17andTreginthelungs.
Background
Influenzaisaninfectiousrespiratorydiseasewhichusuallymanifestsasaseriesofclinicalsymptoms
suchascough,fever,headacheandweakness[1,2].TheInfluenzavirusthatisacommoncauseof
influenzabelongstotheorthomyxoviridaefamily,andtheviralgenomeisasingle-stranded(-)RNAin
sevenoreightfragments.Therearethreeviralserotypes,includingA,B,andC[3].TheinfluenzaA

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virus(IAV),includingPR8,infectsawidevarietyofhosts,particularlymammalsandpoultry[4].
Accordingtopastresearch,multifariousinflammatorycellsinlung,aswellasbronchialepithelial
cells,fibroblasts,smoothmusclecells,couldincreasetheexpressionofMMP-9afterinfectionwith
influenzavirus[5].MMP-9degradescomponentsofthealveolarbasementmembranewhich
contributestothedestructionofstructureinthelung[6].Duringtheinfluenzavirusinfection,both
innateandadaptiveresponsesareinvolvedinhostdefense.Particularly,theratioofTreg/Th17
changesalotininfectedmodels[7],suggestingtheinterplayofTreg/Th17isessentialtoshape
immuneresponseaftervirusinfection.

GuaShaisaneffectivetherapyformanydiseasesintraditionalChinesemedicine.'Gua'means'to
scrapeorscratch',thusGuaShaisamethodtobring'Sha'tothebody'ssurfacebymeansof
scratchingorscraping[8].GuaShatherapyisatherapeuticmodalitythatinvolvesusingasmooth-
edgedinstrumentforskinfrictioningtointentionallycreatetransientredorpurplepetechiaeand
ecchymosis,whichnormallyfadesinafewdays[9].Thistherapyisgenerallywelltolerated,withlittle
ornodiscomfort.Itiswidelyusedandspreadbecauseofitssimpleoperationandavoidingoralside
effectsofmedications[10].AndresearchevidencereportedGuaShahastheeffectontreating
respiratorydiseases[11–13].

ResearchonGuaShatherapyismainlyfocusonclinicaltrialreportsandonlyafewstudiesdiscussed
itsphysiologicaleffectsandpotentialtherapeuticmechanisms[14–16].Somestudiesshowedthat
scrapingcanimprovetheimmunefunctionofthebody.AfteradministrationofGuaSha,thenumber
ofneutrophilsandmacrophagesincreased,andthenkeratinocytesreleasealargenumberof
inflammatorycytokineswhichcouldactivatethemigrationandaccumulationofimmunecells[17].

However,thesefindingslacktheexperimentofapplyingGuaShatherapyaloneandthemechanism
ofGuaShatherapyincontrollingrespiratoryinflammationremainsunclear.Inthepresentstudy,we
demonstrateviralpneumoniamodelcausesseveredamagetolungtissueininfectedmice.AndGua

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ShatherapycouldattenuatethepulmonaryinflammationthroughdecreasingtheexpressionofMMP-
9andreversingtheimbalanceofTreg/Th17inlungtissues.
Methods
1.  M ice,virusanddrugs
MaleICRmiceat6-8weeksofage,werepurchasedfromBeijingVitalRiverLaboratoryAnimal
TechnologyCompany(Beijing,China).Themicewererandomlydividedinto6groups:controlgroup,
modelgroup,prophylacticgroup,therapeuticgroup,shamgroupandribaviringroup.Micewere
monitoredforsurvival,weightloss,andclinicalsignsofillness(e.g.,inactivity,ruffledfur,hunched
posture,poorappetite,rapidshallowbreathingandaudiblecrackling)for14days.Theinfluenza
A/PR/8/H1N1viruswaskindlygiftedbyprofessorYuHaofromtheDepartmentofImmunologyand
Microbiology,BeijingUniversityofChineseMedicine(Beijing,China).TheLD50wasdeterminedin
miceafterserialdilutionofthestock.WechallengedICRmicewith5LD50A/PR8(25mL).Infection
wasestablishedbyintranasalinoculationinmiceafteranesthetizedbyisoflurane.Ribavirinwas
purchasedfromBiokinPharmaceutical(Sichuan,China).Theribaviringroupwasgivenribavirin
(100mg/kg),onceadayfor7days.Allexperimentalprocedureswereconductedaccordingtothe
NationalInstituteofHealthGuidefortheCareandUseofLaboratoryAnimals,andapprovedbythe
AnimalCareCommitteeofBeijingUniversityofChineseMedicine.

2.  G u aShatreatmento n  e x p e r i m e ntalmice
Micewereanesthetizedby5%isofluraneandmaintainedat1.5-2%.Intheprophylacticand
therapeuticgroups,GuaShawasperformedonthesideofthemouse'sbackbyusingabuffalo-horn
GuaShaplate,afterthehairwasshavedwithaclipperadaypriortotheexperiment.Theshavedskin
areawaswipedwith70%ethanolandlefttodry.Then,scrapetheback100-150times/minfromneck
totailinaunidirectionalmanner,withanangleofabout90°betweentheGuaShaplateandthe
mouse'sback.Theforceofscrapewasbasedontheappearanceofredspotsorfreckles.Meanwhile,
theshamgroupwasscrapingatthesamefrequencyandforceintheleftthighofthemouse.


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3.  P a thologicalanalysis
Attheendofexperiment,thewholelungsofthemicewereremoved,washedinphosphatebufferand
fixedin10%formaldehydeatroomtemperature.Thenlungtissuesweredehydratedingraded
concentrationofethanol,embeddedinparaffinandsliced.Tissuesectionsof4μmthicknesswere
stainedwithhematoxylin-eosin(HE).Thentwoexperiencedpathologistsblindedwithmicegroup
observedsectionsoflungtissueunderalightmicroscopeandscoredthelunginjurythroughthe
methodasdescribedbyMikawa[18]:(a)alveolarcongestion,(b)hemorrhage,(c)neutrophil
infiltrationinthealveolarandvascularwall,and(d)alveolarwallthickening/theformationofthe
hyalinemembrane.Eachoftheaboveitemswasgradedintofivelevels:0=nodamage,1=slight
damage,2=moderatedamage,3=severedamage,and4=extremelyseveredamage.Thesumofthe
fouritemswasthefinalscorewithamaximumof16.

4.  I m munohistochemistr y
Immunohistochemistry(IHC)methodwasadoptedtodetecttheexpressionofMMP-9intheparaffin
sectionsofmouselungtissue.Afterbeingsliced,dewaxed,andhydrated,sectionswereplacedin3%
H2O2toincubatefor10min,thenrinsed5minthreetimeswithphosphate-bufferedsaline(PBS).
Thentheywereincubatedwith0.01Mcitratebufferfor15minin95℃waterandflushed5minthree
timeswithPBS.Forprimaryantibodyincubation,anti-mouseMMP-9antibody(fromBioLegend,Inc.,
SanDiego,CA)wasdilutedat1:1000withPBSandaddedintosectionsat4℃overnight.Horseradish
Peroxidase(HPR)-labeledsecondaryantibody(fromZsbioCommerceStore,Beijing,China)was
incubatedat37℃for20min.Peroxidaseactivitywasdetectedbyusing3,3-diaminobenzidine
tetrachloride(DAB;Beijingsolarbiosciencetechnologyco.,ltd.,Beijing,China).Sectionswere
counterstainedbyusinghematoxylinandthenobservedundermicroscope.Tenvisualfieldswere
randomlyselectedforeachgroup,andtheirintegralopticaldensity(OD)wasmeasuredbyuseof
ImageProsoftware,andthensemiquantitativeanalysiswasconductedbymeansofstatistical
software.

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
5.  C e llisolationfromlungtis s u e s
Aftersacrificingonday6,lungtissuesfrommicewereasepticallycollected.Toisolatesinglecell
suspensionfromlungtissues,lungsweremincedanddigestedwith1mg/mltypeIVcollagenase
(Worthington)and50μg/mlDNaseI(Roche)for45minat37°Conarotator.Thendigestedtissues
liquidwerefilteredthrough70-μmcellstrainersandenrichedwith40%Percollgradientafterred
bloodcellswerelysed.Single-cellsuspensionsfromlungswereusedforsubsequentflowcytometry
staining.

6.  F l o wcytometryanaly s i s
Forintracellularcytokinestaining,cellswerestimulatedwithCellStimulationCocktail(eBioscience)
andincubatedfor5hoursat37°C.Cellswerepreincubatedwithanti-mouseCD16/32(BioLegend)to
blockFcreceptorsandwashedbeforefurtherstaining.ThencellswerestainedwithFITCconjugated
anti-CD4(BioLegend)andPercPCy5.5conjugatedanti-CD25(BioLegend),followingbyfixingand
permeabilizingwithBDCytofix/Cytopermbuffer.Atlast,cellswerestainedwithPEconjugatedIL-17A
(BioLegend).Formeasurementoftranscriptionfactors,cellswerefixedandpermeabilizedwiththe
Foxp3/TranscriptionFactorStainingBufferSet(eBioscience)accordingtothemanufacturer's
instructionsandstainedwithantibodiesAPC-FoxP3(BioLegend).CellsweredetectedbyCantoⅡ(BD,
Biosciences)andanalyzedbyFlowJosoftware.

7.  S t a tisticalanalysis
TheSPSS16.0Softwarewasusedtocompletethestatisticalanalysis.Student’st-testwasusedto
comparecontinuousvariablesbetweentwogroups,andANOVAwasusedtocomparecontinuous
variablesacrossmultiplegroups.Mantel-CoxtestwasusedforSurvivaldata.Ap-valuelessthan0.05
wasconsideredstatisticallysignificant.

Results

7
1.  T h eeffectofskinscra p i n g  o n  s u r v ivalandbodyweigh t  o f  P R 8 - i n f e ctedmice.
ProtocolforexperimentalinfluenzainfectionandskinscrapingtreatmentwereshowedinFigure1a.
PR8-infectedmiceshowedthesignsofdehydration,greasyfur,andinactiveconditionfrom3days
afterinfection.RibavirinalleviatedtheconditionofdehydrationandrecoveredtheactivityinPR8-
infectedmice.NoobviousrecoveryinthebehavioralappearancewereobservedinGuaSha
prophylacticandtherapeuticgroups.Miceinmodelgroupshowedsignificantweightlossandsurvival
decreaseduringtheinfluenzainfection.Althoughthebodyweightinribaviringroupbegantolower
fromday3,ribavirinquicklyrecoveredtheirlossinPR8-infectedmicefromday4.Inaddition,
ribavirinprotectedinfectedmicefromdying(Fig.1bandc).It’sworthnotedthat20%and10%
survivalrateexistedrespectivelyinGuaShaprophylacticandtherapeuticgroupsthoughscraping
didn’tpreventthelossofbodyweightinPR8-infectedmice.

2.  S k inscrapingattenua t e d  p u l m o n a ryhyperemiaand  l u n g  i n f l a m m ationinPR8-
infectedmice.
TodetectthepulmonarydamagecausedbyPR8virusinfectioninmice,wemeasuredlung
histopathologyfromanatomicalobservationandH&Estainingoflungsections.Thecontrolgroup
showedanormallungappearancewithpinkcolorandnormalintactalveolistructures(Fig.2aand
b).PR8-infectedmicepossessedadarkredlungwithseverecongestiveedema.H&Estainingofthe
lungsectionsrevealedthatalargeamountofinflammatoryexudatewasbotharoundbronchusandin
thealveoliinterstitiuminPR8-infectedmice,whichcausedtheinterstitialthickeningofthelungand
broketheintegrityofalveolistructure.BothGuaShaprophylacticandtherapeuticgroupsindicated
red-palelungs,significantdecreasingofinflammationinfiltrationandamendmentofdamagefor
alveolistructure.Meanwhile,GuaShaadministrationamelioratedthelunginjuryobviouslyinPR8-
infectedmiceaccordingtoanalysisofpathologicalscores(Fig.2c).Theseresultsindicateskin
scrapinghaspreventiveandcurativeeffectonalleviatingpulmonaryinflammationinPR8-infected
mice.

8

3.  S k inscrapingsuppre s s e d  t h e  e x p ressionofMMP-9in  l u n g  t i s s u e  i n PR8-infectedmice
ThemainfunctionofMMP-9istodegradeIV,Vcollagenandgelatin,whicharethemostimportant
componentsintheextracellularmatrix[19].MMP-9issecretedbybronchialepithelialcells,
neutrophils,eosinophils,mastcellsandalveolarmacrophagessuggestingthatMMP-9canbe
expressedbothinnormallungandthelungtissueinfiltratedbyinflammatoryexudate[20,21].After
theinfectionofinfluenzavirus,remarkablyincreasedexpressionofMMP-9wasfrombothvarious
inflammatorycellsandparenchymalcells,whichwasdetectedbyimmunohistochemistrystaining
(Fig.3a)whileonlyshowninepithelialcellsofthecontrolgroup(p<0.01).GuaShaadministration
significantlyreducedtheexpressionofMMP-9inlungtissuefromPR8-infectedmice.What’smore,
thisexpressionofMMP-9wasrestrictedinlungparenchymacellsinGuaShaprophylacticand
therapeuticgroups.(Fig.3b,c).Theseresultssuggestthatskinscrapingsignificantlyinhibitsthe
expressionofMMP-9inlungtissuecellsfromPR8-infectedmice.

4.  S k inscrapingrectified t h e  r a t i o  o f  T r eg/Th17inPR8-infe c t e d  m i c e .
Th17cellsareasubsetofpro-inflammatoryThelpercellsdefinedbytheirproductionofinterleukin17
(IL-17)andtheirmaineffectorcytokinesareIL-17A,IL-17F,IL-21,andIL-22.Theyplaytheroleofa
double-edgedswordduringinfluenzainfection[22,23].TregcellsareanotheruniqueTlymphocyte
subsetinthebodythatsecretesIL-10,andTGF-β,whichisregulatedbythetranscriptionalfactor
foxheadboxP3(FoxP3)[24,25].Tregcellssuppressactivityofavarietyofimmunecellsand
thereforeinhibitimmuneresponses,whicharecloselyrelatedtoinfluenza[26].ThenumbersofTregs
andTh17cellsfromlungtissuesweredetectedbyflowcytometrysoastoanalyzewhetherandhow
skinscrapinginfluencedthedifferentiationofThelpercellduringPR8infectioninmice.Onday6
afterinfection,singlecellsuspensionsfromlungtissuesofeverygroupwereobtained.Then
intracellularcytokinesstainingwasusedtodetectCD3+CD4+IL-17A+TcellsasTh17afterstimulation
ofPMAplusionomycinwhileTregwasdefinedasCD3+CD4+CD25+FoxP3+byflowcytometry.From

9
Figure4,themodelgroupshowedasignificantlyhigherproportionofTregandlowerTh17compared
withcontrolgroup.However,skinscrapingremarkablyinhibitedtheenhancementofTregand
promotedthedifferentiationofTh17.It’sinterestingthatribavirininducedthehighestproportionof
Th17inPR8-infectedmice.TheratioofTreg/Th17inPR8-infectedmicewas2.16:1,whilethisratioin
prophylacticandtherapeuticgroupwassignificantlylowerthanthatinmodelgroup.Alltheseresults
indicatedthatGuaShahastheefficacyonreducingthepulmonaryinflammationinPR8-infectedmice
possiblyviarestoringtheTreg/Th17balance.
Discussion
GuaShaisatraditionalChinesephysicaltherapy.Undertheguidanceofthetheoryofmeridiansand
acupointsoftraditionalChinesemedicine,redsplotchymarksappearontheskinthatlooklike
scrapesorlightbruising,butinactualityitisavascularresponsecalledtransitorytherapeutic
petechiaeafterGuaShatreatment[27].Weselectedtheerectorspinaemuscleonbothsidesofthe
backspineofthemouseinprophylacticandtherapeuticgroupforscraping,whichisnamed“Urinary
BladderMeridianofFoot-Taiyang”bytraditionalChinesemedicine(TCM),becauseitwasalwaysused
topreventandtreatpulmonarydiseasesinclinic[28].

Inourstudy,wefirstobservedtheeffectofscrapingonthesurvivalrateandbodyweightofinfluenza
mice.Itiswellknownthattheinfluenzavirusattachesairwayepithelialcellsthroughhemagglutinin
(HA)proteinonitssurface,causingdiseasebyalargenumberofreplicationsintheairwaysand
alveolarepithelium[22].Inourexperiments,thoughGuaShaasaphysicaltherapywhichinducedthe
moderateinflammatoryresponseandimmuneresponsesfailedtopreventweightlossandthe
decreasingofsurvivalrateininfectedmice,ithadthesignificantlyprotectiveeffectagainst
pulmonaryinflammatoryexudates.ThisdiscrepancymightbereasonablyexplainedthatGuashais
performedontheskinwhichdeliversitstherapeuticeffectonlung,accordingto“lunggoverningskin
andhair”.However,PR8virusesnotonlyinducedviralpneumoniabyintranasaladministration,but
alsoenteredthebloodandtriggeredsystemicfailure.


10
Inaddition,ImmunohistochemistryrevealedthatthesecretionamountofMMP-9inPR8-infectedmice
washigherthanothergroups.TheseresultstogethershowedthattheexpressionofMMP-9is
associatedwithlungpathologyinducedbyPR8infection.MMP-9,referredtoasazinc-binding
endopeptidasewhosemaincomponentsaretypeIV,Vcollagenandgelatin,whichcandegradea
varietyofextracellularmatrixmolecules,modulatestissueremodelinguponacutelunginjuryand
interstitiallungdisease[6,19].Whenthelungswereinfectedwithinfluenzavirus,MMP-9could
directlyactonthebasementmembraneofthealveolarcapillariestoincreaseitspermeability,andat
thesametimedestroyedtheconnectionofcadherintothevascularendothelialcellsandincreasethe
microvascularpermeability[29].PharmacologicalinhibitionofMMP-9alsohasbeenreportedto
partiallyreducelungpathologyinmicecausedbyinfluenzavirus,andMMP-9deficiencyprotectsmice
fromsevereinfluenzaAviralinfection[30,31].Aninvitrostudyshowedthatinfluenzavirusinfection
increasesMMP-9secretionandpromoteractivityinVerocells[33].Ourdatashowedthatanincrease
ofMMP-9inthelungimmunohistochemicalanalysisofthemodelandtheshamgrouponday6after
infection,andaremarkabledecreaseinGuaShatreatment.Recently,Joselynetal.showedthatthe
increasingpulmonaryadaptiveimmuneresponsetoIAV,withhigherCD4+Tlymphocytesandlower
frequenciesofanti-inflammatoryTregs,appearedinMMP-9-/-miceafterIAVinfection[31].AndMMP-
9mightdecreaseIL-17productioninthelungsbyselectivelyinhibitingIL-23expression[32].
Coincidentalwiththeirs,ourdatashowedthatMMP-9production,whichaffectedthebalanceof
regulatoryTcellandothereffectorCD4+Tlymphocytes,wascriticaltoseverelungpathologycaused
byinfluenzaAvirus.

Itisreportedthatvirus-inducedTregscanbecomeactivatedbyapathogen-derivedpeptideand
downregulateantigen-specificeffectorCD4+andCD8+Tcellaccumulationandcytokineproduction
correlatedwiththeirantigenspecificity,soastomodulateanantiviralimmuneresponseinPR8-
infectedmice[26,34].SomeresearchesdemonstratedthatTregcellslimitTh17cellinflammationby
servingasprincipalamplifiersofnegativeregulatorycircuitsoperatinginimmuneeffectorcells.

11
Interleukin-10(IL-10)signalinginregulatoryTcellsisrequiredforsuppressionofTh17cell-mediated
inflammationduringPR8infection[35,36].Inourstudy,thehigherratioofTreg/Th17emergedinPR8-
infectedmicecomparedwiththecontrolgroup.Meanwhile,thisdominantTregwashighlyrectified
andasignificantproportionofTh17wasinducedafterGuaShaorribavirintreatmentinPR8-infected
mice.Th17wasadouble-edgedswordduringinfluenzainfection.Althoughsomestudieshave
suggestedapathologicalroleforIL-17secretedbyTh17cellsinhostimmunitytoinfluenza,other
studieshavesuggestedaprotectivefunction.Forinstance,ithasbeendocumentedthatIL-17
depletionresultedinincreasedweightlossaswellasreducedsurvivalinmousemodelofinfluenza
[23,37].ThesehigherTh17cellsmightbenecessaryforclearingtheinfectionandpromotingtissue
repair.
Conclusions
Insummary,ourfindingsforthefirsttimesuggestthatGuaShaexhibitsasignificantinhibitionof
inflammatoryinfiltrationwithdown-regulationofMMP-9inlungtissuesfromRR8-infectedmice,which
mightbeassociatedwiththedifferentiationofTh17andTreg.Furtherresearchwillbecarriedtoward
howGuaShafunctionsonmaintainingthehomeostasisofTh17andTreginthelungs.
Abbreviations
Th17:Thelper17cells;Treg:RegulatoryTcells;MMP-9:Matrixmetalloproteinase-9;IAV:InfluenzaA
virus;PR8:A/PR/8/H1N1virus;HE:Hematoxylin-eosin;IHC:Immunohistochemistry;PBS:Phosphate-
bufferedsaline;HPR:HorseradishPeroxidase;OD:opticaldensity;TCM:TraditionalChinesemedicine;
HA:Hemagglutinin.
Declarations
Ethicsapprovalandconsenttoparticipate
AllprocedureswerecarriedoutinaccordancewiththerecommendationsoftheGuidefortheCare
andUseofLaboratoryAnimalsoftheNationalInstitutesofHealth.
Consentforpublication
Allauthorsagreetopublishthispaper.
Availabilityofdataandmaterials
Thedatasetsusedand/oranalyzedduringthecurrentstudyareavailablefromthecorresponding

12
authoronreasonablerequest.
Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
Funding
ThisresearchwasfundedbytheNationalNaturalScienceFoundationofChina(GrantNo.81473656)
andFundamentalResearchFundsforCentralUniversities(GrantNo.2019-JYB-TD014).
Authors’contributions
Eachauthorhascontributedsignificantlytothisstudy.GYP,YLLandJWKconceivedanddesignedthe
study.YLL,YAW,JWKandZRLperformedtheanimalexperimentsandflowcytometrydetection.DYG
andRJDperformedpathologicalandhistochemicalexperiments.YLLandYAWperformedthe
statisticalanalysis.GYP,YLL,YAW,JWKdraftedandrevisedthemanuscript.Allauthorsreadand
approvedthefinalmanuscript.
Acknowledgments
TheauthorswishtothankprofessorYuHaofromtheDepartmentofImmunologyandMicrobiology,
BeijingUniversityofChineseMedicine(Beijing,China)forthevirusstrain.
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Figures

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Figure1
TheeffectofskinscrapingonsurvivalandbodyweightofPR8infectedmice.aThemicein
theprophylacticgroupbegantoscrape3daysbeforePR8infection,onceeveryotherday
for2times.Thetherapeuticgroupandtheshamgroupbegantoscrapeat2hoursafter
infection,onceeveryotherdayfor3times.Thecontrolgroupandtheribaviringroupwere
intragastricallyadministeredat2hoursafterinfection.Theribaviringroupwasgiven
ribavirin(100mg/kg)0.2ml/dayfor7days.Normaldietanddrinkingwaterinmice.bBody
weightwasmonitoredafterscraping.cSurvivalwasmonitoredafterPR8infection.n=10,
Mean±SD.****p0.0001comparedwiththecontrolgroup.ns:notsignificant.

18
Figure1
TheeffectofskinscrapingonsurvivalandbodyweightofPR8infectedmice.aThemicein
theprophylacticgroupbegantoscrape3daysbeforePR8infection,onceeveryotherday
for2times.Thetherapeuticgroupandtheshamgroupbegantoscrapeat2hoursafter
infection,onceeveryotherdayfor3times.Thecontrolgroupandtheribaviringroupwere
intragastricallyadministeredat2hoursafterinfection.Theribaviringroupwasgiven
ribavirin(100mg/kg)0.2ml/dayfor7days.Normaldietanddrinkingwaterinmice.bBody
weightwasmonitoredafterscraping.cSurvivalwasmonitoredafterPR8infection.n=10,
Mean±SD.****p0.0001comparedwiththecontrolgroup.ns:notsignificant.

19
Figure2
SkinscrapingattenuatespulmonaryhyperemiaandlunginflammationinPR8infectedmice.
aThemacroscopicpathologyoflungamongdifferentgroupswereshowedonday6after
PR8infection.bRepresentativepathologicalmicrographsoflungsections(HEstaining,
×200).cLunginjuryscoresofthemiceindifferentgroupsonday6afterPR8infection.
Mean±SD.n=3.***p0.001comparedwiththecontrolgroup.#p0.05,##p0.01
comparedwiththemodelgroup.

20
Figure2
SkinscrapingattenuatespulmonaryhyperemiaandlunginflammationinPR8infectedmice.
aThemacroscopicpathologyoflungamongdifferentgroupswereshowedonday6after
PR8infection.bRepresentativepathologicalmicrographsoflungsections(HEstaining,
×200).cLunginjuryscoresofthemiceindifferentgroupsonday6afterPR8infection.
Mean±SD.n=3.***p0.001comparedwiththecontrolgroup.#p0.05,##p0.01
comparedwiththemodelgroup.

21
Figure3
SkinscrapingcansuppresstheexpressionofMMP-9inlungtissue.aRepresentativeIHC
resultsforMMP-9inmicelungtissueofeachgrouponday6afterinfection(original
magnification,×200).b,cSemiquantitativeanalysisofIHCbythemethodoftheaverage
opticaldensity(AOD).Mean±SD.n=3.**p0.01,comparedwiththecontrolgroup.#p
0.05,##p0.01comparedwiththemodelgroup.

22
Figure3
SkinscrapingcansuppresstheexpressionofMMP-9inlungtissue.aRepresentativeIHC
resultsforMMP-9inmicelungtissueofeachgrouponday6afterinfection(original
magnification,×200).b,cSemiquantitativeanalysisofIHCbythemethodoftheaverage
opticaldensity(AOD).Mean±SD.n=3.**p0.01,comparedwiththecontrolgroup.#p
0.05,##p0.01comparedwiththemodelgroup.

23
Figure4
ChangesinTh17cellsandTregsininfluenzamicebyscraping.Singlecellsuspensionsfrom
lungtissuesofeverygroupwereobtained6dafterPR8infectioninmice.Representative
flowcytometrychartsillustratedpercentageofTh17cellsandFoxP3+regulatoryTcellsin
lungs.Thesewerepre-gatedfromCD3+CD4+cells.

24
Figure4
ChangesinTh17cellsandTregsininfluenzamicebyscraping.Singlecellsuspensionsfrom
lungtissuesofeverygroupwereobtained6dafterPR8infectioninmice.Representative
flowcytometrychartsillustratedpercentageofTh17cellsandFoxP3+regulatoryTcellsin
lungs.Thesewerepre-gatedfromCD3+CD4+cells.