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1、Received:3November2019Revised:10February2021Accepted:15February2021DOI:10.1002/JLB.4AB0221-324RRRRB R I E F CO N C L U S I V E R E P O RTSodiumhouttuyfonateattenuatesdextransulfatesodiumassociatedcolitisprecolonizedwithCandidaalbicansthroughinducing-glucanexposureKelongMa1,2,3MengliChen1JuanjuanLiu1
2、YuzhuGe1TianmingWang1,2,3DaqiangWu1,2,3GuimingYan1,2,3ChangzhongWang1,2,3JingShao1,2,31LaboratoryofInfectionandImmunity,CollegeofIntegratedChineseandWesternMedicine(CollegeofLifeScience),AnhuiUniversityofChineseMedicine,XinzhanDistrict,Hefei,Anhui,China2KeyLaboratoryofXinAnMedicine,MinistryofEducati
3、on,AnhuiAcademyofChineseMedicine,ShushanDistrict,Hefei,Anhui,China3AnhuiProvincialKeyLaboratoryforChineseHerbalCompound,AnhuiAcademyofChineseMedicine,Hefei,ChinaCorrespondenceDr.JingShao,LaboratoryofInfectionandImmunity,CollegeofIntegratedChineseandWesternMedicine(CollegeofLifeScience),AnhuiUniversi
4、tyofChineseMedicine,436Room,ZhijingBuilding,No.1QianjiangRoad,XinzhanDistrict,Hefei230012,Anhui,China.E-mail:AbstractInflammatory bowel disease(IBD)including Crohns disease and ulcerative colitis isa chronic intestinal disease most likely associated with gut dysbiosis.Candida relatedmycobiota has be
5、en demonstrated to play a role in IBD progression.Traditional Chi-nese herbal medicines(TCHMs)with antifungal activity have a potential in preventionand treatment of fungi-related IBD.Sodium houttuyfonate(SH)is a promising anti-CandidaTCHMs.Inthisstudy,adextransulfatesodiuminducedcolitismodelwithCan
6、-dida albicans precolonization is established.SH gavage can significantly decrease thefungalburdensinfecesandcolontissues,reducediseaseactivityindexscore,elongatecolon length,and attenuate colonic damages.Moreover,SH markedly inhibits the lev-elsofanti-Saccharomycescerevisiaeantibodies,-glucan,andpr
7、oinflammatorycytokine(IL-1,IL-6,IL-8,TNF-),and increases anti-inflammatory factor IL-10 level in serumand colon tissue.Further experiments demonstrate that SH could induce-glucanexposure,primingintestinalmacrophagestogetridofcolonizedC.albicansthroughthecollaboration of Dectin-1 and TLR2/4.With the
8、decreased fungal burden,the proteinlevels of Dectin-1,TLR2,TLR4,and NF-Bp65 are fallen back,indicating the primedmacrophages calm down and the colitis is alleviated.Collectively,these results mani-fest that SH can attenuate C.albicans associated colitis via-glucan exposure,deepen-ing our understandi
9、ng of TCHMs in the prevention and treatment of fungi associatedIBD.KEYWORDS-glucan,Candidaalbicans,Dectin-1,macrophage,sodiumhouttuyfonate,ulcerativecolitis1INTRODUCTIONInflammatory bowel disease(IBD),comprising Crohns disease andulcerative colitis(UC),is clinically featured by chronic inflammatoryA
10、bbreviations:ASCA,Anti-Saccharomycescerevisiaeantibodies;DAI,Diseaseactivityindex;DSS,Dextransulfatesodium;IBD,Inflammatoryboweldisease;MES,Mesalazine;SH,Sodiumhouttuyfonate;TCHM,TraditionalChineseherbalmedicine;UC,Ulcerativecolitis;XTT,2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5carboxa
11、nilide.progression in the internal tract with complicated causes.1GenerallyUC presents continuous,relatively superficial mucosa,and submucosainflammation,and occurs mainly in the colon with increasing morbid-ity in the developed countries as well as in China.1,2Among the envi-ronmental and genetic f
12、actors,Candida spp.,the representative myco-biota in human gastrointestinal tract,are being considered to playa nonnegligible role in UC pathogenesis,35although it is estimatedJLeukocBiol.2021;110.2021SocietyforLeukocyteBiology1www.jleukbio.org2MA ET AL.FIGURE 1Sodiumhouttuyfonate(SH)alleviatesdextr
13、ansulfatesodium(DSS)-inducedcolitiswithCandidaalbicansaddition.(A)Schemeofexperimentaldesign.DSS-inducedcolitiswithC.albicansprecolonizationisperformedbyintragastricaladministrationwithC.albicansSC5314(1108CFU/mouse/d)fromdays0to4,followedby3%DSSgavage(withSHormesalazineMESadministration)fromdays5to
14、11.Atday12,allmicearesacrificed.(B)Dailyweightfromdays1to12.(C)Dailydiseaseactivityindex(DAI)basedonweightloss,stoolconsistency,andoccultbloodfromdays1to12.(D)Representativephotographsofmicecolonatday12.(E)Thelengthofmicecolonsmeasuredaftersacrifice.(F)RepresentativeprofilesofH&Estainingofmicecolon.
15、Thecontrolgroupisfedwithnormalsalinewithoutextraadministration.TheDSSgroupisprocessedwithnormalsalinefromdays1to4andDSSfromdays5to11.ThemodelgroupisdisposedwithC.albicansfromdays1to4andDSSfromdays5to11.TheSHandMESgroupsaretreatedwithC.albicansfromdays1to4andDSSplus25mg/kg/dSHor200mg/kg/dMESfromdays5
16、to11.DataareshownasthemeanvaluesSD.*,P0.05;*,P0.01.Scalebarrepresents20mthat fungi make up only approximately 0.1%of the whole intestinalmicroorganisms.6Candida albicans is the most commonly isolated opportunistic fungicharacterized by its dimorphic morphologies:the yeast-form for dis-persion and th
17、e hypha-form for invasion,resulting in manifestationsranging from mucosal disorders to deep-seated infections.7C.albi-cans is a naturally commensal resident in the human intestinal tract.8Increasing evidence has correlated the dysbiosis of C.albicans with theMA ET AL.3FIGURE 2Sodiumhouttuyfonate(SH)
18、inhibitsCandidaalbicanscolonizationincolitis.(A)Fungalburdeninstoolseveryotherdayfromdays1to11.(B)Fungalburdenoftissuehomogenates(liver,spleen,kidney,stomach,colon)postmicesacrifice.(C)Serumlevelsof-glucanaftersacrifice.(D)Serumlevelsofanti-Saccharomycescerevisiaeantibodies(ASCA)atday12.Dataareshown
19、asthemeanvaluesSD.*,P0.05;*,P0.01;*,P0.001.Scalebarrepresents20mhigh incidence of UC.912Restoring flora disequilibrium of intestinalfungi seems an alternative and potential approach for the preventionandtreatmentofUC.Severely,most clinically isolated C.albicans strains resist the con-ventional antif
20、ungals.13Traditional Chinese herbal medicine(TCHM)is a valuable reservoir for antimycotic purpose.Sodium houttuy-fonate(SH,CH3CH28COCH2CHOHSO3Na)derivesfromhouttuynin(i.e.,decanoyl acetaldehyde,CH3CH28COCH2CHO),which is oneof the main effective phytoanticipins extracted from Houttuynia cor-data Thun
21、b(Saururaceae family).14Previously,we demonstratedthe antifungal activity of SH and its potential of inducing-glucanexposure.15,16DuetoitsinvisibilitycoveredbyC.albicanscellwallman-nan,-glucan is hard to be recognized by Dectin-1 receptor triggeringinnate immune response to fungal infection.17In sev
22、eral Chinese doc-uments,SH has already been used for UC treatment.18,19Up till now,however,theeffectofSHonthetreatmentofC.albicansassociatedUChasnotbeenreported.In this study,a dextran sulfate sodium(DSS)induced UC modelwith C.albicans precolonization is established.SH is used to assess itseffect on
23、 the pathologic and histologic changes of micecolon,cytokinesecretions,fungal burdens,-glucan,and anti-Saccharomyces cerevisiaeantibodies(ASCA)levels and crucial receptor protein expressions.Theunderlying mechanism associated with Dectin-1/TLR2/TLR4 signalingisalsodiscussed.2MATERIALS AND METHODS2.1
24、Strains and animalsC.albicans SC5314 were kindly provided by Prof.Yuanying Jiang,SchoolofPharmacy,SecondMilitaryMedicalUniversity.Fifty810wkfemale C57BL/6J mice were purchased from Pengyue Experimen-tal Animal Breeding(SCXK20190003).The animal experiment wasapproved by the Experimental Animal Ethics
25、 Committee of Anhui Uni-versityofChineseMedicine.2.2Establishment of colitis modelMice in the model group was managed by intragastrical admin-istration of C.albicans SC5314(1 108CFU/mouse/d)for 4 dfollowed by 7 d of 3.0%DSS(MP Biomedicals).In mesalazine(MES)and SH groups,200 mg/kg/d MES(H20143164,Ai
26、defa)4MA ET AL.FIGURE 3Sodiumhouttuyfonate(SH)modulatesthesecretionofinflammatorycytokinesinserum(leftpanel)andcolontissues(rightpanel).(A)ThelevelofTNF-.(B)ThelevelofIL-1.(C)ThelevelofIL-6.(D)ThelevelofIL-8.(E)ThelevelofIL-10.DataareshownasthemeanvaluesSD.*,P0.05;*,P0.01MA ET AL.5FIGURE 4Sodiumhout
27、tuyfonate(SH)-induced-glucanexposurepromotesCandidaalbicansclearancebyTHP1-transformedmacrophage.(A)Representativefluorescentimagesof-glucanexposurebyovernightincubationwith8g/mlSHandthenstainingwithanti-glucanantibody(red).(B)Fluorescentintensityassessmentbyflowcytometry.(C)FungalclearancebyTHP1-tr
28、ansformedmacrophageat1h(leftpanel)and24h(rightpanel)incubationswithC.albicanspretreatedwithSHovernight.Thefungalsurvival(%)iscalculatedastheratiobetweenODtreatedandODuntreatedusingthe2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5carboxanilide(XTT)methodat492nm.TheODtreatedistheODvalueoffun
29、galcellstreatedwithSHand/orTHP1,whereasODuntreatedistheODvalueoffungalcellswithnotreatment.(D)MacrophageactivitybyimmunofluorescentstainingwithF4/80(red)andDAPI(blue)incolontissues.DataareshownasthemeanvaluesSD.*,P0.05;*,P0.01.Scalebarrepresents20mand 25 mg/kg/d SH(A03148,Kailai)were given along wit
30、h DSS.MES was set as positive control,which could inhibit the synthe-sis of prostaglandins and leukotriene,relieving intestinal mucosalinflammation.20At day 12,all mice were sacrificed by cervical disloca-tion(Fig.1A).Disease activity index(DAI)was calculated as previouslyreported.52.3Histopathology
31、 and immunohistochemistryThe tissues were fixed with 4%paraformaldehyde,sectioned andstained with H&E(DH006,Leagene)as described previously.5Mean-time,the tissue sections were deparaffinized with xylene,hydratedwith gradient ethanol,and then incubated with anti-p65 and anti-TLR4antibodies(26j7543and
32、16c5074,AffinityBioscience,OH,USA).After being developed by diaminobenzidine(K186621D,Zhong ShanGolden Bridge),the sections were observed using an optical micro-scope(OlympusBX51).2.4Fungal enumerationThe homogenates of feces and tissues are collected at days 1,3,5,7,9,11,and12,platedonChromagar(Sha
33、nghaiCentralBio-engineering)containing 100 U/ml penicillin and 0.1 mg/ml streptomycin(Beyotime)for48hat37C.106MA ET AL.FIGURE 5RAW264.7cellsrecognizeexposed-glucaninducedbysodiumhouttuyfonate(SH)viaDectin1,TLR2andTLR4receptorsandareactivatedinresponsetoCandidaalbicansviaelevatedTNF-level.(A)TNF-leve
34、lsdetectedinthepresenceandabsenceoflaminarin(Dectin-1inhibitor)afterincubationwithRAW264.7for24h.(B)TNF-levelsdetectedinthepresenceandabsenceofC29(TLR2inhibitor)afterincubationwithRAW264.7for4h.(C)TNF-levelsdetectedinthepresenceandabsenceofC34(TLR4inhibitor)afterincubationwithRAW264.7for4h.(D)TNF-le
35、velsdetectedinthepresenceandabsenceoflaminarin+C29afterincubationwithRAW264.7for24h,(E)TNF-levelsdetectedinthepresenceandabsenceoflaminarin+C34afterincubationwithRAW264.7for24h.DataareshownasmeanvalueSD.*,P0.05;*,P0.01;*,P0.001MA ET AL.7FIGURE 6TheproteinlevelsofDectin1,TLR2,TLR4,andtheirassociatedd
36、ownstreameffectorNF-Bp65aredeclinedpostsodiumhouttuyfonate(SH)management.(A)RepresentativeprofilesofimmunohistochemistryofTLR4andNF-Bp65incolontissues.(B)IntensitiesofTLR4andNF-Bp65inimmunohistochemistryofcolontissues.(C)RepresentativeimagesofDectin-1,TLR2,andTLR4bands.(D)IntensitiesofthebandsofDect
37、in-1,TLR2,andTLR4.DataareshownasmeanvalueSD.*,P0.05;*,P0.01.Scalebarrepresents20m2.5-glucan exposureC.albicans SC5314 and the colonic tissue homogenates were used for-glucan exposure.Prior to being stained with-1,3-glucan antibody(400-2,Bioscience Supplies)and Cy3 labeled IgG(143702a,Abbkine),C.albi
38、cans SC5314(2 103CFU/ml)was incubated with SH(8 g/ml)overnight,and the homogenates was firstly blocked by BSA albuminfraction V(9048-46-8,Biofroxx).The observation was viewed by a flu-orescentmicroscopy(OlympusIX81)andtheintensitywasanalyzedbyflowcytometry(BDAccuriC6).16,212.6Macrophage stimulation
39、and phagocytosisFor interaction with THP-1,the cells(SCSP-567,Chinese Academyof Sciences Shanghai Institute of Life Sciences Cell Resources)weredifferentiated into macrophage with 100 ng/ml PMA(P1585,Sigma)and cultured with HBSS(H1020,Solarbio)supplemented with FCS(JC53576,Clarkbio).Candida inoculum
40、(1 106CFU/ml)was incu-batedwithSH(24g/ml)overnightandculturedwithphagocytes(mul-tiplicity of infection=1)at 37C under 5%CO2for 1 and 24 h.Thefungal damages were assessed with the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5carboxanilide(XTT)metabolic assay.Asfor interaction with RAM 264.
41、7,the macrophages(1 105cells/ml)were cultivated in DEME high sugar medium(k210802,BasalMedia)at 37C under 5%CO2.SH(8 g/ml)was incubated with the fungi(1 104cells/ml)overnight prior to coculture with RAW 264.7 in the pres-ence and absence of 250 g/ml laminarin(Dectin1 inhibitor,9008-22-4,Sigma)/50 M
42、C29(TLR2 inhibitor,GC33820,Glpbio)/15 M C34(TLR4 inhibitor,GC11275,Glpbio)used alone and in combination forindicated times.The stimulated macrophage was evaluated by TNF-proteinlevel.22,238MA ET AL.FIGURE 7Theworkingmodelsummarizingthefindingsinthisstudy2.7Immunofluorescent stainingThe embedded inte
43、stinal tissue sample was sectioned,fixed in ace-tone and 4%paraformaldehyde,blocked with 10%goat serum,andincubated with anti-F4/80 antibody(123110,BioLegend)and DAPI(C1005,Beyotime).22.8ELISA assayThe levels of TNF-(CK-E20852),IL-1(CK-E20174),IL-6(CK-E20188),IL-8(CK-E20191),IL-10(CK-E20162),and ASC
44、A(CK-E22252)wereanalyzedbyELISAkits(Ruixin)accordingtotheinstruc-tions.The ELISA kit for-glucan(EHT-96896 m)was purchased fromXiamenHuijiaBiologicalTechnologyCo.Ltd.2.9Western blotExtracted proteins by radio-immunoprecipitation assay(RIPA)kit(R0010,Solarbio)were separated by SDS-PAGE.After electro-t
45、ransfer,the polyvinylidene fluoride(PVDF)membrane is incubatedwithanti-TLR2(H11071747,Wanleibio),anti-TLR4(16c5074,Affinity Bioscience),anti-Dectin-1(E0317,Santa Cruz),and anti-TNF-(ab215188,Abcam)antibodies prior to treatment withHRP conjugated secondary antibody(GR3299244-7,Abcam).Thebands were de
46、veloped with electrochemiluminescence(05A089,Wanleibio)and imaged by protein imaging system(LAS4000,GE).2.10Statistical analysisThe data were calculated by SPSS 17.0(SPSS,Inc.,Chicago,IL,USA),DSS(MP Biomedicals,LLC,Aurora,OH,USA),MES(H20143164,Aidefa,Shanghai,China),SH(A03148,Kailai,xi An,China),hem
47、atoxylinand eosin(DH006,Leagene,Beijing,China),DAB(K186621D,ZhongShan Golden Bridge,Beijing,China),Chromagar(Shanghai CentralBio-engineering,Shanghai,China),streptomycin(Beyotime Biotech-nology,Shanghai,China),-1,3-glucan antibody(400-2,biosciencesupplies,Bundoora,Australia),Cy3 labeled IgG(143702a,
48、Abbkine,Shanghai,China),BSA(9048-46-8,Biofroxx,Shanghai,China),PMA(P1585,Sigma,USA),HBSS(H1020,Solarbio,Beijing,China),FCM(JC53576,Clarkbio,Shanghai,China),DEME high sugar medium(k210802,BasalMedia,Shanghai,China),laminarin(Dectin1 inhibitor,9008-22-4,Sigma,USA),C29(TLR2 inhibitor,GC33820,Glpbio,USA
49、),(TLR4 inhibitor,GC11275,Glpbio,USA),anti-F4/80 antibody(123110,BioLegend,San Diego,CA,USA),DAPI(C1005,Beyotime,Shanghai,China),RIPA kit(R0010,Solarbio,Beijing,China),anti-TLR2(H11071747,Wanleibio,Beijing,China),anti-TLR4(16c5074,Affinity Bioscience,OH,USA),anti-Dectin-1(E0317,Santa Cruz,USA),anti-
50、TNF-(ab215188,abcam,USA),electrochemiluminescenceMA ET AL.9(05A089,Wanleibio,Beijing,China),and analyzed by 1-way ANOVAwithP20%),increased DAI score,and short-ened colon length at the 12th day.The addition of SH could signifi-cantly restore mice weight,decrease DAI score,and elongate colon(Fig.1BE).