益生菌衍生的可溶性因子对于宿主防御的调节机制：对消化系统的健康影响

FangYan,M.D.,Ph.D.DepartmentofPediatricsDivisionofGastroenterology,HepatologyandNutritionVanderbiltUniversitySchoolofMedicineNashville,TNSupport:NationalInstituteofDiabetesandDigestiveDiseasesandKidneyDiseases,Crohn’sandColitisFoundationofAmerica,VanderbiltDigestiveDiseaseResearchCenter,theNestleNutritionGrant,andAtticus-BrownFamilyTrustProbiotic-derivedsolublefactorsregulatehostdefensemechanisms:impactondigestivehealth糖尿病、消化系统疾病和肾病国家研究所、美国克洛病和结肠炎基金会、范德比特消化系统疾病研究中心、雀巢营养公司的授权、布朗家族信托益生菌衍生的可溶性因子对于宿主防御的调节机制：对消化系统的健康影响范德比特大学医学院肠道学、肝胆学和营养学部门儿科田纳西州纳什维尔Researchprojects:

1.Preventionandtreatmentofintestinalinflammation

-Probiotic(LactobacillusGG)-derivedsolubleproteins

-Berberine(analkaloidisolatedfromplants)2.TherolesofEGFandTNFsignalingpathwaysinregulationofinjuryand repairmechanismsinintestinalinflammationandcarcinogenesis

3.ThemechanismsofH.pylori-inducedgastricinflammationandcancer development

预防和治疗肠道炎症-LGG的分泌蛋白-黄连素（一种从植物中分离的生物碱）-可治疗腹泻

EGF和TNF的信号通路在肠道炎症及癌变中对损伤和修复机制的调节作用。幽门螺杆菌诱导胃炎症和癌变的机制

Intestinalepithelialcellhomeostasisandfunctions

肠上皮细胞的动态平衡和功能

Digestionandabsorption

WaterandelectrolytetransportProtectivedefensemechanismsBarrierfunction(tightjunctions)Anti-bacterialsubstancesInnateandadaptiveimmunity

肠细胞保护防御机制屏障功能腺管小肠绒毛杯状细胞潘氏细胞内分泌细胞干细胞先天免疫后天免疫抗菌物质Thegastrointestinalmicrobiota1014GIbacteria1013cellsinthebody1012skinbacteriaOralcavity200species

StomachH.PyloriSmallintestine108bacteria/mlColon1010-1011bacteria/g400-500speciesBacteriodesEubacteriumBifidobacteriumRuminococcusBacillusClostridiumLactobacillusEnterococcusEnterobacter杆菌属真菌属双歧杆菌属瘤胃球菌属芽孢杆菌属梭菌属乳杆菌属肠球菌属肠杆菌属胃肠道细菌幽门螺杆菌Commensalbacteriainthegastrointestinaltract胃肠道中的共生细菌Beneficialeffects:

Promoteintestinaldevelopment.

Benefithosthealthbyenhancingpolysaccharidedigestion,uptakeofnutrients,and antimicrobialactivity.Modulateintestinalimmuneresponses.

Regulateintestinalepithelialcells.YanandPolk,CurrOpinGastroenterol20:565-571,2004扁平细胞树突状细胞基底膜上皮紧密连接粘液促进肠道发育提高多糖的消化和营养物质的吸收，提高抗菌活性，从而有益于宿主健康调节肠道免疫反应调节肠道上皮细胞ThemechanismsofprobioticactionBlockingpathogenicbacterialeffects.

Modulatingmucosalimmuneresponses.

Promotingintestinalepithelialintegrity,survivalanddefenseresponses.

Vanderpooletal.,InflamBowelDis,2008Probiotics:Livemicroorganismswhichwhenconsumedinadequateamounts aspartoffood,conferahealthbenefittothehost.阻止致病菌的入侵调节粘膜免疫反应促进肠道上皮的完整性、存活以及防御反应LactobacillusrhamnosusGG(LGG)

IsolatedfromahealthyhumanOneofthebest-studiedprobioticbacteriainclinicaltrialtopreventand/ortreat: Diarrhea,Clostridiumdifficile,RotavirusandEnterococciinfections Ulcerativecolitis,AtopicdermatitisLGG-derivedsolublefactors: Intestinalepithelialcells: Survivalandproliferation Barrierfunction Synthesisofcytoprotectiveproteins Macrophages: DecreaseLPS-inducedTNFproduction

IncreaseG-CSF

LGG巨噬细胞肠上皮细胞鼠李糖乳杆菌GG株在预防和治疗轮状病毒腹泻、梭状芽孢杆菌、肠球菌感染的溃疡性结肠炎，过敏性皮炎的临床试验中，它是研究的最多的益生菌之一。细胞保护蛋白的合成细菌脂多糖粒细胞集落刺激因子肿瘤坏死因子LGGandfactorsrecoveredfromLGGbrothculturesupernatantregulateintestinalepithelialcellsignalingpathwaystoblockapoptosis

LGG和从LGG肉汤培养基的上清液分离的因子通过调节肠上皮细胞的信号转导

途径来阻止细胞凋亡YanandPolk,J.Biol.Chem.,52,50959,2002.

(pro-apoptotic)(Anti-apoptotic)ApoptosisTNFIFNIL-1ColonepithelialcellCytokineReceptorLGGLGGsolubleproteinsAktp38Cytokinereceptor细胞因子受体Colonepithelialcell结肠上皮细胞Apoptosis细胞凋亡Pro-apoptotic促凋亡Anti-apoptotic抗凋亡Apoptosis细胞凋亡Whyisitimportanttodevelopbacteria-derivedsolubleproteinsforclinicalapplications?为什么开发细菌衍生的可溶性蛋白的临床应用是很重要的？Concernsforuseofprobioticbacteriaintherapies:益生菌用于治疗的关注点1、Bioavailability–survival,locationandfunctionofprobioticbacteriainthegastrointestinaltractrelativelyunknown

生物利用率——益生菌在胃肠道中的存活率，位置和功能都是未知的2、Biosafety(youngandimmuno-compromisedpatients)

安全性（小孩和免疫系统有缺陷的人群）Approachtoaddresstheseconcerns:-Developingprobioticbacteria-derivedproteinsfortargeteddelivery

开发能够定向投放的益生菌衍生蛋白 TodefinemechanismsthroughwhichLGG-secretedsolubleproteinsregulateintestinalepithelialcellfunction

确定LGG分泌的哪种可溶性蛋白调节肠道上皮细胞功能的机制Aim1Purificationofp40andp75fromLGGbrothculturesupernatant(LGG-s)p40p75Yanetal.,Gastroenterology,132,562,2007p40andp75activationofAkt颗粒过滤器LGG分泌蛋白阳离子交换色谱NaCl洗脱p40andp75preventTNF-inducedapoptosisincolonepithelialcells.在结肠上皮细胞p40和p75阻止TNF-α诱导细胞凋亡Yanetal.,Gastroenterology,132,562,2007

*p<0.01comparedtoTNFtreatment1、TUNEL法：[terminaldexynucleotidyltransferase(TdT)-mediateddUTPnickendlabeling](末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法)也称DNA断裂的原位末端标记法，这一方法能对，DNA分子断裂缺口中的3’-OH进行原位标记，借助一种可观测的标记物，如荧光素，能对凋亡细胞的核DNA中产生的3’-OH末端进行原位标记，用荧光显微镜即可进行观察。2、DAPI：DAPI即4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole)，是一种能够与DNA强力结合的荧光染料，常用与荧光显微镜观测1。因为DAPI可以透过完整的细胞膜，它可以用于活细胞和固定细胞的染色。EGFreceptor(EGFR)signalingpathwayregulatesAktactivationandcellularresponses.

表皮生长因子受体（EGFR）信号通路的调节Akt的活化和细胞的反应

EGFR:Type1receptortyrosinekinaseExpressedbasolaterallyintheintestinalepitheliumcolonepithelialcell结肠上皮细胞proliferation增殖migration迁移differentiation区别survival生存RTKsGPCRG蛋白欧联受体Cytokine细胞因子Receptor接收器srcMMPs基质金属蛋白酶Ligands配体trans-membraneligands跨膜配体EGFR表皮生长因子受体丝裂原活化蛋白激酶胞内磷脂酰肌醇激酶，与v．src和v．ras等癌基因的产物相关，且PI3K本身具有丝氨酸/苏氨酸(Ser/Thr)激酶的活性，也具有磷脂酰肌醇激酶的活性EGFR为I型跨膜酪氨酸激酶生长因子受体．定位于细胞膜．与HER2／ErbB．2／Neu／pl85、HER3／ErbB一3、HER4／ErbB-4等同归入HER／Eerb家族。由胞外区、跨膜区及胞内区三部分组成。其配体包括表皮生长因子(epidermalgrowthfactor。EGF)、转化生长因子Of．(transforminggrowthfactor-or，TGF—d)、8一细胞素(betacellulin，BTC)、双调蛋白(amphiregulin)、表皮素(epiregulin)、肝素结合的表皮生长因子(heparin．bindingEGF，HB．EGF)等，以EGF和TGF．d最为重要。EGFR与其配体胞外区结合后相互作用形成同源二聚体或异源二聚体。与同源二聚体相比，异源二聚体在介导细胞增殖、分化、迁移等信号传递中起着更为重要的作用。二聚体的形成导致胞内酪氨酸激酶区的激活，进而通过转磷酸化和磷酸化作用，促使受体酪氨酸残基磷酸化．启动ras．MAPK、P13K、PLC'y／PKC、STAT等一系列级联反应．将信号传到细胞核内．最终引起一系列相关基因活化．促进细胞从G，期过渡到S期141．从而对核内基因表达和细胞生长分化产生调节作用。p40activatesAktinanEGFRdependentmannerinintestinalepithelialcells.在肠上皮细胞p40依靠EGFR（表皮生长因子）受体激活Akt蛋白EGFRisrequiredforp40preventionofcytokine-inducedapoptosisinintestinalepithelialcells在肠上皮细胞中预防细胞因子诱导的凋亡，EGFR对P40来说是必需的p40orp75preventH2O2disruptionoftightjunctioninCaco2cells.p40或p75可以防止H2O2中断Caco2细胞紧密连接

H2O2H2O2/p40H2O2/p40/AG1478H2O2/p75H2O2/p75/AG1478ZO-1OccludinAG1478:EGFreceptorkinaseinhibitorAG1478：表皮生长因子受体激酶抑制剂Sethetal.AmJPhysGastrointestLiverPhysiol2008p40requiresEGFRkinaseactivitytostimulateAktactivationinmousecolonexplants.在小鼠结肠外植体中，p40需要EGFR激酶活性刺激Akt的活性wt(C57BL/6)EGFRwa5p40(10ng/ml)ColonexplantDMEM0.5%FBSColonicmucosallysatesEGFRkinaseinhibitorAG1478(10nM)结肠黏膜裂解p40inhibitionofTNF-inducedapoptosisrequiresEGFRactivityincolonicorganculture.在结肠器官培养物中，p40抑制TNF-α诱导的细胞凋亡需要EGFR的活性EGFRmediatesp40preventionofTNF-inducedredistributionofoccludininintestinalepithelialcells.在肠上皮细胞中，EGFR介导p40预防TNF-α诱导的密封蛋白的再分配Red:Occludin,

Blue:DAPIControlTNFTNF+p40wtEGFRwa5红色代表Occludin密封蛋白蓝色：DAPIp40preventscytokine-inducedapoptosisanddisruptionofintegritythroughactivationofEGFreceptor(EGFR)andAktinintestinalepithelialcells.在肠上皮细胞p40通过激活表皮生长因子受体（EGFR）和Akt，阻止细胞因子诱导的细胞凋亡和破坏其完整性。Yanetal.Gastroenterology32:562-75,2007,JClinInvest121:2242-53,2011.ColonepithelialcellAktActivationEGFRTNFIFNIL-1CytokineReceptorApoptosisDisruptionofbarrierp40?Aim2Toinvestigatewhetherp40stimulatesEGFreceptorligandreleaseasamechanismfortransactivationofEGFreceptorbyp40intheintestinalepithelialcells.p40isanovelprotein

p40是一种新的蛋白

Full-lengthp40proteinsequence(412aa)

MKFNKAMITLVAAVTLAGSVSALTPVFADTSASIASNKSETNDLLKQIEAANTEVINLNKQIDAKNGEISDATAKISATDAKIASLSGEITAAQKNVAARKNNLKDQLISLQKKAGSSVSGNVYIDFVLNSQSLSDLIARTMTVGKLSQASKDALDAVTVAKDKLAALKSEQETARQTLVSTKASLETQKSQLETLQKTASDKQDALNKEIADHKDELVALQSQFAQEQSEAAKATQAALKTAAASTASSSTSSTSNKSANSSVLSTGTSSTNTSSNSGASSTVISSNTASGSGSHADYSGSGNTYPWGQCTWYVKSVASWAGNGWGNGAEWGASAAAAGFTVNHTPAAGSIIVFAAGQSVGGQWTADGSYGHVAYVQSVSGDSVTITQGGMGFSSPTGPNTQTISGASSYVGreen:N-terminalsequencedetectedbyEdmandegradation.Orange:internalsequencedetectedbyMALDI-TOF/MS/MSandLC/MS/MSanalysis.Purple:Leadersequenceforproteinsecretion.绿：N-末端序列由Edman降解法检测。橙：由MALDI-TOF/MS/MS和LC/MS/MS分析检测到的内部序列。紫色：前导序列的蛋白质分泌。Yanetal.,Gastroenterology,132,562,2007Predictionofp40tertiarystructureforstructure-functionanalysisp40的三级结构的结构与功能分析预测p40aminoacidsequencehomologyanalysisHigh-confidencetemplatesforp40domainsUncharacterizeddomain-sheetcoiled-coilNH2175150225300375412COOHp40的氨基酸序列的同源性分析高信任度的p40域模板p40stimulatesHB-EGFrelease,butnotTGForamphiregulin,inyoungadultmousecolon(YAMC)epithelialcells.在年轻成年小鼠结肠（YAMC的）上皮细胞，p40刺激HB-EGF的释放，而不是TGF或双调蛋白。(10ng/ml)YAMCp40Supernatant:ELISALysates:WesternblotHB-EGF上清p40stimulatesHB-EGFrelease,butnotTGForamphiregulin,inmice在小鼠实验中，P40刺激HB-EGF的释放，而不是TGF或双调蛋白Gavagep40-pectin/zeinbeadsBloodELISAKnock-downHB-EGFsuppressesactivationofEGFRandinhibitionofapoptosisbyp40inYAMCcells.在YAMC细胞中去除HB-EGF通过p40抑制EGFR的活性并抑制细胞凋亡Metalloproteinaseinhibitorsblockp40-stimulatedEGFRactivationinYAMCcells.在YAMC细胞中，金属蛋白酶抑制剂阻止p40刺激表皮生长因子的活性ADAM:ADisintegrinAndMetalloproteinasep40:10ng/ml,1hrEGF:10ng/ml,5minMetalloproteinaseinhibitors:GM6001(10nM)andTAPI-1(10nM)Anti-EGFRantibody:C225,1:1000dilutionInactive

MembraneboundligandactivePropeptideMetalloproteaseDisintegrinCystein-richTransmembranecytoplasmic

ADisintegrinAndMetalloproteinase去整合素和金属蛋白酶inactive无活性propeptide肽Metalloprotease金属蛋白酶Disintegrin整合素cystein-rich富含半胱氨酸Transmembrane跨膜Cytoplasmic细胞质membraneboundligand膜结合配体p40stimulatesTACE(ADAM17)activityincolonepithelialcellsinvitroandinvivo.在体内或体外的结肠上皮细胞中，p40刺激TACE（肝动脉栓塞？）（ADAM17）的活性YAMCp40Lysates:TACEactivityassayGavagep40-pectin/zeinbeadsColonicepithelialcellsTACEactivityassayHouraftergavageTACEmediatesp40regulatedanti-apoptoticresponseincolonepithelialcells.在结肠上皮细胞上，TACE介导P40的抗凋亡性反应p40transactivationofEGFRdependsonTACE.TACEmediatesinhibitionofapoptosisbyp40.P40的表皮生长因子受体的转录依赖于TACE通过p40，TACE介导抑制细胞凋亡Summaryp40-stimulatedTACEactivity,leadingtoHB-EGFrelease,servesasamechanismunderlyingEGFreceptortransactivationinintestinalepithelialcells在肠上皮细胞，P40-刺激TACE的活性，导致HB-EGF释放，从而使其在表皮生长因子受体下转录Aim3Totestthehypothesisthatp40preventsexperimentalcolitisinmiceinanEGFR-dependentmanner.为了检验这个假设，p40依赖表皮生长因子受体来抑制小鼠的实验性结肠炎Preparationofpectin/zeinbeadsforspecificdeliveryofp40tothecoloninvivo.制备果胶/玉米醇溶蛋白的药丸（包埋有p40），能够使其在体内到达结肠位置。Gavagedwithpectin/zeinbeads(2beads/mouse)containingFITC-His-p40orHis-p40-Sacrificedafter4hours.FITC-labeledHis-p40beadsHis-p40beadsGreen:FITC-His-p40Red:E-cadherinBlue:DAPIStomachSmallintestineColonColonpectin/zeinbeadHis-p40:5g/bead1cmYanetal.,JCI,121,2242,2011-灌胃果胶/玉米醇溶蛋白的药丸（每只小鼠两颗），药丸里面包含有FITC-His-p40（标记的）或His-p40（未标记的）-药丸在4h后完全消化上表皮钙粘着蛋白p40activatesEGFRandAktincolonepithelialcellsinvivo.在体内的结肠上皮细胞中，p40激活EGFR和AktYanetal.,JCI,121,2242,2011磷酸化表皮生长因子受体p40preventsDSS-inducedcolitisinwt,butnotEGFRwa2mice.在wt小鼠模型中p40防止DSS诱导结肠炎，但在EGFRwa2小鼠模型中不起作用Yanetal.,JCI,121,2242,2011损伤或炎症得分牺牲/处理P40treatsDSS-inducedcolitisinwt,butnotEGFRwa2mice.在wt小鼠模型中p40可以治疗DSS诱导的结肠炎，但在EGFRwa2小鼠模型中不起作用Yanetal.,JCI,121,2242,2011p40blocksDSS-inducedcolonepithelialcellapoptosisinanEGFR-dependentmanner.

p40需要依赖EGFR来抑制DSS诱导结肠上皮细胞凋亡Yanetal.,JCI,121,2242,2011WaterDSS-7DDSS+p40半胱氨酸天冬氨酸蛋白酶-3：被激活后可以通过对caspase靶蛋白的水解，导致程序性细胞死亡Bcl-2基因（即B细胞淋巴瘤/白血病-2基因）是一种原癌基因，它具有抑制凋亡的作用p40preventsDSS-induceddisruptionofintestinalepithelialtightjunctionsinanEGFR-dependentmanner.

p40需要依赖EGFR来抑制DSS诱导肠上皮细胞紧密连接的中断Yanetal.,JCI,121,2242,2011体内渗透检测血清FITC-葡聚糖紧密连接蛋白ZO-1GenerationoftransgenicmicewithEGFreceptorspecificallydeletedintheintestinalepithelium.

新一代的转基因小鼠的肠上皮细胞中专门去除EGFRYanetal.,JCI,121,2242,2011上皮基质EGFreceptorexpressioninthecolonepitheliumisrequiredforp40treatingDSS-inducedcolitisinmice.p40治疗DSS诱导的小鼠结肠炎需要EGFR在结肠上皮细胞的表达Day:0473%DSS

Water10gofp40/day/mouseYanetal.,JCI,121,2242,2011外伤/炎症评分血清中FITC标记的聚葡萄糖凋亡细胞所占百分比p40amelioratingoxazolone-inducedcolitisrequiresEGFreceptorexpressioninthecolonepithelium.p40改善恶唑酮诱导的小鼠结肠炎需要EGFR在结肠上皮细胞的表达乙醇p40activationofEGFreceptorinintestinalepithelialcellsmediatesameliorationofintestinalinflammationinmice.

在肠上皮细胞中p40激活EGFR的活性，间接地改善了小鼠肠道炎症Yanetal.,JClinInvest121:2242-53,2011破坏屏障炎症Aim4Todetectimmuneresponsesregulatedbyp40duringcolitisinmice.检测小鼠结肠炎期间由p40调控的免疫反应p40treatmentdecreasemacrophageandneutrophilinfiltrationinDSS-inducedcolitisinmice.在具有DSS诱导的结肠炎的小鼠中，p40处理后可以减少巨噬细胞和嗜中性白血球的渗透活动嗜中性白血球巨噬细胞p40treatmentdecreasesTNF,IFN-γ,IL-6,KC,andIL-17mRNAlevelsinDSStreatedmousecolonmucosa.在DSS处理的结肠粘膜的小鼠中，p40处理后可以减少TNF,IFN-γ,IL-6,KC,和IL-17mRNA巨噬细胞产生TNF,IL-6和KC，TH1细胞产生IFN-p40treatmentdoesnotaffectIL-10,IL-1βmRNAlevelsinDSStreatedmousecolonmucosa.在DSS处理的结肠粘膜的小鼠中，p40处理后不影响IL-10，IL-1βmRNA水平IL-10、IL-13主要由th2细胞产生p40doesnotaffectIL-13productioninoxazolone-inducedcolitisinmice.在具有恶唑酮诱导的结肠炎的小鼠中，p40处理后不影响IL-13的量ColonmucosalIL-13mRNAlevelRealtimePCRColonmucosalIL-13proteinlevelFlowcytometry实时定量PCR流式细胞仪结肠黏膜IL-13蛋白水平结肠黏膜IL-13mRNA水平IL-13由th2细胞产生Summary

Basedonthecytokineprofilesthatareregulatedbyp40,itispossiblethatp40playsaroleinregulationofinnateimmunityandaTh1immuneresponseincolitis,suchasregulationofTNF,IL-6andKCproductionbymacrophagesandIFN-productionbyTh1cells.

根据上述资料，p40可能在结肠炎中发挥先天免疫和Th1型的免疫应答的调节作用，如调节巨噬细胞产生TNF,IL-6和KC，TH1细胞产生IFN-。

Ourfindingssupportdevelopmentofprobiotic-derivedproteinsasnovelreagentsformoreconvenientanddirecttherapeuticinterventioninpreventingand/ortreatingulcerativeintestinalinflammatorydisorder