齐鲁医学关于氨基酸和神经肽与疼痛关系的研究

TatLeangLEEDepartmentofAnaesthesiaYongLooLinSchoolofMedicineNationalUniversityofSingaporeAdvancesinAminoAcidsandNeuropeptidesResearchandPain关于氨基酸和神经肽与疼痛关系的研究2021/9/301Rationaltreatmentofpainrequiresidentificationofpainmechanismsastargetsofdrugtherapy

致病因素临床实体疾病机制症状体征疼痛机制基于疼痛机制的治疗与疼痛相关的症状体征特定机制结果的评测2021/9/302Rationaltreatmentofpainrequiresidentificationofpainmechanismsastargetsofdrugtherapy

致病因素临床实体疾病机制症状体征疼痛机制与疼痛相关的症状体征基于疼痛机制的治疗特定机制结果的评测2021/9/303Keymolecularelementsinthetransduction,transmissionandsignalprocessingofnociceptiveinputintheperipheralandcentralnervoussystemrepresentpotentialtargetsforthedevelopmentofnewanalgesics.

Mechanism-basedpaindiagnosis:issuesforanalgesicdrugdevelopmentWoolfCJ,MaxMB.

Anesthesiology2001;95:241-9Pain:movingfromsymptomcontroltowardmechanism-specificpharmacologicmanagementWoolfCJ.AnnInternMed.2004;140:441-5Canweconquerpain?ScholJ,WoolfCJ.Natureneurosciencesuppl.2002;5:1062-7基于病理机制的疼痛诊断:镇痛药发展的问题我们能否征服疼痛?疼痛:从症状控制向特异机制的药理学治疗转移2021/9/304Background背景Cerebralspinalfluid(CSF)reflectsmetabolicstateofbrain

脑脊液反映疼痛的新陈代谢状态AnalysisofCSFaminoacidsandpeptides–highlyrelevantinpainperceptionandalsoneurologicaldiseases

脑脊液中氨基酸和缩氨酸与疼痛感知和神经系统疾病高度相关ChronicPain–inflammatory,neuropathyandcancer

慢性疼痛分类—发炎，神经系病，癌症Mechanismsverychallengingandnotyetunderstood

对于疼痛机制，目前还没有被足够的了解，非常具有挑战性Lackofeffectivetherapeutics

缺乏有效的治疗法2021/9/305BackgroundExcitatoryandinhibitoryaminoacidsandalsonitricoxide(NO)areinvolvedinpainmodulation.

兴奋性，抑制性氨基酸和一氧化氮涉及到对疼痛的调节Neuropeptidesareimplicatedinpainmodulation.

神经肽影响对疼痛的调节2021/9/306Objectives

AtwoprongedstudyonCSF对脑脊液的两次研究ToquantitativelyanalyzephysiologicalaminoacidsandpainrelatedcompoundsinCSFsamplesfrompainpatients

对疼痛病人脑脊液中生理氨基酸和疼痛相关的化合物定量研究Tofindnewpain-relatedpeptides(molecularmass<15,000Da)fromCSFsamplesofchronicpainpatients

从慢性疼痛病人的脑脊液中寻找与疼痛相关的肽（分子量小于15000）Resultsshouldleadtobetterunderstandingofpainmechanismsandnewtreatmentstrategy

实验结果帮助我们更深的了解疼痛机制和寻找新的治疗方法2021/9/307AminoacidsinCSFandPain

脑脊液中氨基酸和疼痛关系的研究2021/9/308液相色谱法对生理氨基酸在未被预处理的脑脊液中的定量测量2021/9/309TypicalChromatogram典型色谱层析图

STDAA氨基酸分离标准图1-aspartate天(门)冬氨酸盐2-glutamate

谷氨酸3-asparagine天冬酰胺酸4-glutamine谷酰胺5-citrulline瓜氨酸6-serine丝氨酸

7-threonine苏氨酸

8-glycine甘氨酸9-alanine丙胺酸10-arginine精氨酸11-taurine牛磺酸12-GABA伽马氨基丁酸13-proline脯氨酸14-valine缬氨酸15-methionine蛋氨酸16-isoleucine异亮氨酸17-leucine亮氨酸18-tryptophan色氨酸19-phenylalanine苯基丙氨酸

20-cystine胱氨酸21-Ammonia氨22-lysine赖氨酸23-histidine组氨酸24-tyrosine酪氨酸2021/9/30101-aspartate天(门)冬氨酸盐2-glutamate

谷氨酸3-asparagine天冬酰胺酸4-glutamine谷酰胺5-citrulline瓜氨酸6-serine丝氨酸

7-threonine苏氨酸

8-glycine甘氨酸9-alanine丙胺酸10-arginine精氨酸11-taurine牛磺酸12-GABA伽马氨基丁酸13-proline脯氨酸14-valine缬氨酸15-methionine蛋氨酸16-isoleucine异亮氨酸17-leucine亮氨酸18-tryptophan色氨酸19-phenylalanine苯基丙氨酸

20-cystine胱氨酸21-Ammonia氨22-lysine赖氨酸23-histidine组氨酸24-tyrosine酪氨酸

CSFsample2021/9/3011Salientfeaturesofthemethod

方法特色UseofTritonX-100（TritonX-100的应用）

Nopre-treatmentofCSF脑脊液不被预处理Enhancedrecoveryofhydrophilicaminoacids提高亲水性氨基酸的恢复Detectionofcitrullineimproved提高对瓜氨酸的检测RequiressmallvolumeofCSF-10-20L使用较少量的脑脊液

20LofCSFsampleyields140Lofdabsylatedsample.20微升脑脊液样本经过dabsylation产生140微升样本Injectionvolume–20L一次样本液相色谱分析只需20微升6repeatedanalysisofsamesampleoncedabsylatedispossible

能够对同一Dabsylate

过的样本进行6次重复分析Qeffectivelyseparated-100timeshigherconcentrationinCSF谷酰胺被有效分离—在脑脊液中浓度是其它氨基酸的100倍Stabilityofdabsylderivates

Dabsyl衍生物稳定性

Dabsylatedsamplestableforoneweekin4Candfor48hrsatroomtemperature

经过Dabsylation的样本能够在4度保持一周，室温下保持48小时2021/9/3012Analysisofaminoacidsandnitricoxideinlaborpain

对产前疼痛病人脑脊液中氨基酸和一氧化氮的分析Differentialaminoacidprofileinthecerebrospinalfluidofparturientswithandwithoutlaborpain对产前疼痛和非疼痛产妇脑脊液中氨基酸量不同的分析LeeTL,SethuramanR,ChuiJW,TachibanaSAnnualmeetingoftheAmericanSocietyofAnesthesiologists,23-27Oct2004,LasVegas,USA2021/9/3013ConcentrationsofpainrelatedaminoacidsinCSFofpregnantwomen

怀孕妇女脑脊液中和疼痛相关的氨基酸浓度Aminoacids氨基酸Age(mean±SD)年龄（平均数±方差）Controlgroupa(µmol/L)(n=30)33.4±5.0标准组无产前疼痛Laborpaingroupb(µmol/L)(n=38)28.6±4.5

急性产前疼痛组Arginine精氨酸13.34±0.6513.51±0.56Asparagine天冬酰胺酸12.63±0.7910.19±0.51*Aspartate天冬氨酸盐4.42±0.636.63±0.41**Citrulline瓜氨酸‡0.032.11±0.42***GABA伽马氨基丁酸3.94±0.345.71±0.73*Glutamate谷氨酸2.13±0.222.48±0.18Glutamine谷酰胺981.03±46.49583.65±16.36***Glycine甘氨酸4.39±0.278.18±1.26**Taurine牛黄酸13.74±0.899.85±1.01**Valuesareexpressedasmean±SEM;aPregnantwomenwithnolaborpain;bPregnantwomeninacutelaborpain(P<0.05)**(P<0.01)***(P<0.001)–Statisticallysignificantwhencomparedtothecontrolgroup.产前急性疼痛b与产前无疼痛a相比具有统计价值2021/9/3014StatisticalcorrelationbetweenPainintensity(PI)andtheconcentrationofpainrelatedaminoacidsincerebrospinalfluidofthelaborpaingroupascomparedagainstthenopaincontrol.疼痛强度和与疼痛相关氨基酸在产妇脑脊液中浓度的统计相关性LaborPaingroup(n=38)PIvs.arginine精氨酸

Correlationcoefficient相关系数PvalueP值0.0350.706PIvs.asparagine天冬酰胺酸

CorrelationcoefficientPvalue-0.207*0.026PIvs.aspartate天(门)冬氨酸盐

CorrelationcoefficientPvalue0.285**0.002PIvs.citrulline瓜氨酸

CorrelationcoefficientPvalue0.501***<0.001PIvs.GABA伽马氨基丁酸

CorrelationcoefficientPvalue0.0190.835PIvs.glutamate谷氨酸

CorrelationcoefficientPvalue0.0600.518PIvs.glutamineCorrelationcoefficientPvalue-0.601***<0.001PIvs.glycineCorrelationcoefficientPvalue0.332***<0.001PIvs.taurineCorrelationcoefficientPvalue-0.307**0.001PI–Painintensity–gradedasMild轻度(n=11),Moderate中度(n=18),Severe严重(n=9)*(P<0.05)**(P<0.01)***

(P<0.001)–Statisticallysignificantcorrelationrelativetothecontrolgroup.2021/9/3015CSFconcentrationofotheraminoacidsnotrelatedtopain

与疼痛不相关的氨基酸在脑脊液中浓度AminoacidsAge(mean±SD)Controlgroupa(µmol/L)(n=30)33.4±5.0Laborpaingroupb(µmol/L)(n=38)28.6±4.5Alanine丙胺酸

29.41±1.3333.41±1.53Cystine胱氨酸

‡0.6±0.120.2±0.06**Histidine组氨酸

14.87±1.1317.39±0.84Isoleucine异亮氨酸

3.77±0.154.03±0.19Leucine亮氨酸

8.56±0.379.09±0.35Lysine赖氨酸

18.72±1.3819.34±1.31Methionine蛋氨酸

1.85±0.132.19±0.10*Phenylalanine苯基丙氨酸

7.26±0.297.94±0.27Proline脯氨酸

0.89±0.091.22±0.09**Serine丝氨酸

32.05±1.2724.55±0.87***Threonine苏氨酸58.68±2.7359.94±3.16Tryptophan色氨酸1.87±0.102.26±0.10**Tyrosine酪氨酸3.94±0.434.95±0.44Valine缬氨酸9.59±0.5010.02±0.38Valuesareexpressedasmean±SEMaPregnantwomenwithnolaborpain

bPregnantwomeninacutelaborpain*(P<0.05)**(P<0.01)***

(P<0.001)–Statisticallysignificantwhencomparedtothecontrolgroup.2021/9/3016Results

结果Aspartateisinvolvedinlaborpainbutnotglutamate

天冬氨酸盐与产前疼痛相关，谷氨酸不相关Inhibitoryaminoacidsglycine,GABA,taurineinvolvedinlaborpain

抑制性氨基酸如甘氨酸，伽马氨基丁酸，牛黄酸与产前疼痛相关NOmightbeinvolvedinlaborpain–mayhavedifferentialroles

一氧化氮可能涉及至产前疼痛，但可能有不同作用2021/9/3017Acomparativestudyontherolesofaminoacidsandnitricoxideinchronicandacutepain氨基酸和一氧化氮在慢性和急性疼痛中的作用对比研究Relationshipbetweenphysiologicalaminoacidsincerebrospinalfluidandpain.

脑脊液中生理氨基酸和疼痛的联系

TachibanaS,SethuramanRandLeeTL

In:AnnaCapasso(Ed)RecentDevelopmentsinPainResearch,ResearchSignpost;2005,ppISBN81-308-0012-8.2021/9/3018Concentrationofpain-relatedaminoacidsinCSF–ChronicpainVsControlgroupandChronicpainVsAcutepaingroup和疼痛相关的氨基酸在脑脊液中浓度—慢性疼痛组和标准组，慢性疼痛组和急性疼痛组Aminoacids氨基酸Age(mean±SD)年龄（平均数±方差）Controla(µmol/L)(n=35)37.6±12.2标准组Chronicpainb(µmol/L)(n=29)64.7±12慢性疼痛组Acutepainc(µmol/L)(n=46)36.8±19.1急性疼痛组Arginine精氨酸15.52±1.21***32.32±2.70‡‡‡15.27±1.07Asparagine天冬酰氨酸13.28±0.99**21.22±1.90‡‡‡10.84±0.55Aspartate天门冬氨酸盐4.94±0.69***12.05±1.69‡‡6.83±0.59Citrulline瓜氨酸¥0.13±0.08***1.65±0.362.00±0.37GABA伽马氨基丁酸4.20±0.373.37±0.60‡5.14±0.64Glutamate谷氨酸2.27±0.23**4.42±0.682.86±0.46Glutamine谷酰胺1010.27±49.01994.7±91.31‡‡‡597.49±22.12Glycine甘氨酸4.86±0.34**10.50±1.428.18±1.11Taurine牛黄酸13.95±0.8512.51±1.859.90±0.95Valuesareexpressedasmean±SEM;*,‡(P<0.05)**,‡‡(P<0.01)***,‡‡‡(P<0.001)–Statisticallysignificantwhencomparedtothechronicpaingroup与慢性疼痛组相比有统计.¥Citrullinedetectedinonly3outof35controlCSF,17outof29chronicpainCSFand22outof46acutepainCSF.在标准组（35样本）中，仅有3个样本中含瓜氨酸；29个慢性疼痛病人中17个含有瓜氨酸；46个急性疼痛病人中有22个含瓜氨酸2021/9/3019************ComparisonbetweenPainrelatedaminoacidsinCSFofChronicandAcutepainpatients-DifferentialrolesfortheseaminoacidsinthetwotypesofPainstates和疼痛相关的氨基酸在慢性和急性病人脑脊液浓度对比—这些氨基酸在两种不同疼痛状态中的作用*(P<0.05)**(P<0.01)***(P<0.001)–StatisticallysignificantdifferencesbetweenthetwogroupscomparedGlutamine(Gln)concentrationsareexpressedasx10-2µmol/L2021/9/3020ResultsChronicpainVsNopaingroup慢性疼痛和无疼痛 Aspartate,glutamate,glycineandNOinvolvedinchronicpainperception

天门冬氨酸盐，谷氨酸，甘氨酸和一氧化氮与疼痛的感知有关ChronicpainVsAcutepain–Differentialrolesofaminoacidsinchronicandacutepainstates

慢性疼痛和急性疼痛—氨基酸在慢性和急性疼痛状态的不同作用Aspartate-akeyplayerinpathogenesisofchronicpain

天门冬氨酸盐—导致慢性疼痛的重要因素

2021/9/3021FutureScope前景ThissimpleCSFaminoacidanalysismethodcanbeappliedtostudyvariousotherpainstatesandCNSdiseases.

这种简易的氨基酸分析方法可以用于其它疼痛状态和神经系疾病分析ThismethodofCSFanalysiswithoutpretreatmentinthepresenceofTritoncanalsobeappliedtootheranalysisprocedures.

这种含有Triton，无预处理脑脊液分析方法可以用于其它分析方法中Withmorenumberofagematchedsubjects(increase“n”)andwithwelldefinedpainconditionspotentialmarkerscanbeidentifiedforeachtypeofpain.

同样年龄组中如果有足够样本，在有明确的对疼痛定义的前提下，对各种疼痛类型，可以鉴别出潜在的生物标记2021/9/3022

PurificationandcharacterizationofpeptidesfromCSFofchronicpainpatients

慢性疼痛病人脑脊液中肽的提纯和特性研究2021/9/3023PeptidePurificationStrategy

肽的提纯方法PooledCSF

PreparativeHPLCFractionAFractionBFractionCUltrafiltrationAnalyticalHPLCMicroboreHPLCStaining,Digestion,MALDI-TOFMSMSsequencingStaining2021/9/3024Identification鉴别Characterisation特性Quantification量化Localisation

位置CNSeffects在中枢神经中的影响Antagonists对抗剂Painmodels疼痛原型2021/9/3025TheStory……2021/9/3026

1973–morphinebindingsites/opiatereceptorsLTerenius,SSnyder,ESimon1975–

enkephalinJHughes,HKosterlitz

Papaver

somniferum–pain,diarrhoea&dysentery1806–Morphine

FriedrichSertÜrner

疼痛,腹泻及痢疾吗啡吗啡结合部位/阿片受体脑啡肽2021/9/30272021/9/3028OrphanGProtein-CoupledReceptors孤儿G蛋白偶联受体2021/9/30292021/9/3030OrphanGProtein-CoupledReceptorsORL1(humans),ROR-C&LC132(rats),KOR-3(mice)Highdegreeofaminoacidsequencehomologytothepreviouslycloned,,and-opioidreceptorsDidnotshowhighaffinitiesfortheknownopioidligands孤儿G蛋白偶联受体ORL1(人类),ROR-C和LC132(大鼠),KOR-3(小鼠)-与以往克隆的,,和-阿片受体的氨基酸序列高度同源性-对以知的阿片配体未显示高亲和力2021/9/3031

Endogenousligendfor

OpioidReceptor-Like(ORL1)OrphanReceptorMeunieretal.

Nature1995;377:532-35

Reinscheidetal.

Science1995;270:792-94NOCICEPTIN(Noc)ORPHANINFQ(OFQ)阿片孤儿受体（ORL-1）的内源性配体孤啡肽孤啡肽Ratbrain–nociceptinMice(i.c.v.)–hyperalgesiaPorcinebrain–OrphaninFQMice(i.c.v.)–

hyperalgesia(tail-flickassay) decreaselocomotoractivity大鼠脑猪脑痛觉过敏(甩尾试验)减少运动的活动性2021/9/30322021/9/3033Noc/OFQHeptadecapeptideStructurallysimilartodynorphinAi.t.ori.c.v.administration: -hyperalgesia(hotplate&tail-flicktests) -allodyniatoinnocuoustactilestimuli -analgesia(tail-flicktests)十七肽结构上类似于强啡肽A用药后:痛觉过敏(热板法及甩尾试验)-异常痛觉(对无损伤的触觉刺激)-痛觉缺失(甩尾试验)2021/9/3034Noc/OFQpPNoc/OFQandtheNoc/OFQreceptorareexpressedwidelyinthenervoussystemLocomotion,motivation,cognitiveprocesses,feeding,andneuroendocrinesecretion

pPNoc/OFQ和Noc/OFQ受体在神经系统内广泛地表达行动,动机,认知过程,摄食和神经内分泌2021/9/30352021/9/30362021/9/30372021/9/30382021/9/3039Nocistatin(NST)i.t.NST

hyperalgesiaandallodyniaevokedbyNoc/OFQandPGE2i.t.anti–NSTantibodyallodyniainducedbyNoc/OFQDoesnotaffectnociceptivethresholdsOkuda-AshitakaEetal1998痛稳素-不影响感受伤害的阈值i.t.痛稳素可减轻由Noc/OFQ和PGE2所诱发的痛觉过敏和异常痛觉-i.t.抗痛稳素抗体可增强由Noc/OFQ所诱发的异常痛觉2021/9/3040(b)Hyperalgesia(a)Normalpain(c)AnalgesiaInnocuousNoxiousStimulusintensityLesspainMorepainAllodyniaNocistatinMorphineNociceptin2021/9/30412021/9/30422021/9/3043Nocistatin(NST)i.t.NSTformalin-inducedpaininmiceandratsAntinociceptiveeffectwasnotantagonisedbynaloxonei.c.v.NSThyperalgesiaassociatedwithcarrageenan/kaolininjectedinratpawsNakano2000,Yamamoto1999Nakagawaetal1999痛稳素在大鼠和小鼠中,减轻由甲醛诱发的疼痛抗伤害刺激反应的效应不被纳络酮对抗减轻在大鼠爪部注射爱兰苔胶/白陶土所致的痛觉过敏2021/9/3044Immunofluorescentconfocalimagesofmousespinalcordwithanti-NST/anti-Nocantiserum.Atransversesectionofmouses.cwasincubatedwithanti-NST(b,d)/anti-Noc(c,e)antibodyfollowedbyCy3-conjugatedanti-rabbitIgG.Okuda-AshitakaEetal.1998NSTNSTNocNoc2021/9/3045Immunofluorescentconfocalimagesofsectionsofmousebrainincubatedwithanti-nocistatinantiserum.Atransversesectionofmousebrainwasincubatedwithanti-mPNP-3antibodyfollowedbyCy3-conjugatedanti-rabbitIgG.Bar,250m(a,b,c,d);100m(c’,d’).Okuda-AshitakaEetal.2000SpinaltrigeminaltractparamedianraphenucleusVentromedialnucleusofhypothalamus2021/9/3046NSTreceptor(?)

Doesnotactontheclassicopiatereceptors

DoesnotactonORL1receptor痛稳素受体(?)不作用于典型的阿片受体不作用于阿片孤儿受体(ORL1

)受体2021/9/3047TheroleoftheORL1receptorinthemodulationofspinalNeurotransmissionbynociceptin/orphaninFQandnocistatinAhmadiS,LiebelJT,ZeilhoferHU.Eur.J.Pharmacl.2001;412:39-44Modulationofsynaptictransmissionbynociceptin/orphaninFQandnocistatininthespinalcorddorsalhornofmutantmicelackingthenociceptin/orphaninFQreceptorAhmadiS,KotallaC,GuhringH,TakeshimaH,PahlA,ZeilhoferHUMolecularPharmacology2001;59:612-82021/9/3048PossiblepathwaysinvolvedinNoc/OFQ-inducedhyperalgesiaandallodyniainthespinalcordItoSetal.20002021/9/30492021/9/3050ClinicalSignificanceAnti-nociceptiveResponsesProducedbyHumanPutativeCounterpartofNocistatinMinamiTetalBrJPharmacol1998;124:1016-18IdentificationofNocistatininHuman,RatandMouseBrainandHumanCerebrospinalFluidLeeTLetal.NeuroReport1999;10:1537临床意义对抗由人们公认的痛稳素类似体所引起的伤害性反应人,大鼠,小鼠大脑及人脑脊液中痛稳素的鉴别2021/9/30512021/9/3052IdentificationofNocistatininHuman,RatandMouseBrainandHumanCerebrospinalFluidLeeTL,FungF,TachibanaSetal.NeuroReport1999;10:1537PresentinhumannervoussystemDifferentNSTformsAnimalmodels人,大鼠,小鼠大脑及人脑脊液中痛稳素的鉴别存在于人类的神经系统中痛稳素的不同结构动物模型2021/9/3053LeeTLetal.19992021/9/3054Pathophysiologicalrolesofthetwoisoformsofnocistatinin