全 文 ：天然产物研究与开发 NatProdResDev2008, 20:614-616, 638
文章编号:1001-6880(2008)04-0614-04
　
　
　ReceivedJune4, 2007;AcceptedSeptember11, 2007
　FoundationItem:ThisworkwassupportedbyScienceandTechnology
FoundationofYunnanEducationAdministration.
＊CorespondingauthorTel:86-871-5338904;E-mail:mszhaoyn@yahoo.
com.cn
粗根荨麻水提取部分对佐剂性关节炎
大鼠腹腔巨噬细胞分泌 TNF-α及 PGE2 的影响
李晓红 ,赵永娜＊ ,邵晓霞 ,李顺英 ,张荣平
昆明医学院药学院 ,昆明 650031
摘　要:观察滇产粗根荨麻水提取部分对佐剂性关节炎(adjuvantarthritis, AA)大鼠腹腔巨噬细胞(peritoneal
macrophages, PMφ)分泌肿瘤坏死因子-α(tumornecrosisfactor-alpha, TNF-α)及前列腺素 E2(prostaglandinE2 ,
PGE2)的影响。建立大鼠佐剂性关节炎模型 , Ur水提取部分连续灌胃给药 14或 21 d后分次获取大鼠腹腔巨噬
细胞 , 脂多糖(lipopolysaccharide, LPS)诱导大鼠腹腔巨噬细胞 , 用酶联免疫吸附法检测培养上清液中 TNF-α及
PGE2水平。 AA大鼠腹腔巨噬细胞 TNF-α及 PGE2分泌较正常组升高 , Ur水提取部分(400, 200 mg/kg)对 LPS
诱导的 AA大鼠腹腔巨噬细胞分泌 TNF-α及 PGE2水平有明显抑制作用。滇产粗根荨麻水提取部分对佐剂性
关节炎的治疗作用可能与其抑制腹腔巨噬细胞分泌 TNF-α及 PGE
2
有关。
关键词:佐剂性关节炎;粗根荨麻;肿瘤坏死因子-α;前列腺素 E2
中图分类号:R285;Q946.91 文献标识码:A
TheEfectsofAqueousFractionofUrticamacrorrhiza
Hand-MazzonProductionofTNF-α, PGE2 ReleasefromPeritoneal
MacrophagesInducedbyLPSinAdjuvantArthritisRats
LIXiao-hong, ZHAOYong-na＊ , SHAOXiao-xia, LIShun-ying, ZHANGRong-ping
FacultyofPharmaceuticalSciences, KunmingMedicalCollege, Kunming650031 , China
Abstract:ToinvestigatetheefectsofaqueousfractionofUrticamacrorhizaHand-Mazz(Ur)onmodulatingtumornec-
rosisfactor-alpha(TNF-α)andprostaglandinE2(PGE2)productioninducedbylipopolysaccharide(LPS)inperitoneal
macrophagesinadjuvantarthritisratsandelucidatethepossiblemechanismsofanti-inflammatoryandantirheumatoid
effectsofUr, adjuvantarthritis(AA)ratwasusedasthemodel.ThePMφsamplesweretakenatdiferenttimeafter
medication.TNF-α, PGE2 levelsweremeasuredbyELISAmethod.ProductionofTNF-α, andPGE2 increasedinthecul-
turesupernatantofPMφinAAmodelrats.Ur(400 and200 mg/kg)couldinhibitTNF-αandPGE2 releaseinducedby
LPSfromPMφinAArats.Theanti-inflammatorymechanismsofUrinAAratsmightberelatedtoitsinhibitoryefects
onthelevelofTNF-αandPGE2 fromPMφinvivo.
Keywords:adjuvantarthritis;UrticamacrorrhizaHand-Mazz;tumornecrosisfactor-alpha;prostaglandinE2
Introduction
Urticamacrorrhizaisakindofherbmedicine.Several
speciesofUrticawasusedinthefolkmedicineinChi-
naasantipyretic, antinflammatoryandpainrelief.Urti-
cadioicapossessesanti-inflammatoryefectbyinhibi-
tingofNF-κBandantiproliferativeefectonhuman
cancercels[ 1] .Urticamacrorhizawasusedtorelease
painduetorheumaticconditionandhypertension.Urti-
camacrorrhizashowedanti-inflammatoryefectonear
edemainducedbydimethylbenzeneinmice.Uralso
couldrelievethecarageenininducedpawedemain
rats[ 2] , buttheefectsofUronactivitieslevelofcyto-
kines, tumornecrosisfactor-α(TNF-α)andPGE2 re-
leasedfromperitonealmacrophages(PMφ)remainun-
known.Theaimofthispaperistoinvestigatethepossi-
blemechanismofanti-rheumaticefectofUr.
DOI :10.16333/j.1001-6880.2008.04.018
Experimental
Plantmaterial
UrticamacrorrhizaHand-MazzwascolectedduringJu-
ly2005 inWudingcountryofYunnanProvinceChina
andauthenticatedbyProf.X.W.Li(KunmingInstitute
ofBotany, Kunming, China).Thewholegrass(150 g)
wasextractedwith70% ethanol, theextractwascon-
centratedtodrynessunderreducedpressure, andthe
residuewasdissolvedandsuspendinwaterandparti-
tionedwithpetroleumether, ethylacetateandn-butanol
successively.Theaqueouspartwasevaporatedtoget
theaqueousextractofUrticamacrorrhizaHand-Maz,
theresidue(9.83 g)wasgained.Themainextractis
amylose.
Chemicals
PrednisoneacetateTablet(Pre), ZhejiangXianjuMedi-
cineCo., China, 060904;Lipopolysaccharides(LPS, E
coil, 011:B4), Trypanblue(T-0776), Hank sbal-
ancedsalts(016K8317)andFreund scompleteadju-
vant(FCA)were from Sigma Co., RPMI-1640
(Nrg0016)medium, Bovineserum(Hyclonecalf),
NewZealandsourced(DQA0194)werefromHyclone
Co., Enzymeimmunoasayadliteranmdiagnosticlabo-
ratories(goatanti-ratprostaglandinE2)waspurchased
fromADLCo., TNF-αEnzymeimmunoassayadliter-
anmdiagnosticlaboratorieswaspurchasedfromJingmei
BiotechCo., China.MicrotiterplatereaderBIO-RD,
505, USA.
Animals
FiftySprague-Dawleyrats, weighing(180±20)g, were
providedbyAnimalCenterofKunmingMedicalCol-
lege.Eachgrouphas5 rats, and50 ratswereusedin
theexperiment.
BuildingAAratsmodel[ 3]
Eachratwasinjectedinthehindfootpadwith0.1 mL
ofFCA[ 3] .Sevendaysafterinjection, medicinewere
continuedtoadministratefor14or21 d.
Celculture[ 4]
Afteradministrationfor14 or21 d, 15 mLvolumeof
precoolingD-Hank sbalancedsaltssolutionwasused
todoucheabdominalcavityforseveraltimes.Thecels
werewashed2 timesinRPMI-1640 medium.Thecel
suspensionwasadjustedto2×106 /mL(Theactivities
ofcelswere>95% whichmeasuredwithTrypan
blue.)inRPMI-1640mediumcontaining10%serum,
anddispensedat200 μL/welin96-welplates.After
2hincubationat37 ℃ in5% CO2 , thenonadherent
celswereremovedbyrinsingwithRPMI-1640 medi-
um, andtheremainingcelswereculturedwithRPMI-
1640 medium 200 μLwhichhadbeenaddedLPS5
mg/Lfor24 h.Supernatantwascolected, thelevelsof
TNF-αandPGE2 weremeasuredbytheELISAassay.
Statisticalanalysis
Dataareexpressedasthex±s.Thestatisticalanalysis
wasperformedusingvarianceanalysis.Adiferencewas
consideredsignificationatP<0.05.
Results
DynamicefectsoftheaqueousofUrticamacrorrhi-
zaHand-MazzonTNF-αlevelsecretedfromPMφ
inAArats
ThelevelofTNF-αincreasedinAAmodel(P<0.01,
comparedwithnormalgroup).Ur400 mg/kgcouldin-
hibitLPS-inducedTNF-αreleaseafteroraladministra-
tionofUrfor14or21 d(P<0.01, comparedwithAA
group), Ur200 mg/kgcouldinhibitLPS-inducedTNF-
αreleaseafteroraladministrationofUrfor14 d(P<
0.05, comparedwithAAgroup, Table1).
Table1　DynamiceffectsoftheaqueousfractionfromUrti-
camacrorrhizaonTNF-αlevelsecretedfromPMφ
inAArats(x±s, n=5)
Group Dose(mg/kg) TNF-αlevel(μg/L)
14d 21d
AA - 51.69±19.95■■ 54.83±21.13■■
Pre 5 24.61±6.07＊＊ 28.15±6.25＊＊
Ur 400 29.02±8.91＊＊ 35.64±8.20＊＊
200 37.53±12.37＊ 45.23±13.33
N - 17.61±4.71＊＊ 21.26±5.72＊＊
　　Note:＊P<0.05;＊＊P<0.01comparedwithAAmodelgroup;■■
P<0.01comparedwithnormalgroup.
DynamicefectsoftheaqueousofUrticamacrorrhi-
zaHand-MazzonPGE2 levelsecretedfromPMφin
AArats
ThelevelofPGE2 increasedextremelyinAAmodel(P
<0.01, comparedwithnormalgroup).Ur400and200
615Vol.20　　　　　　　LIXiao-hong, etal:TheEfectsofAqueousFractionofUrticamacrorrhizaHand-MazzonProductionofTNF-α, PGE2 　
mg/kgcouldinhibittheproductionofPGE2 fromperi-
tonealmacrophagesinducedbyLPSinAAratsaftero-
raladministrationfor14 or21 d(P<0.01, compared
withAAgroup, Table2).
Table2　DynamiceffectsoftheaqueousfractionfromUrti-
camacrorrhizaonPGE2 levelsecretedfromPMφ
inAArats(x±s, n=5)
Group Dose(mg/kg)
PGE2 level(μg/L)
14d 21d
AA - 11.70±1.34■■ 11.78±1.51■■
Pre 5 6.07±0.56＊＊ 5.60±1.51＊＊
Ur 400 7.55±1.67＊＊ 6.88±0.79＊＊
200 6.52±2.09＊＊ 5.94±0.71＊＊
N - 4.52±1.46 ＊＊ 5.25±1.22＊＊
　　Note:＊P<0.05;＊＊P<0.01comparedwithmodelgroup;■■P<
0.01 comparedwithnormalgroup.
Discussion
Inthepresentexperiments, theAAratmodelwasused
toinvestigatethepossiblemechanismsofantirheumatic
efectsofUr.Thismodelproducedbysensitizingani-
malswithscinjectionsofadjuvant.InAAmodel, ar-
thritisdevelopswithin2 weeksandischaracterizedby
pawedemaandarthritisindexofinflammatorylesions.
Afterinjectionofadjuvantfor7d, oraladministrationof
Urfor14 or21 d, bothdose(200 , 400 mg/kg)could
inhibitthepawedemaandarthritisindexwhichcharac-
terizedthesecondarysymptom[ 5] .
Rheumatoidarthritis(RA), anautoimmunedisorderof
unknownetiology, ischaracterizedbychronicinflam-
mationofsynovialtissuesandinfiltrationoftheafected
jointsinbloodcels.Mononuclearcelsoftheperipher-
alblood, thesynovialcelsofthejoints, andmacropha-
gesareinactivestateintheRApatients.Thesecels
highlyexpressingIL-1β, PGE2 andTNF-α.IL-1β, TNF-
αistheinducingfactorsofCOX-2, iNOSandother
genes, whichhighexpresionwilstimulatethesynthe-
sisofPGE2 , NOinsynoviocyteandcartilagecel.These
cytokinesplayanimportantroleinthedevelopmentof
theacutesynovialinflammation, synovialfibrosisand
destructionoftheboneandcartilage[ 6, 7] .Anti-TNF-α
monocloneantibody, COX-2 selectiveinhibitorwere
usefulintreatmentofRA[ 8] .ExtractumUrticasedi-
oicasefoliorumcaninhibitthesecretionofTNF-αand
IL-1β inhumanwholebloodinducedbyLPSeitherin
vitroorinvivo[ 9] .Inpresentstudy, wefoundthatthe
levelsofPGE2 andTNF-αincreasedfromperitoneal
macrophages(PMφ)inducedbyLPSinadjuvantar-
thritisrats.Ur400 and200 mg/kgcouldinhibitthe
productionofTNF-αinducedbyLPSafteroraladmin-
istrationofUrfor14 d.ThelevelofTNF-αinducedby
LPSwasdecreasedbyoraladministrationofUrina
dosedependentmanner.TheproductionofPGE2 in-
ducedbyLPSinperitonealmacrophagesinAArats
couldbeinhibitedbyoraladministrationofUr(400
and200mg/kg)for14 or21d.Inourpreviousstudy,
wefoundthatUrhadanti-inflammatoryefect[ 10, 11] .
Themechanismofanti-inflammatoryefectofUrtica
macrorrhizaHand-Mazzprobablyrelatedtoitsinhibito-
ryefectsonTNF-αandPGE2 releaseinAArats.
References
1　RiehemannK, BehnkeB, Schuke-OsthoffK.Plantextracts
fromstingingnetle(Urticadioica), anantirheumaticreme-
dy, inhibittheproinflammatorytranscriptionfactorNF-κB.
FEBS, 1999, 442:89-94.
2　WangMY, WeiYF.Preliminarypharmacodynamiceffect
studyofthreenettlesdecoction.LishizhenMedMatRes,
2001, 12:676-677.
3　XuSY, BianRL, ChenX, etal.MethodologyinPharmacolog-
icalExperiment.Beijing:PeoplesMedicalPublishingHouse,
2002, 2:911-913.
4　ChenMZ, WeiW, ZhangH, etal.MethodologyinPharmaco-
logicalofImmunopotentiating.Beijing:People sMedical
PublishingHouse, 1991.1424.
5　LiXH, ZhaoYN, LiSY, etal.Efectsoftheaqueousfraction
fromUrticamacrorrhizaHand-Mazzontreatingadjuvantar-
thritisinrats.NatProdResDev, 2007(Suppl.), inpress.
6　HuangQC, ChenJF, ChenGX, etalEffectofsinomenineon
apotosisofsynovincytesinCIArats.ZhongguoLinchuang
Kangfu, 2002, (5):2541.
7　DayerJM.Theprocessofidentifyingandunderstandingcyto-
kines:frombasicstudiestotreationgrheumaticdiseases.Best
PractResClinRheumatol, 2004, 18:31-45.
8　SeitzM, LoetscherP, DewaldB, etal.Invivomodulationof
cytokineinhibitor, andprostaglandinEreleasefromblood
mononuclearcellandsynovialfibroblastsbyantirheumatic
drugs.TheJournalofRheumatology, 1997, 24:1471-1476.
(下转第 638页)
616 NatProdResDev　　　　　　　　　　　　　　　　　　　　 　Vol.20
13)78.7(C-14), 39.5(C-15), 84.9(C-16), 62.1(C-
17), 80.5(C-18), 53.8(C-19), 49.3(N-CH2 -CH3),
13.6(N-CH2-CH3), 167.0(OCOCH3), 21.8(OCOCH3),
166.3(CO-C6H5), 122.8(C-1′), 131.9(C-2′, 6′),
113.9(C-3′, 5′), 163.6(C-4′), 59.2, 58.9, 57.9,
56.2, (4×OCH3), 55.6(As-OCH3)。以上数据与文
献 [ 6]报道的粗茎乌碱甲 (crassicaulineA)基本一
致 。
化合物 6　无色针状晶体 , mp.136 ～ 137 ℃,与
β -谷甾醇标准品进行对照 ,混合熔点不下降;在多种
体系中进行薄层层析(TLC)检测 , Rf值一致 ,且显
色相同 ,故确定化合物 6为 β-谷甾醇(β-sitosterol)。
化合物 7　白色粉末 ,与 β-胡萝卜苷准品进行
对照 ,混合熔点不下降;在多种体系中进行薄层层析
(TLC)检测 , Rf值一致 ,且显色相同 ,故确定化合物
7为 β-胡萝卜苷(β-daucosterol)。
参考文献
1　中国植物志编辑委员会.FloraofChina(中国植物志).
Beijing:SciencePress, 1979, 27:281-282.
2　PeletierSW, DjarmatiZ.Carbon-13 nuclearmagneticreso-
nance:aconitine-typediterpenoidalkaloidsfrom Aconitum
andDelphiniumspecies.JAmChemSoc, 1976, 98:2626-
2636.
3　KonnoC, ShirasakaM, HikinoH.StructureofsenbusineA, B
andC, diterpenealkaloidsofAconitumcarmichaelirootsfrom
China.JNatProd, 1982, 45:128-133.
4　WangJL(王建莉), ZhouXL(周先礼), WangFP(王锋
鹏).ChemicalstudiesonthediterpenoidalkaloidsofDel-
phiniumgiraldil.NatProdResDev(天然产物研究与开
发), 2003, 6:498-501.
5　KhetwalKS, DesaiHK, JoshiBS, etal.Norditerpenoidalka-
loidsfromaerialpartsofAcomitumbalfouriistapf.Heterocy-
cles, 1994, 38:833-842.
6　WangFP, FangQC.AlkaloidsfromrootsofAconitumcrassi-
caue.PlantaMedica, 1981, 42:375-379.
(上接第 616页)
9　ObertreisB, RuttkowskiT, TeucherT, etal.Ex-vivoin-vitro
inhibitionoflipopolysaccharidestimulatedtumornecrosis
factor-αandinterleukin-1β secretioninhumanwholeblood
byex-tractumUrticaedioicaefoliorum.ArzneimForsch/Drug
Res, 1996, 46:389-394.
10 ZhaoYN, WantanaR, PisitB, etal.Studyonanti-inflamma-
tory, antinociceptiveoftraditionalmeddicinalplant:Urtica
macrorrhizaHand-Mazz.NatProdResDev, 2005, 17:69-71.
11 LiXH, ZhaoYN, LiSY, etal.Antinociceptiveandanti-in-
flammatoryefectsofdifferentfractionsofUrticamacrorrhiza
Hand-Mazzinexperimentalanimals.NatProdResDev,
2007, inpress.
638 天然产物研究与开发　　　　　　　　　　　　　　　　　　　　　 　Vol.20