Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 626038 12 pageshttpdxdoiorg1011552013626038

Research ArticleTriptolide Prevents Bone Destruction inthe Collagen-Induced Arthritis Model of Rheumatoid Arthritisby Targeting RANKLRANKOPG Signal Pathway

Chunfang Liu1 Yanqiong Zhang1 Xiangying Kong1 Liuluan Zhu2 Jian Pang3 Ying Xu1

Weiheng Chen4 Hongsheng Zhan3 Aiping Lu5 and Na Lin1

1 Department of Properties Theory of Traditional Chinese Medicine Institute of Chinese Materia MedicaChina Academy of Chinese Medical Sciences No 16 Nanxiaojie Dongzhimennei Beijing 100700 China

2 Institute of Infectious Diseases Beijing Ditan Hospital Capital Medical University Beijing 100015 China3 Shirsquos Center of Orthopedics and Traumatology Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese MedicineShanghai 201203 China

4Wangjing Hospital China Academy of Chinese Medical Sciences Beijing 100700 China5 Institute of Basic Research of Clinical Medicine China Academy of Chinese Medical Sciences Beijing 100700 China

Correspondence should be addressed to Na Lin linna888163com

Received 3 January 2013 Accepted 18 February 2013

Academic Editor Hyung-In Moon

Copyright copy 2013 Chunfang Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Focal bone destruction within inflamed joints is the most specific hallmark of rheumatoid arthritis (RA) Our previous studyindicated that the therapeutic efficiency of triptolide in RA may be due partially to its chondroprotective and anti-inflammatoryeffects However its roles in bone destruction are still unclear In this study our data firstly showed the therapeutic effectsof triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice Then by micro-CTquantification triptolide treatment significantly increased bone mineral density bone volume fraction and trabecular thicknessand decreased trabecular separation of inflamed joints Interestingly triptolide treatment could prevent the bone destruction byreducing the number of osteoclasts in inflamed joints reducing the expression of receptor activator of NF-120581B (RANK) ligand(RANKL) and RANK increasing the expression of osteoprotegerin (OPG) at both mRNA and protein levels and decreasingthe ratio of RANKL to OPG in sera and inflamed joints of CIA mice which were further confirmed in the coculture system ofhuman fibroblast-like synovial and peripheral blood mononuclear cells These findings offer the convincing evidence for the firsttime that triptolide may attenuate RA partially by preventing the bone destruction and inhibit osteoclast formation by regulatingRANKLRANKOPG signal pathway

1 Introduction

Rheumatoid arthritis (RA) represents a chronic autoim-mune disease characterized by the presence of inflamma-tory synovitis the predominance of proerosive mediatorsand the progressive destruction of cartilage and bone [1]Focal bone destruction within inflamed joints is the mostspecific hallmark of RA and leads to deformation laxityand functional disability [2] Osteoclasts the primary boneresorptive cells play an important role in bone destruction[3] It has been demonstrated that osteoclasts are mainlylocated in the synovial inflammatory tissue and the bone

destruction occurred in RA belongs to osteoclast-mediatedbone destruction that is regulated by the receptor activator ofnuclear factor-120581B (RANK) ligand (RANKL) [4] Both RANKand its ligand RANKL are crucial regulators of osteoclastdifferentiation Under the physiological condition RANKL isexpressed on osteoblasts and activated T cells [5] It triggersosteoclast maturation and bone resorption by binding withits receptor RANK on osteoclasts As a member of the tumornecrosis factor (TNF)-receptor family and a soluble decoyreceptor for RANKL osteoprotegerin (OPG) is normallyexpressed by osteoblasts and inhibits bone resorption by

2 Evidence-Based Complementary and Alternative Medicine

binding with RANKL which subsequently prevents RANKLbinding to RANK [6] Under the pathological conditionRANKLRANKOPG signal pathway plays a crucial rolein the process of bone destruction [7] RANKL inducesosteoclast-mediated bone destruction and OPG protectsagainst bone destruction by inhibiting the binding of RANKLwith its receptor RANK [8] Bone resorption is regulatedlocally by the relative levels of expression of RANKL andOPG Thus RANKLRANKOPG signal pathway has beenconsidered as a potential target for preventing systemic jointdestruction in RA patients

In the current treatments for RA there are three kindsof therapeutic agents disease modifying antirheumaticdrugs (DMARDs) such as methotrexate leflunomidehydroxychloroquine and glucocorticoids nonsteroidalanti-inflammatory drugs (NSAIDs) such as dichlorophenloxoprofen and nabumetone steroid and biological responsemodifiers which are all clinically used to relieve the severityof RA slow this disease progression and prevent thesubsequent joint damage [9 10] However the clinical useof these therapies has been limited because of their adverseeffects with a high frequency and high cost of treatmentExtracts of the herb TripterygiumWilfordii Hook f (TWHF)also known as ldquoLei Gong Tengrdquo are clinically used as one ofthe most common systemic treatments for (auto) immunedisorders including RA immune complex nephritis andsystemic lupus erythematosus [11 12] It has been extensivelyused for centuries in China because of its favorable cost-benefit ratio In 2009 Goldbach-Mansky et al [13] performedclinical trials to validate the disease-modifying effects ofextracts of TWHF on patients with RA The treatment withthis extract administered over 24 weeks may be both effectiveand safe in treating patients with active RA The rapidimprovement in function and pain and the profound effecton inflammation may make it an attractive and affordablealternative to currently available agents As an activecompound of TWHF triptolide is immunosuppressivecartilage protective and anti-inflammatory in vivo andeffective on both humans and animals inflicted by a rangeof inflammatory and autoimmune diseases such as RA[14ndash16] A large number of recent studies has found itsmechanisms on the treatment of RA [17ndash19] However theroles of triptolide in bone destruction which is a majorproblem of RA are still unclear

Collagen-induced arthritis (CIA) by the immunizationof DAB1 mice with type II collagen in complete Freundrsquosadjuvant is widely used for the study of autoimmune arthritis[20] This model exhibits joint swelling synovitis periostealnew bone formation articular bone erosion and osteopenia[21] Because this model mimics many of the clinical andpathological features of human RA we here investigated theprotective effect of triptolide on bone destruction in CIAmice

2 Materials and Methods

The study was approved by the Research Ethics Committeeof Institute of Chinese Materia Medica China Academy of

Chinese Medical Sciences Beijing China All animals weretreated in accordance with the guidelines and regulations forthe use and care of animals of the Center for LaboratoryAnimal Care China Academy of Chinese Medical Sciences

21 Animals Seventy-two male DBA1 mice (6sim8 weeksold) were purchased from Charles River Laboratory JapanKanagawa Japan All mice were maintained in a roomequippedwith an air-filtering system and the cages andwaterwere sterilized

22 Induction of CIA CIA was induced as our previouslyreported study [22 23] Briefly bovine type II collagen(Chondrex Redmond WA USA) was dissolved in 01Macetic acid overnight at 4∘C This was emulsified in an equalvolume of complete Freundrsquos adjuvant (Chondrex RedmondWA USA) The mice were immunized intradermally atthe base of the tail with 100120583L of emulsion containing100 120583g of type II collagen On day 21 mice were boostedintraperitoneally with 100 120583g type II collagen dissolved inphosphate buffered saline (PBS)

23 Treatment and Groups Triptolide (purity gt 9998) waskindly provided by Professor Sui Lin (Fujian Institute ofMedical Sciences Fuzhou China) and this is commerciallyavailable from Alexix Biochemicals (San Diego CA USA)Methotrexate was purchased from Sigma (St Louis MOUSA) Bothwere dissolved in 005DMSO (Sigma St LouisMOUSA)The routes of triptolide andmethotrexate deliverywere both oral administration Treatment was given dailyfor a period of 21 days The dosage selection for triptolide[8ndash32 120583g(kgsdotday)] was according to our previous study [22]and corresponded to 0625ndash25 of LD50 for triptolide(1278mgkg)

Seventy-two DBA1 mice were divided into 6 groupswith the equal number (119899 = 12) normal control group(Normal) CIA model control group (Vehicle) CIA micetreated with 8 120583g(kgsdotday) triptolide (Trip 8) 16 120583g(kgsdotday)triptolide (Trip 16) 32 120583g(kgsdotday) triptolide (Trip 32) and01mgkg methotrexate (MTX)

24 Severity Assessment of Arthritis Mice were observedonce every 2-3 days after primary immunization Arthritisseverity was evaluated by arthritis index arthritis incidenceand percentage of arthritic limbs which were performed bytwo independent blinded observers All four limbs of themice were evaluated and scored from 0 to 4 according tothe following scale 0 normal 1 detectable arthritis witherythema at least some digits 2 significant swelling andredness 3 severe swelling and redness from joint to digit and4 maximal swelling with ankylosis The total score was thecumulative value for the four limbs with a maximum of 16for each mouse Arthritis was considered to be present if thescore for a paw was gt2 The incidence of arthritis is definedas the percentage of animals within each group exhibiting anysign of disease regardless of severity In addition the numberof arthritic limbs of individual mice were counted and addedto represent the number of arthritic limbs in a group The

Evidence-Based Complementary and Alternative Medicine 3

percentage of arthritic limbs in a group was calculated as thefollowing formula

percentage of arthritic limbs

=

number of arthritic limbs in a groupnumber of all limbs in a group

times 100(1)

25 Change in Body Weight after Onset of Arthritis Becausethe loss of body mass is associated with aggressive progres-sion of RA in clinics [24 25] we detected the changes in bodyweight after the onset of arthritis in order to evaluate the sideeffect of triptolide in the treatment of RAThe change in bodyweight () of each individual CIA mice after the onset ofarthritis was calculated as the following formula

change in bodyweight

= (

bodyweight (day 22 of arthritis)bodyweight (day 1 of arthritis)

minus

bodyweight (day 1 of arthritis)bodyweight (day 1 of arthritis)

) times 100

(2)

26 Histopathology and TRAP Staining Mice were sacrificedby cervical dislocation on day 22 of arthritis The righthind limbs including the paws ankles and knees weredissected fixed immediately for 2 h in 4 paraformaldehydedecalcified in 10 EDTA for up to 1 month at 4∘C andembedded in paraffin Tissue sections (5120583m) were mountedon common slides for staining with hematoxylin and eosinAll sections were randomized and evaluated by two trainedobservers who were blinded to the treatment groups and thearthritis severity of each mouse The data were expressed asmean bone destruction scores which were based on a scaleof 0ndash3 as our previous study [22] Minor differences betweenobservers were resolved by mutual agreement To identifyosteoclasts sections were stained for tartrate-resistant acidphosphatase (TRAP) using an acid phosphatase kit (SigmaSt LouisMOUSA) TRAP-positivemultinucleated cells thatcontained more than 3 nuclei were identified as osteoclastsand these were counted by light microscopy

27 Micro-CT Imaging Three-dimensional reconstructionand sagittal images of the right knee and ankle joints wereobtained by microfocal computed tomography (micro-CTExplore Locus SP GE USA) at the day 22 of arthritis Brieflyafter the mice in different groups being killed using etheranesthesia the hind limbs were removed and fixed in 4paraformaldehyde for 2 h The samples were scaned withmicro-CT Using the constructed sagittal image from micro-CT scanning data histomorphometric analysis of the proxi-mal tibial epiphysis exclude the cortical bone was performedin the measurement area facing the articular cavity from thebegining of growth plate Bone volume bonemineral density(BMD) trabecular thickness and trabecular separation at

the tibial spongiosa were measured using an image analyzingsystem (Microview ABA22 software GE USA)

28 Osteoclast Formation in Coculture System of SynovialFibroblasts and Peripheral Blood Mononuclear Cells Thecoculture system of human fibroblast-like synovial cells(HFLS) and peripheral blood mononuclear cells (PBMCs)were constructed according to the protocol of Nakano etal [26] Briefly HFLS derived from RA patients aftersynovectomy were purchased from Cell Applications (SanDiego CA USA) and were routinely cultured in synoviocytegrowth medium (Cell Applications) Blood was collectedfrom healthy volunteers and their PBMCs were isolated bycentrifugation over FicollPaque at 400 g for 30 minutesAfter isolation PBMCs (2 times 105 cellswell) were resus-pended in 120572-minimum essential medium containing 10fetal calf serum (FCS) and 50 ngmL of macrophage colony-stimulating factor (M-CSF PeproTech Inc Rocky Hill NJUSA) and then seeded in 48-well tissue culture plates (CostarCorning Inc Corning NY USA) Three days later adherentcells were used for subsequent cocultures with HFLS whichwere then added into 48-well tissue culture plates (2 times104 cellswell) with PBMCs and cocultured for 9 days in

MEM containing 10 FCS 50 ngmL of M-CSF 10minus7M 125-dihydroxyvitamin D

3(125[OH]

2D3) and triptolide (028

28 and 28 nmolL resp) After that some dishes were usedfor TRAP staining as aforementioned in Section 26 TRAP-positive multinucleated cells that contained more than 3nuclei were identified as osteoclasts and these were countedby light microscopy

29 RNA Isolation and Real-Time PCR The left hind paws(including the paw and ankle) were dissected from mice onday 22 of arthritis snap-frozen in liquid nitrogen ground intopowder and homogenized This procedure was done underRNase-free conditionsTheRNA isolation and real-time PCRassay were carried out following the protocol of our previousstudy [22 23] Briefly total RNA was extracted with TRIzolreagent (Invitrogen Carlsbad CA USA) from the tissuehomogenates according to the manufacturerrsquos instructionsThe total RNA (1 120583g) was reverse transcribed to cDNA usingthe QuantiTect Reverse Transcription Kit (QIAGEN KKTokyo Japan) according to the instructions manual Thespecific transcripts were quantified by quantitative real-timePCR using QuantiTect SYBR Green PCR Kit (QIAGEN KKTokyo Japan) and analyzed with ABI 7500 real-time PCRsystem (Applied Biosystems USA) Gene-specific primerswere used for RANKL (51015840-CAG CAT CGC TCT GTT CCTGTA-31015840 as forward and 51015840-CTG CGT TTT CAT GGA GTCTCA-31015840 as reverse) RANK (51015840-GCC CAG TCT CAT CGTTCT GC-31015840 as forward and 51015840-GCA AGC ATC ATT GACCCA ATT C-31015840 as reverse) OPG (51015840-ACC CAG AAA CTGGTC ATC AGC-31015840 as forward and 51015840-CTG CAA TAC ACACACTCATCACT-31015840 as reverse) and 120573-actin (51015840-GGC TGTATT CCC CTC CAT CG-31015840 as forward and 51015840-CCA GTTGGTAACAATGCCATGT-31015840 as reverse)ThemRNA levelsof RANKL RANK and OPG were normalized to 120573-actinmRNA level PCRwas performed as 40 cycles at 94∘C for 15 s

4 Evidence-Based Complementary and Alternative Medicine

55∘C for 30 s and 72∘C for 30 sThe relativemRNAexpressionwas calculated with comparative 119862

119879method

210 Immunohistochemical Staining Paraffin sections (5120583m)of tissue from the knee and ankle joints were mounted onpoly-L-lysine-coated slides Immunolocalizations of RANKLand OPG in the joints were carried out with commer-cial Polink-2 plus Polymer HRP Detection System ForGoat Primary Antibody kits (Golden Bridge InternationalInc Mukilteo WA USA) according to the manufacturerrsquosinstructions The paraffin sections were dewaxed by routinemethod and incubated for 10min with 3 H

2O2 Each

section was incubated with normal goat serum for 20minat room temperature and then with primary antibodiesagainst mouse RANKL (goat polyclonal antibody dilution1 50 Santa Cruz Biotechnology Inc Santa Cruz CA USA)and OPG (goat polyclonal antibody dilution 1 50 SantaCruz Biotechnology Inc SantaCruz CAUSA) respectivelyovernight at 4∘C After incubation with polymer helper for20min at 37∘C sections were reacted with poly-HRP anti-goat IgG for 20min at 37∘C The sections were then stainedwith 33-diaminobenzidine (Sigma St Louis MO USA) andcounterstained with hematoxylin For the control stainingPBS was used instead of the primary antibodies

Specimens were examined using a Leica image analyzerand analyzed by computer image analysis (LeicaMicrosystemWetzlar Gmbh Wetzlar Germany) in a blinded mannerTo localize and identify areas with positively stained cellsten digital images per specimen of synovium from a kneeor ankle joint were recorded and quantitative analysis wasperformed according to the color cell separation The resultsare expressed as the mean region of interest representing thepercentage of area covered with positively stained cells perimage at a magnification of times400

211 Enzyme-Linked Immunosorbant Assay Sera from themice on day 22 of arthritis and conditioned media fromHFLS cultured in the presence of triptolide (028 28 and28 nmolL resp) for 3 d 7 d 10 d 14 d 17 d and 21 dwas obtained and stored at minus80∘C until use The amounts ofRANKL and OPG in serum and OPG in supertant of HFLSwere detected by ELISA assay (RampD system MinneapolisMN USA) according to the manufacturerrsquos protocol andabsorbance was measured at 450 nm All experiments weredone in triplicate

212 Western Blot Analysis Isolated PBMCs(3 times 106 cellswell) or HFLS (105 cellswell) were cultured in6-well plates for 3 weeks in the presence of triptolide (02828 and 28 nmolL resp) The cells were washed twice withPBS and treated with lysis buffer (10mM HEPES-KOH [pH78] 15mM KCl 2MmMgCl

2 1 Igepal 2120583gmL leupeptin

and 1mM phenylmethylsulfonyl fluoride) The protein wasobstained to detect the levels of RNAKL in HFLS and RNAKin PBMCs by Western blot The Western blot protocol andsemiquantitative analysis were carried out following theprotocol of our previous study [23] The following antibodieswere used RANKL antibody (goat polyclonal antibody

dilution 1 200 Santa Cruz Biotechnology Inc Santa CruzCA USA) RANK antibody (goat polyclonal antibodydilution 1 200 Santa Cruz Biotechnology Inc SantaCruz CA USA) and GAPDH antibody (internal controlrabbit polyclonal antibody dilution 1 200 Santa CruzBiotechnology Inc Santa Cruz CA USA) All experimentswere done in triplicate

213 Statistical Analysis The software of SPSS version 130for Windows (SPSS Inc Chicago IL USA) and SAS 91(SAS Institute Cary NC) was used for statistical analysisContinuous variables were expressed as 119883 plusmn 119904 Arthritisincidence and percentage of arthritic limbs were analyzedby a chi-square test Arthritis index pathological scoresand number of TRAP positive cells were analyzed withnonparametric statistics (Kruskal-Wallis test) Other datawere analyzed by one-way ANOVA followed by LSD testDifferences were considered statistically significant when 119875was less than 005

3 Results

31 Effects of Triptolide on Severity of Arthritis and ArthritisProgression To investigate the effect of triptolide on arthritisthe CIAmodel inDBA1mice was used Although the diseasemanifested itself on different days after immunization wedid not observe a relation between clinical response andtime of onset of disease Consistent with the previous studies[22 27] oral administration of triptolide once a day startedat the beginning of the fist rheumatoid arthritis signs such aserythema andor oedema in joint(s) of CIAmice and continuedfor 21 days As shown in Figure 1(a) macroscopic evidence ofarthritis such as erythema or swelling wasmarkedly observedin vehicle-treated CIA mice while a dose of 32 120583g(kgsdotday)triptolide significantly attenuated arthritis severity in CIAmice Additionally the mean arthritis index (all 119875 lt 005Figure 1(b)) arthritis incidence (all119875 lt 005 Figure 1(c)) andthe percentage of arthritis limbs (all 119875 lt 005 Figure 1(d))in triptolide-treated mice were significantly lower than thosein methotrexate-treated and vehicle-treated CIA mice with adose-dependent manner In the groups receiving triptolidewith doses of 16sim32 120583g(kgsdotday) the rate of incidence andthe percentage of arthritis limbs weremarkedly reduced fromday 7 of arthritis Remarkably triptolide treatment couldeffectively suppress the loss of body weight of CIA mice (forTrip 16 group versus Vehicle 119875 lt 005 for Trip 32 groupversus Vehicle 119875 lt 005 Figure 1(e)) but methotrexatetreatment did not have this effect (Figure 1(e))

32 Triptolide Prevents Bone Destruction in CIAMice Micro-CT scan was performed to validate the efficiency of triptolidein CIA mice Figures 2(a) 2(b) and 3(a) respectivelyshowed the three-dimensional reconstructed bones of kneejoints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday)triptolide markedly reduced the extent of joint destructionin triptolide-treated CIA mice In addition four parametersincluding bone volume BMD trabecular thickness and

Evidence-Based Complementary and Alternative Medicine 5

Vehicle Trip 32 120583gkg

(a)

lowastlowast

lowastlowast

0

2

4

6

8

10

12

14

16

7 10 13 16 19 22

Art

hriti

s ind

ex

Day of arthritis

lowast

+ +

+

+

+lowast

lowast

1 4

lowast + ⋆

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(b)

lowast lowast

lowast

lowast

lowastlowast

lowast

++

lowast

lowast

lowast

lowast

++

⋆

lowast

lowast

lowast0

20

40

60

80

100

120

1 4 7 10 13 16 19 22

Art

hriti

s inc

iden

ce (

)

Day of arthritis

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(c)

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

lowast

lowastlowast lowast

+

lowast

lowast

lowast

lowastlowast

+ + ++

⋆

lowast

lowast

lowast

0

20

40

60

80

100

1 4 7 10 13 16 19 22

Art

hriti

s lim

b (

)

Day of arthritis

(d)

Normal

Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

minus15

minus10

minus5

0

5

10

15

20

Chan

ge in

bod

ywei

ght (

)

lowast

$lowast$

(e)

Figure 1 Effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice Mice were orallyadministered triptolide (Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onsetof the clinical symptoms of arthritis At the end of the experiment the arthritis index arthritis incidence the percentage of arthritic limbsand changes in bodyweight were evaluated Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 005 in comparison with the normalcontrol lowast119875 lt 005 +119875 lt 001 and lowast119875 lt 0001 in comparison with the vehicle control $119875 lt 005 in comparison with methotrexate-treatedCIA mice

6 Evidence-Based Complementary and Alternative Medicine

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

(b)

Figure 2 Micro-CT scan validates efficiency of triptolide in collagen-induced arthritis (CIA) mice Mice were orally administered triptolide(Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptomsof arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice (a) and (b)respectively showed the three-dimensional reconstructed bones of ankle and knee joints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treatedCIA mice

trabecular separation of inflamed joints in different groupswere detected to quantify the extent of joint destructionAs shown in Figure 3(b) compared with vehicle-treatedCIA mice doses of 8sim32120583g(kgsdotday) triptolide significantlyincreased BMD (all 119875 lt 005) bone volume fraction (all119875 lt 001) and trabecular thickness of inflamed joints (all119875 lt 005) and decreased trabecular separation (for Trip 16group versus Vehicle 119875 lt 005 for Trip 32 group versusVehicle 119875 lt 0001) suggesting a protective role of triptolideon volume and quality of preserved trabecular bone despitejoint inflammation Notably dose of 16 or 32 120583g(kgsdotday)triptolide could alsomore effectively change these parametersthan 01mgkgmethotrexate could (all119875 lt 005 Figure 3(b))

Histopathological evaluation of knee joint sections ofvehicle-treated CIA mice revealed inflammatory cell infil-tration synovial hyperplasia and partial bone destructionIn contrast oral administration of triptolide could distinctlyreduce the extent of inflammatory cell infiltration and bonedestruction (Figure 4(a)) To elucidate the effects of trip-tolide treatment on bone destruction at the histologic levelinflamed joints were scored with semiquantitative gradingscales As shown in Figure 4(b) the bone destruction scores

in triptolide-treated CIA mice were significantly decreasedwith a dose-dependent tendency in comparison with vehicle-treated CIA mice (for Trip 16 group versus vehicle group119875 lt 005 for Trip 32 group versus vehicle group 119875 lt 001)MTX also reduced significantly the bone destruction scoresof inflamed joints compared with vehicle-treated CIA mice(119875 lt 005 Figure 4(b)) although this value remained higherthan those for triptolide-treated groups

To confirm the effect of triptolide on the number ofosteoclasts knee joint sections were stained with TRAPOnly TRAP-positive multinucleated cells located at the bonesurfacewithin the bone destructionwere considered as osteo-clasts (Figure 4(a)) Comparedwith vehicle-treatedCIAmicethe number of osteoclasts in the areas of bone destructionwassignificantly decreased in triptolide-treatedmice with a dose-dependent tendency (all 119875 lt 001 Figure 4(c)) Methotrexatealso reduced significantly the number of osteoclasts in theareas of bone destruction compared with vehicle control(119875 lt 005 Figure 4(c)) although this value remained higherthan those for triptolide-treated groups (all 119875 lt 0001Figure 4(c))

Evidence-Based Complementary and Alternative Medicine 7

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

lowast

+lowast

++

0100200300400500600700

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

BMD

(mg

cc)

120583gkg

+

01020304050607

BVF

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

0

lowast

+++

lowast

0001002003004005006007

TbTh

(m

m)

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowast + + +

0

004

008

012

016

TbS

p (m

m)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowast

(b)

Figure 3 Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice Mice wereorally administered triptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of theonset of the clinical symptoms of arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolidein CIAmice (a)The two-dimensional reconstructed bones of knee joints in different groups Compared with vehicle-treated CIAmice doseof 32120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIAmice (b)The values of four parametersincluding bone volume bone mineral density (BMD) trabecular thickness (TbTh) and trabecular separation (TbSp) of proximal end of thetibia facing the articular cavity in different groups Compared with vehicle-treated andmethotrexate-treated CIAmice triptolide significantlyincreased BMD bone volume fraction (BVF) and TbTh of inflamed joints and decreased TbSp of inflamed joints Data are represented asthe mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normal control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison withthe vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 in comparison with methotrexate-treated CIA mice

33 Triptolide Inhibits Osteoclast Differentiation by TargetingRANKLRANKOPG Signal Pathway To obtain insights intothemechanisms of the inhibitory effects of triptolide on bonedestruction in inflamed joints of CIA mice the expressionof RANKL RANK and OPG at mRNA and protein levelsin inflamed joints were respectively detected by quantitativereal-time RT-PCR (Figure 5(a)) and immunohistochemistry(Figure 5(b)) and the serum levels of RANKL and OPG pro-teins were detected by ELISA assay (Figure 5(c)) Comparedwith vehicle-treated CIA mice doses of 8sim32 120583g(kgsdotday)

triptolide significantly reduced the expression of RANKL (all119875 lt 0001 only except for Trip 8 group at protein level injoints Figure 5) and RANK (all 119875 lt 005 Figure 5(a)) andenhanced the expression of OPG (all 119875 lt 0001 only exceptfor Trip 8 group at protein level in joints and sera Figure 5)More interestingly triptolide treatments markedly reducedthe ratio of RANKL to OPG in the sera and inflamed jointsof CIA mice with a dose-dependent tendency (all 119875 lt 001Figure 5) Methotrexate also reduced significantly the ratioof RANKL to OPG in the sera and inflamed joints of CIA

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

50 120583m

5 120583m

(a)

lowastlowast

0

02

04

06

08

1

12

14

Bone

des

truc

tion

scor

e

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

(b)

+ ++

lowast

0

10

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Num

ber o

f oste

ocla

st

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

lowastlowastlowast lowastlowastlowast

(c)

Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

binding with RANKL which subsequently prevents RANKLbinding to RANK [6] Under the pathological conditionRANKLRANKOPG signal pathway plays a crucial rolein the process of bone destruction [7] RANKL inducesosteoclast-mediated bone destruction and OPG protectsagainst bone destruction by inhibiting the binding of RANKLwith its receptor RANK [8] Bone resorption is regulatedlocally by the relative levels of expression of RANKL andOPG Thus RANKLRANKOPG signal pathway has beenconsidered as a potential target for preventing systemic jointdestruction in RA patients

In the current treatments for RA there are three kindsof therapeutic agents disease modifying antirheumaticdrugs (DMARDs) such as methotrexate leflunomidehydroxychloroquine and glucocorticoids nonsteroidalanti-inflammatory drugs (NSAIDs) such as dichlorophenloxoprofen and nabumetone steroid and biological responsemodifiers which are all clinically used to relieve the severityof RA slow this disease progression and prevent thesubsequent joint damage [9 10] However the clinical useof these therapies has been limited because of their adverseeffects with a high frequency and high cost of treatmentExtracts of the herb TripterygiumWilfordii Hook f (TWHF)also known as ldquoLei Gong Tengrdquo are clinically used as one ofthe most common systemic treatments for (auto) immunedisorders including RA immune complex nephritis andsystemic lupus erythematosus [11 12] It has been extensivelyused for centuries in China because of its favorable cost-benefit ratio In 2009 Goldbach-Mansky et al [13] performedclinical trials to validate the disease-modifying effects ofextracts of TWHF on patients with RA The treatment withthis extract administered over 24 weeks may be both effectiveand safe in treating patients with active RA The rapidimprovement in function and pain and the profound effecton inflammation may make it an attractive and affordablealternative to currently available agents As an activecompound of TWHF triptolide is immunosuppressivecartilage protective and anti-inflammatory in vivo andeffective on both humans and animals inflicted by a rangeof inflammatory and autoimmune diseases such as RA[14ndash16] A large number of recent studies has found itsmechanisms on the treatment of RA [17ndash19] However theroles of triptolide in bone destruction which is a majorproblem of RA are still unclear

Collagen-induced arthritis (CIA) by the immunizationof DAB1 mice with type II collagen in complete Freundrsquosadjuvant is widely used for the study of autoimmune arthritis[20] This model exhibits joint swelling synovitis periostealnew bone formation articular bone erosion and osteopenia[21] Because this model mimics many of the clinical andpathological features of human RA we here investigated theprotective effect of triptolide on bone destruction in CIAmice

2 Materials and Methods

The study was approved by the Research Ethics Committeeof Institute of Chinese Materia Medica China Academy of

Chinese Medical Sciences Beijing China All animals weretreated in accordance with the guidelines and regulations forthe use and care of animals of the Center for LaboratoryAnimal Care China Academy of Chinese Medical Sciences

21 Animals Seventy-two male DBA1 mice (6sim8 weeksold) were purchased from Charles River Laboratory JapanKanagawa Japan All mice were maintained in a roomequippedwith an air-filtering system and the cages andwaterwere sterilized

22 Induction of CIA CIA was induced as our previouslyreported study [22 23] Briefly bovine type II collagen(Chondrex Redmond WA USA) was dissolved in 01Macetic acid overnight at 4∘C This was emulsified in an equalvolume of complete Freundrsquos adjuvant (Chondrex RedmondWA USA) The mice were immunized intradermally atthe base of the tail with 100120583L of emulsion containing100 120583g of type II collagen On day 21 mice were boostedintraperitoneally with 100 120583g type II collagen dissolved inphosphate buffered saline (PBS)

23 Treatment and Groups Triptolide (purity gt 9998) waskindly provided by Professor Sui Lin (Fujian Institute ofMedical Sciences Fuzhou China) and this is commerciallyavailable from Alexix Biochemicals (San Diego CA USA)Methotrexate was purchased from Sigma (St Louis MOUSA) Bothwere dissolved in 005DMSO (Sigma St LouisMOUSA)The routes of triptolide andmethotrexate deliverywere both oral administration Treatment was given dailyfor a period of 21 days The dosage selection for triptolide[8ndash32 120583g(kgsdotday)] was according to our previous study [22]and corresponded to 0625ndash25 of LD50 for triptolide(1278mgkg)

Seventy-two DBA1 mice were divided into 6 groupswith the equal number (119899 = 12) normal control group(Normal) CIA model control group (Vehicle) CIA micetreated with 8 120583g(kgsdotday) triptolide (Trip 8) 16 120583g(kgsdotday)triptolide (Trip 16) 32 120583g(kgsdotday) triptolide (Trip 32) and01mgkg methotrexate (MTX)

24 Severity Assessment of Arthritis Mice were observedonce every 2-3 days after primary immunization Arthritisseverity was evaluated by arthritis index arthritis incidenceand percentage of arthritic limbs which were performed bytwo independent blinded observers All four limbs of themice were evaluated and scored from 0 to 4 according tothe following scale 0 normal 1 detectable arthritis witherythema at least some digits 2 significant swelling andredness 3 severe swelling and redness from joint to digit and4 maximal swelling with ankylosis The total score was thecumulative value for the four limbs with a maximum of 16for each mouse Arthritis was considered to be present if thescore for a paw was gt2 The incidence of arthritis is definedas the percentage of animals within each group exhibiting anysign of disease regardless of severity In addition the numberof arthritic limbs of individual mice were counted and addedto represent the number of arthritic limbs in a group The

Evidence-Based Complementary and Alternative Medicine 3

percentage of arthritic limbs in a group was calculated as thefollowing formula

percentage of arthritic limbs

=

number of arthritic limbs in a groupnumber of all limbs in a group

times 100(1)

25 Change in Body Weight after Onset of Arthritis Becausethe loss of body mass is associated with aggressive progres-sion of RA in clinics [24 25] we detected the changes in bodyweight after the onset of arthritis in order to evaluate the sideeffect of triptolide in the treatment of RAThe change in bodyweight () of each individual CIA mice after the onset ofarthritis was calculated as the following formula

change in bodyweight

= (

bodyweight (day 22 of arthritis)bodyweight (day 1 of arthritis)

minus

bodyweight (day 1 of arthritis)bodyweight (day 1 of arthritis)

) times 100

(2)

26 Histopathology and TRAP Staining Mice were sacrificedby cervical dislocation on day 22 of arthritis The righthind limbs including the paws ankles and knees weredissected fixed immediately for 2 h in 4 paraformaldehydedecalcified in 10 EDTA for up to 1 month at 4∘C andembedded in paraffin Tissue sections (5120583m) were mountedon common slides for staining with hematoxylin and eosinAll sections were randomized and evaluated by two trainedobservers who were blinded to the treatment groups and thearthritis severity of each mouse The data were expressed asmean bone destruction scores which were based on a scaleof 0ndash3 as our previous study [22] Minor differences betweenobservers were resolved by mutual agreement To identifyosteoclasts sections were stained for tartrate-resistant acidphosphatase (TRAP) using an acid phosphatase kit (SigmaSt LouisMOUSA) TRAP-positivemultinucleated cells thatcontained more than 3 nuclei were identified as osteoclastsand these were counted by light microscopy

27 Micro-CT Imaging Three-dimensional reconstructionand sagittal images of the right knee and ankle joints wereobtained by microfocal computed tomography (micro-CTExplore Locus SP GE USA) at the day 22 of arthritis Brieflyafter the mice in different groups being killed using etheranesthesia the hind limbs were removed and fixed in 4paraformaldehyde for 2 h The samples were scaned withmicro-CT Using the constructed sagittal image from micro-CT scanning data histomorphometric analysis of the proxi-mal tibial epiphysis exclude the cortical bone was performedin the measurement area facing the articular cavity from thebegining of growth plate Bone volume bonemineral density(BMD) trabecular thickness and trabecular separation at

the tibial spongiosa were measured using an image analyzingsystem (Microview ABA22 software GE USA)

28 Osteoclast Formation in Coculture System of SynovialFibroblasts and Peripheral Blood Mononuclear Cells Thecoculture system of human fibroblast-like synovial cells(HFLS) and peripheral blood mononuclear cells (PBMCs)were constructed according to the protocol of Nakano etal [26] Briefly HFLS derived from RA patients aftersynovectomy were purchased from Cell Applications (SanDiego CA USA) and were routinely cultured in synoviocytegrowth medium (Cell Applications) Blood was collectedfrom healthy volunteers and their PBMCs were isolated bycentrifugation over FicollPaque at 400 g for 30 minutesAfter isolation PBMCs (2 times 105 cellswell) were resus-pended in 120572-minimum essential medium containing 10fetal calf serum (FCS) and 50 ngmL of macrophage colony-stimulating factor (M-CSF PeproTech Inc Rocky Hill NJUSA) and then seeded in 48-well tissue culture plates (CostarCorning Inc Corning NY USA) Three days later adherentcells were used for subsequent cocultures with HFLS whichwere then added into 48-well tissue culture plates (2 times104 cellswell) with PBMCs and cocultured for 9 days in

MEM containing 10 FCS 50 ngmL of M-CSF 10minus7M 125-dihydroxyvitamin D

3(125[OH]

2D3) and triptolide (028

28 and 28 nmolL resp) After that some dishes were usedfor TRAP staining as aforementioned in Section 26 TRAP-positive multinucleated cells that contained more than 3nuclei were identified as osteoclasts and these were countedby light microscopy

29 RNA Isolation and Real-Time PCR The left hind paws(including the paw and ankle) were dissected from mice onday 22 of arthritis snap-frozen in liquid nitrogen ground intopowder and homogenized This procedure was done underRNase-free conditionsTheRNA isolation and real-time PCRassay were carried out following the protocol of our previousstudy [22 23] Briefly total RNA was extracted with TRIzolreagent (Invitrogen Carlsbad CA USA) from the tissuehomogenates according to the manufacturerrsquos instructionsThe total RNA (1 120583g) was reverse transcribed to cDNA usingthe QuantiTect Reverse Transcription Kit (QIAGEN KKTokyo Japan) according to the instructions manual Thespecific transcripts were quantified by quantitative real-timePCR using QuantiTect SYBR Green PCR Kit (QIAGEN KKTokyo Japan) and analyzed with ABI 7500 real-time PCRsystem (Applied Biosystems USA) Gene-specific primerswere used for RANKL (51015840-CAG CAT CGC TCT GTT CCTGTA-31015840 as forward and 51015840-CTG CGT TTT CAT GGA GTCTCA-31015840 as reverse) RANK (51015840-GCC CAG TCT CAT CGTTCT GC-31015840 as forward and 51015840-GCA AGC ATC ATT GACCCA ATT C-31015840 as reverse) OPG (51015840-ACC CAG AAA CTGGTC ATC AGC-31015840 as forward and 51015840-CTG CAA TAC ACACACTCATCACT-31015840 as reverse) and 120573-actin (51015840-GGC TGTATT CCC CTC CAT CG-31015840 as forward and 51015840-CCA GTTGGTAACAATGCCATGT-31015840 as reverse)ThemRNA levelsof RANKL RANK and OPG were normalized to 120573-actinmRNA level PCRwas performed as 40 cycles at 94∘C for 15 s

4 Evidence-Based Complementary and Alternative Medicine

55∘C for 30 s and 72∘C for 30 sThe relativemRNAexpressionwas calculated with comparative 119862

119879method

210 Immunohistochemical Staining Paraffin sections (5120583m)of tissue from the knee and ankle joints were mounted onpoly-L-lysine-coated slides Immunolocalizations of RANKLand OPG in the joints were carried out with commer-cial Polink-2 plus Polymer HRP Detection System ForGoat Primary Antibody kits (Golden Bridge InternationalInc Mukilteo WA USA) according to the manufacturerrsquosinstructions The paraffin sections were dewaxed by routinemethod and incubated for 10min with 3 H

2O2 Each

section was incubated with normal goat serum for 20minat room temperature and then with primary antibodiesagainst mouse RANKL (goat polyclonal antibody dilution1 50 Santa Cruz Biotechnology Inc Santa Cruz CA USA)and OPG (goat polyclonal antibody dilution 1 50 SantaCruz Biotechnology Inc SantaCruz CAUSA) respectivelyovernight at 4∘C After incubation with polymer helper for20min at 37∘C sections were reacted with poly-HRP anti-goat IgG for 20min at 37∘C The sections were then stainedwith 33-diaminobenzidine (Sigma St Louis MO USA) andcounterstained with hematoxylin For the control stainingPBS was used instead of the primary antibodies

Specimens were examined using a Leica image analyzerand analyzed by computer image analysis (LeicaMicrosystemWetzlar Gmbh Wetzlar Germany) in a blinded mannerTo localize and identify areas with positively stained cellsten digital images per specimen of synovium from a kneeor ankle joint were recorded and quantitative analysis wasperformed according to the color cell separation The resultsare expressed as the mean region of interest representing thepercentage of area covered with positively stained cells perimage at a magnification of times400

211 Enzyme-Linked Immunosorbant Assay Sera from themice on day 22 of arthritis and conditioned media fromHFLS cultured in the presence of triptolide (028 28 and28 nmolL resp) for 3 d 7 d 10 d 14 d 17 d and 21 dwas obtained and stored at minus80∘C until use The amounts ofRANKL and OPG in serum and OPG in supertant of HFLSwere detected by ELISA assay (RampD system MinneapolisMN USA) according to the manufacturerrsquos protocol andabsorbance was measured at 450 nm All experiments weredone in triplicate

212 Western Blot Analysis Isolated PBMCs(3 times 106 cellswell) or HFLS (105 cellswell) were cultured in6-well plates for 3 weeks in the presence of triptolide (02828 and 28 nmolL resp) The cells were washed twice withPBS and treated with lysis buffer (10mM HEPES-KOH [pH78] 15mM KCl 2MmMgCl

2 1 Igepal 2120583gmL leupeptin

and 1mM phenylmethylsulfonyl fluoride) The protein wasobstained to detect the levels of RNAKL in HFLS and RNAKin PBMCs by Western blot The Western blot protocol andsemiquantitative analysis were carried out following theprotocol of our previous study [23] The following antibodieswere used RANKL antibody (goat polyclonal antibody

dilution 1 200 Santa Cruz Biotechnology Inc Santa CruzCA USA) RANK antibody (goat polyclonal antibodydilution 1 200 Santa Cruz Biotechnology Inc SantaCruz CA USA) and GAPDH antibody (internal controlrabbit polyclonal antibody dilution 1 200 Santa CruzBiotechnology Inc Santa Cruz CA USA) All experimentswere done in triplicate

213 Statistical Analysis The software of SPSS version 130for Windows (SPSS Inc Chicago IL USA) and SAS 91(SAS Institute Cary NC) was used for statistical analysisContinuous variables were expressed as 119883 plusmn 119904 Arthritisincidence and percentage of arthritic limbs were analyzedby a chi-square test Arthritis index pathological scoresand number of TRAP positive cells were analyzed withnonparametric statistics (Kruskal-Wallis test) Other datawere analyzed by one-way ANOVA followed by LSD testDifferences were considered statistically significant when 119875was less than 005

3 Results

31 Effects of Triptolide on Severity of Arthritis and ArthritisProgression To investigate the effect of triptolide on arthritisthe CIAmodel inDBA1mice was used Although the diseasemanifested itself on different days after immunization wedid not observe a relation between clinical response andtime of onset of disease Consistent with the previous studies[22 27] oral administration of triptolide once a day startedat the beginning of the fist rheumatoid arthritis signs such aserythema andor oedema in joint(s) of CIAmice and continuedfor 21 days As shown in Figure 1(a) macroscopic evidence ofarthritis such as erythema or swelling wasmarkedly observedin vehicle-treated CIA mice while a dose of 32 120583g(kgsdotday)triptolide significantly attenuated arthritis severity in CIAmice Additionally the mean arthritis index (all 119875 lt 005Figure 1(b)) arthritis incidence (all119875 lt 005 Figure 1(c)) andthe percentage of arthritis limbs (all 119875 lt 005 Figure 1(d))in triptolide-treated mice were significantly lower than thosein methotrexate-treated and vehicle-treated CIA mice with adose-dependent manner In the groups receiving triptolidewith doses of 16sim32 120583g(kgsdotday) the rate of incidence andthe percentage of arthritis limbs weremarkedly reduced fromday 7 of arthritis Remarkably triptolide treatment couldeffectively suppress the loss of body weight of CIA mice (forTrip 16 group versus Vehicle 119875 lt 005 for Trip 32 groupversus Vehicle 119875 lt 005 Figure 1(e)) but methotrexatetreatment did not have this effect (Figure 1(e))

32 Triptolide Prevents Bone Destruction in CIAMice Micro-CT scan was performed to validate the efficiency of triptolidein CIA mice Figures 2(a) 2(b) and 3(a) respectivelyshowed the three-dimensional reconstructed bones of kneejoints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday)triptolide markedly reduced the extent of joint destructionin triptolide-treated CIA mice In addition four parametersincluding bone volume BMD trabecular thickness and

Evidence-Based Complementary and Alternative Medicine 5

Vehicle Trip 32 120583gkg

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Figure 1 Effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice Mice were orallyadministered triptolide (Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onsetof the clinical symptoms of arthritis At the end of the experiment the arthritis index arthritis incidence the percentage of arthritic limbsand changes in bodyweight were evaluated Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 005 in comparison with the normalcontrol lowast119875 lt 005 +119875 lt 001 and lowast119875 lt 0001 in comparison with the vehicle control $119875 lt 005 in comparison with methotrexate-treatedCIA mice

6 Evidence-Based Complementary and Alternative Medicine

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

(b)

Figure 2 Micro-CT scan validates efficiency of triptolide in collagen-induced arthritis (CIA) mice Mice were orally administered triptolide(Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptomsof arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice (a) and (b)respectively showed the three-dimensional reconstructed bones of ankle and knee joints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treatedCIA mice

trabecular separation of inflamed joints in different groupswere detected to quantify the extent of joint destructionAs shown in Figure 3(b) compared with vehicle-treatedCIA mice doses of 8sim32120583g(kgsdotday) triptolide significantlyincreased BMD (all 119875 lt 005) bone volume fraction (all119875 lt 001) and trabecular thickness of inflamed joints (all119875 lt 005) and decreased trabecular separation (for Trip 16group versus Vehicle 119875 lt 005 for Trip 32 group versusVehicle 119875 lt 0001) suggesting a protective role of triptolideon volume and quality of preserved trabecular bone despitejoint inflammation Notably dose of 16 or 32 120583g(kgsdotday)triptolide could alsomore effectively change these parametersthan 01mgkgmethotrexate could (all119875 lt 005 Figure 3(b))

Histopathological evaluation of knee joint sections ofvehicle-treated CIA mice revealed inflammatory cell infil-tration synovial hyperplasia and partial bone destructionIn contrast oral administration of triptolide could distinctlyreduce the extent of inflammatory cell infiltration and bonedestruction (Figure 4(a)) To elucidate the effects of trip-tolide treatment on bone destruction at the histologic levelinflamed joints were scored with semiquantitative gradingscales As shown in Figure 4(b) the bone destruction scores

in triptolide-treated CIA mice were significantly decreasedwith a dose-dependent tendency in comparison with vehicle-treated CIA mice (for Trip 16 group versus vehicle group119875 lt 005 for Trip 32 group versus vehicle group 119875 lt 001)MTX also reduced significantly the bone destruction scoresof inflamed joints compared with vehicle-treated CIA mice(119875 lt 005 Figure 4(b)) although this value remained higherthan those for triptolide-treated groups

To confirm the effect of triptolide on the number ofosteoclasts knee joint sections were stained with TRAPOnly TRAP-positive multinucleated cells located at the bonesurfacewithin the bone destructionwere considered as osteo-clasts (Figure 4(a)) Comparedwith vehicle-treatedCIAmicethe number of osteoclasts in the areas of bone destructionwassignificantly decreased in triptolide-treatedmice with a dose-dependent tendency (all 119875 lt 001 Figure 4(c)) Methotrexatealso reduced significantly the number of osteoclasts in theareas of bone destruction compared with vehicle control(119875 lt 005 Figure 4(c)) although this value remained higherthan those for triptolide-treated groups (all 119875 lt 0001Figure 4(c))

Evidence-Based Complementary and Alternative Medicine 7

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

lowast

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(mg

cc)

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+

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0

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(b)

Figure 3 Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice Mice wereorally administered triptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of theonset of the clinical symptoms of arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolidein CIAmice (a)The two-dimensional reconstructed bones of knee joints in different groups Compared with vehicle-treated CIAmice doseof 32120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIAmice (b)The values of four parametersincluding bone volume bone mineral density (BMD) trabecular thickness (TbTh) and trabecular separation (TbSp) of proximal end of thetibia facing the articular cavity in different groups Compared with vehicle-treated andmethotrexate-treated CIAmice triptolide significantlyincreased BMD bone volume fraction (BVF) and TbTh of inflamed joints and decreased TbSp of inflamed joints Data are represented asthe mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normal control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison withthe vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 in comparison with methotrexate-treated CIA mice

33 Triptolide Inhibits Osteoclast Differentiation by TargetingRANKLRANKOPG Signal Pathway To obtain insights intothemechanisms of the inhibitory effects of triptolide on bonedestruction in inflamed joints of CIA mice the expressionof RANKL RANK and OPG at mRNA and protein levelsin inflamed joints were respectively detected by quantitativereal-time RT-PCR (Figure 5(a)) and immunohistochemistry(Figure 5(b)) and the serum levels of RANKL and OPG pro-teins were detected by ELISA assay (Figure 5(c)) Comparedwith vehicle-treated CIA mice doses of 8sim32 120583g(kgsdotday)

triptolide significantly reduced the expression of RANKL (all119875 lt 0001 only except for Trip 8 group at protein level injoints Figure 5) and RANK (all 119875 lt 005 Figure 5(a)) andenhanced the expression of OPG (all 119875 lt 0001 only exceptfor Trip 8 group at protein level in joints and sera Figure 5)More interestingly triptolide treatments markedly reducedthe ratio of RANKL to OPG in the sera and inflamed jointsof CIA mice with a dose-dependent tendency (all 119875 lt 001Figure 5) Methotrexate also reduced significantly the ratioof RANKL to OPG in the sera and inflamed joints of CIA

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

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Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

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0

5

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lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

percentage of arthritic limbs in a group was calculated as thefollowing formula

percentage of arthritic limbs

=

number of arthritic limbs in a groupnumber of all limbs in a group

times 100(1)

25 Change in Body Weight after Onset of Arthritis Becausethe loss of body mass is associated with aggressive progres-sion of RA in clinics [24 25] we detected the changes in bodyweight after the onset of arthritis in order to evaluate the sideeffect of triptolide in the treatment of RAThe change in bodyweight () of each individual CIA mice after the onset ofarthritis was calculated as the following formula

change in bodyweight

= (

bodyweight (day 22 of arthritis)bodyweight (day 1 of arthritis)

minus

bodyweight (day 1 of arthritis)bodyweight (day 1 of arthritis)

) times 100

(2)

26 Histopathology and TRAP Staining Mice were sacrificedby cervical dislocation on day 22 of arthritis The righthind limbs including the paws ankles and knees weredissected fixed immediately for 2 h in 4 paraformaldehydedecalcified in 10 EDTA for up to 1 month at 4∘C andembedded in paraffin Tissue sections (5120583m) were mountedon common slides for staining with hematoxylin and eosinAll sections were randomized and evaluated by two trainedobservers who were blinded to the treatment groups and thearthritis severity of each mouse The data were expressed asmean bone destruction scores which were based on a scaleof 0ndash3 as our previous study [22] Minor differences betweenobservers were resolved by mutual agreement To identifyosteoclasts sections were stained for tartrate-resistant acidphosphatase (TRAP) using an acid phosphatase kit (SigmaSt LouisMOUSA) TRAP-positivemultinucleated cells thatcontained more than 3 nuclei were identified as osteoclastsand these were counted by light microscopy

27 Micro-CT Imaging Three-dimensional reconstructionand sagittal images of the right knee and ankle joints wereobtained by microfocal computed tomography (micro-CTExplore Locus SP GE USA) at the day 22 of arthritis Brieflyafter the mice in different groups being killed using etheranesthesia the hind limbs were removed and fixed in 4paraformaldehyde for 2 h The samples were scaned withmicro-CT Using the constructed sagittal image from micro-CT scanning data histomorphometric analysis of the proxi-mal tibial epiphysis exclude the cortical bone was performedin the measurement area facing the articular cavity from thebegining of growth plate Bone volume bonemineral density(BMD) trabecular thickness and trabecular separation at

the tibial spongiosa were measured using an image analyzingsystem (Microview ABA22 software GE USA)

28 Osteoclast Formation in Coculture System of SynovialFibroblasts and Peripheral Blood Mononuclear Cells Thecoculture system of human fibroblast-like synovial cells(HFLS) and peripheral blood mononuclear cells (PBMCs)were constructed according to the protocol of Nakano etal [26] Briefly HFLS derived from RA patients aftersynovectomy were purchased from Cell Applications (SanDiego CA USA) and were routinely cultured in synoviocytegrowth medium (Cell Applications) Blood was collectedfrom healthy volunteers and their PBMCs were isolated bycentrifugation over FicollPaque at 400 g for 30 minutesAfter isolation PBMCs (2 times 105 cellswell) were resus-pended in 120572-minimum essential medium containing 10fetal calf serum (FCS) and 50 ngmL of macrophage colony-stimulating factor (M-CSF PeproTech Inc Rocky Hill NJUSA) and then seeded in 48-well tissue culture plates (CostarCorning Inc Corning NY USA) Three days later adherentcells were used for subsequent cocultures with HFLS whichwere then added into 48-well tissue culture plates (2 times104 cellswell) with PBMCs and cocultured for 9 days in

MEM containing 10 FCS 50 ngmL of M-CSF 10minus7M 125-dihydroxyvitamin D

3(125[OH]

2D3) and triptolide (028

28 and 28 nmolL resp) After that some dishes were usedfor TRAP staining as aforementioned in Section 26 TRAP-positive multinucleated cells that contained more than 3nuclei were identified as osteoclasts and these were countedby light microscopy

29 RNA Isolation and Real-Time PCR The left hind paws(including the paw and ankle) were dissected from mice onday 22 of arthritis snap-frozen in liquid nitrogen ground intopowder and homogenized This procedure was done underRNase-free conditionsTheRNA isolation and real-time PCRassay were carried out following the protocol of our previousstudy [22 23] Briefly total RNA was extracted with TRIzolreagent (Invitrogen Carlsbad CA USA) from the tissuehomogenates according to the manufacturerrsquos instructionsThe total RNA (1 120583g) was reverse transcribed to cDNA usingthe QuantiTect Reverse Transcription Kit (QIAGEN KKTokyo Japan) according to the instructions manual Thespecific transcripts were quantified by quantitative real-timePCR using QuantiTect SYBR Green PCR Kit (QIAGEN KKTokyo Japan) and analyzed with ABI 7500 real-time PCRsystem (Applied Biosystems USA) Gene-specific primerswere used for RANKL (51015840-CAG CAT CGC TCT GTT CCTGTA-31015840 as forward and 51015840-CTG CGT TTT CAT GGA GTCTCA-31015840 as reverse) RANK (51015840-GCC CAG TCT CAT CGTTCT GC-31015840 as forward and 51015840-GCA AGC ATC ATT GACCCA ATT C-31015840 as reverse) OPG (51015840-ACC CAG AAA CTGGTC ATC AGC-31015840 as forward and 51015840-CTG CAA TAC ACACACTCATCACT-31015840 as reverse) and 120573-actin (51015840-GGC TGTATT CCC CTC CAT CG-31015840 as forward and 51015840-CCA GTTGGTAACAATGCCATGT-31015840 as reverse)ThemRNA levelsof RANKL RANK and OPG were normalized to 120573-actinmRNA level PCRwas performed as 40 cycles at 94∘C for 15 s

4 Evidence-Based Complementary and Alternative Medicine

55∘C for 30 s and 72∘C for 30 sThe relativemRNAexpressionwas calculated with comparative 119862

119879method

210 Immunohistochemical Staining Paraffin sections (5120583m)of tissue from the knee and ankle joints were mounted onpoly-L-lysine-coated slides Immunolocalizations of RANKLand OPG in the joints were carried out with commer-cial Polink-2 plus Polymer HRP Detection System ForGoat Primary Antibody kits (Golden Bridge InternationalInc Mukilteo WA USA) according to the manufacturerrsquosinstructions The paraffin sections were dewaxed by routinemethod and incubated for 10min with 3 H

2O2 Each

section was incubated with normal goat serum for 20minat room temperature and then with primary antibodiesagainst mouse RANKL (goat polyclonal antibody dilution1 50 Santa Cruz Biotechnology Inc Santa Cruz CA USA)and OPG (goat polyclonal antibody dilution 1 50 SantaCruz Biotechnology Inc SantaCruz CAUSA) respectivelyovernight at 4∘C After incubation with polymer helper for20min at 37∘C sections were reacted with poly-HRP anti-goat IgG for 20min at 37∘C The sections were then stainedwith 33-diaminobenzidine (Sigma St Louis MO USA) andcounterstained with hematoxylin For the control stainingPBS was used instead of the primary antibodies

Specimens were examined using a Leica image analyzerand analyzed by computer image analysis (LeicaMicrosystemWetzlar Gmbh Wetzlar Germany) in a blinded mannerTo localize and identify areas with positively stained cellsten digital images per specimen of synovium from a kneeor ankle joint were recorded and quantitative analysis wasperformed according to the color cell separation The resultsare expressed as the mean region of interest representing thepercentage of area covered with positively stained cells perimage at a magnification of times400

211 Enzyme-Linked Immunosorbant Assay Sera from themice on day 22 of arthritis and conditioned media fromHFLS cultured in the presence of triptolide (028 28 and28 nmolL resp) for 3 d 7 d 10 d 14 d 17 d and 21 dwas obtained and stored at minus80∘C until use The amounts ofRANKL and OPG in serum and OPG in supertant of HFLSwere detected by ELISA assay (RampD system MinneapolisMN USA) according to the manufacturerrsquos protocol andabsorbance was measured at 450 nm All experiments weredone in triplicate

212 Western Blot Analysis Isolated PBMCs(3 times 106 cellswell) or HFLS (105 cellswell) were cultured in6-well plates for 3 weeks in the presence of triptolide (02828 and 28 nmolL resp) The cells were washed twice withPBS and treated with lysis buffer (10mM HEPES-KOH [pH78] 15mM KCl 2MmMgCl

2 1 Igepal 2120583gmL leupeptin

and 1mM phenylmethylsulfonyl fluoride) The protein wasobstained to detect the levels of RNAKL in HFLS and RNAKin PBMCs by Western blot The Western blot protocol andsemiquantitative analysis were carried out following theprotocol of our previous study [23] The following antibodieswere used RANKL antibody (goat polyclonal antibody

dilution 1 200 Santa Cruz Biotechnology Inc Santa CruzCA USA) RANK antibody (goat polyclonal antibodydilution 1 200 Santa Cruz Biotechnology Inc SantaCruz CA USA) and GAPDH antibody (internal controlrabbit polyclonal antibody dilution 1 200 Santa CruzBiotechnology Inc Santa Cruz CA USA) All experimentswere done in triplicate

213 Statistical Analysis The software of SPSS version 130for Windows (SPSS Inc Chicago IL USA) and SAS 91(SAS Institute Cary NC) was used for statistical analysisContinuous variables were expressed as 119883 plusmn 119904 Arthritisincidence and percentage of arthritic limbs were analyzedby a chi-square test Arthritis index pathological scoresand number of TRAP positive cells were analyzed withnonparametric statistics (Kruskal-Wallis test) Other datawere analyzed by one-way ANOVA followed by LSD testDifferences were considered statistically significant when 119875was less than 005

3 Results

31 Effects of Triptolide on Severity of Arthritis and ArthritisProgression To investigate the effect of triptolide on arthritisthe CIAmodel inDBA1mice was used Although the diseasemanifested itself on different days after immunization wedid not observe a relation between clinical response andtime of onset of disease Consistent with the previous studies[22 27] oral administration of triptolide once a day startedat the beginning of the fist rheumatoid arthritis signs such aserythema andor oedema in joint(s) of CIAmice and continuedfor 21 days As shown in Figure 1(a) macroscopic evidence ofarthritis such as erythema or swelling wasmarkedly observedin vehicle-treated CIA mice while a dose of 32 120583g(kgsdotday)triptolide significantly attenuated arthritis severity in CIAmice Additionally the mean arthritis index (all 119875 lt 005Figure 1(b)) arthritis incidence (all119875 lt 005 Figure 1(c)) andthe percentage of arthritis limbs (all 119875 lt 005 Figure 1(d))in triptolide-treated mice were significantly lower than thosein methotrexate-treated and vehicle-treated CIA mice with adose-dependent manner In the groups receiving triptolidewith doses of 16sim32 120583g(kgsdotday) the rate of incidence andthe percentage of arthritis limbs weremarkedly reduced fromday 7 of arthritis Remarkably triptolide treatment couldeffectively suppress the loss of body weight of CIA mice (forTrip 16 group versus Vehicle 119875 lt 005 for Trip 32 groupversus Vehicle 119875 lt 005 Figure 1(e)) but methotrexatetreatment did not have this effect (Figure 1(e))

32 Triptolide Prevents Bone Destruction in CIAMice Micro-CT scan was performed to validate the efficiency of triptolidein CIA mice Figures 2(a) 2(b) and 3(a) respectivelyshowed the three-dimensional reconstructed bones of kneejoints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday)triptolide markedly reduced the extent of joint destructionin triptolide-treated CIA mice In addition four parametersincluding bone volume BMD trabecular thickness and

Evidence-Based Complementary and Alternative Medicine 5

Vehicle Trip 32 120583gkg

(a)

lowastlowast

lowastlowast

0

2

4

6

8

10

12

14

16

7 10 13 16 19 22

Art

hriti

s ind

ex

Day of arthritis

lowast

+ +

+

+

+lowast

lowast

1 4

lowast + ⋆

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(b)

lowast lowast

lowast

lowast

lowastlowast

lowast

++

lowast

lowast

lowast

lowast

++

⋆

lowast

lowast

lowast0

20

40

60

80

100

120

1 4 7 10 13 16 19 22

Art

hriti

s inc

iden

ce (

)

Day of arthritis

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(c)

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

lowast

lowastlowast lowast

+

lowast

lowast

lowast

lowastlowast

+ + ++

⋆

lowast

lowast

lowast

0

20

40

60

80

100

1 4 7 10 13 16 19 22

Art

hriti

s lim

b (

)

Day of arthritis

(d)

Normal

Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

minus15

minus10

minus5

0

5

10

15

20

Chan

ge in

bod

ywei

ght (

)

lowast

$lowast$

(e)

Figure 1 Effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice Mice were orallyadministered triptolide (Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onsetof the clinical symptoms of arthritis At the end of the experiment the arthritis index arthritis incidence the percentage of arthritic limbsand changes in bodyweight were evaluated Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 005 in comparison with the normalcontrol lowast119875 lt 005 +119875 lt 001 and lowast119875 lt 0001 in comparison with the vehicle control $119875 lt 005 in comparison with methotrexate-treatedCIA mice

6 Evidence-Based Complementary and Alternative Medicine

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

(b)

Figure 2 Micro-CT scan validates efficiency of triptolide in collagen-induced arthritis (CIA) mice Mice were orally administered triptolide(Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptomsof arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice (a) and (b)respectively showed the three-dimensional reconstructed bones of ankle and knee joints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treatedCIA mice

trabecular separation of inflamed joints in different groupswere detected to quantify the extent of joint destructionAs shown in Figure 3(b) compared with vehicle-treatedCIA mice doses of 8sim32120583g(kgsdotday) triptolide significantlyincreased BMD (all 119875 lt 005) bone volume fraction (all119875 lt 001) and trabecular thickness of inflamed joints (all119875 lt 005) and decreased trabecular separation (for Trip 16group versus Vehicle 119875 lt 005 for Trip 32 group versusVehicle 119875 lt 0001) suggesting a protective role of triptolideon volume and quality of preserved trabecular bone despitejoint inflammation Notably dose of 16 or 32 120583g(kgsdotday)triptolide could alsomore effectively change these parametersthan 01mgkgmethotrexate could (all119875 lt 005 Figure 3(b))

Histopathological evaluation of knee joint sections ofvehicle-treated CIA mice revealed inflammatory cell infil-tration synovial hyperplasia and partial bone destructionIn contrast oral administration of triptolide could distinctlyreduce the extent of inflammatory cell infiltration and bonedestruction (Figure 4(a)) To elucidate the effects of trip-tolide treatment on bone destruction at the histologic levelinflamed joints were scored with semiquantitative gradingscales As shown in Figure 4(b) the bone destruction scores

in triptolide-treated CIA mice were significantly decreasedwith a dose-dependent tendency in comparison with vehicle-treated CIA mice (for Trip 16 group versus vehicle group119875 lt 005 for Trip 32 group versus vehicle group 119875 lt 001)MTX also reduced significantly the bone destruction scoresof inflamed joints compared with vehicle-treated CIA mice(119875 lt 005 Figure 4(b)) although this value remained higherthan those for triptolide-treated groups

To confirm the effect of triptolide on the number ofosteoclasts knee joint sections were stained with TRAPOnly TRAP-positive multinucleated cells located at the bonesurfacewithin the bone destructionwere considered as osteo-clasts (Figure 4(a)) Comparedwith vehicle-treatedCIAmicethe number of osteoclasts in the areas of bone destructionwassignificantly decreased in triptolide-treatedmice with a dose-dependent tendency (all 119875 lt 001 Figure 4(c)) Methotrexatealso reduced significantly the number of osteoclasts in theareas of bone destruction compared with vehicle control(119875 lt 005 Figure 4(c)) although this value remained higherthan those for triptolide-treated groups (all 119875 lt 0001Figure 4(c))

Evidence-Based Complementary and Alternative Medicine 7

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

lowast

+lowast

++

0100200300400500600700

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

BMD

(mg

cc)

120583gkg

+

01020304050607

BVF

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

0

lowast

+++

lowast

0001002003004005006007

TbTh

(m

m)

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowast + + +

0

004

008

012

016

TbS

p (m

m)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowast

(b)

Figure 3 Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice Mice wereorally administered triptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of theonset of the clinical symptoms of arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolidein CIAmice (a)The two-dimensional reconstructed bones of knee joints in different groups Compared with vehicle-treated CIAmice doseof 32120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIAmice (b)The values of four parametersincluding bone volume bone mineral density (BMD) trabecular thickness (TbTh) and trabecular separation (TbSp) of proximal end of thetibia facing the articular cavity in different groups Compared with vehicle-treated andmethotrexate-treated CIAmice triptolide significantlyincreased BMD bone volume fraction (BVF) and TbTh of inflamed joints and decreased TbSp of inflamed joints Data are represented asthe mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normal control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison withthe vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 in comparison with methotrexate-treated CIA mice

33 Triptolide Inhibits Osteoclast Differentiation by TargetingRANKLRANKOPG Signal Pathway To obtain insights intothemechanisms of the inhibitory effects of triptolide on bonedestruction in inflamed joints of CIA mice the expressionof RANKL RANK and OPG at mRNA and protein levelsin inflamed joints were respectively detected by quantitativereal-time RT-PCR (Figure 5(a)) and immunohistochemistry(Figure 5(b)) and the serum levels of RANKL and OPG pro-teins were detected by ELISA assay (Figure 5(c)) Comparedwith vehicle-treated CIA mice doses of 8sim32 120583g(kgsdotday)

triptolide significantly reduced the expression of RANKL (all119875 lt 0001 only except for Trip 8 group at protein level injoints Figure 5) and RANK (all 119875 lt 005 Figure 5(a)) andenhanced the expression of OPG (all 119875 lt 0001 only exceptfor Trip 8 group at protein level in joints and sera Figure 5)More interestingly triptolide treatments markedly reducedthe ratio of RANKL to OPG in the sera and inflamed jointsof CIA mice with a dose-dependent tendency (all 119875 lt 001Figure 5) Methotrexate also reduced significantly the ratioof RANKL to OPG in the sera and inflamed joints of CIA

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

50 120583m

5 120583m

(a)

lowastlowast

0

02

04

06

08

1

12

14

Bone

des

truc

tion

scor

e

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

(b)

+ ++

lowast

0

10

20

30

40

50

Num

ber o

f oste

ocla

st

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

lowastlowastlowast lowastlowastlowast

(c)

Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

55∘C for 30 s and 72∘C for 30 sThe relativemRNAexpressionwas calculated with comparative 119862

119879method

210 Immunohistochemical Staining Paraffin sections (5120583m)of tissue from the knee and ankle joints were mounted onpoly-L-lysine-coated slides Immunolocalizations of RANKLand OPG in the joints were carried out with commer-cial Polink-2 plus Polymer HRP Detection System ForGoat Primary Antibody kits (Golden Bridge InternationalInc Mukilteo WA USA) according to the manufacturerrsquosinstructions The paraffin sections were dewaxed by routinemethod and incubated for 10min with 3 H

2O2 Each

section was incubated with normal goat serum for 20minat room temperature and then with primary antibodiesagainst mouse RANKL (goat polyclonal antibody dilution1 50 Santa Cruz Biotechnology Inc Santa Cruz CA USA)and OPG (goat polyclonal antibody dilution 1 50 SantaCruz Biotechnology Inc SantaCruz CAUSA) respectivelyovernight at 4∘C After incubation with polymer helper for20min at 37∘C sections were reacted with poly-HRP anti-goat IgG for 20min at 37∘C The sections were then stainedwith 33-diaminobenzidine (Sigma St Louis MO USA) andcounterstained with hematoxylin For the control stainingPBS was used instead of the primary antibodies

Specimens were examined using a Leica image analyzerand analyzed by computer image analysis (LeicaMicrosystemWetzlar Gmbh Wetzlar Germany) in a blinded mannerTo localize and identify areas with positively stained cellsten digital images per specimen of synovium from a kneeor ankle joint were recorded and quantitative analysis wasperformed according to the color cell separation The resultsare expressed as the mean region of interest representing thepercentage of area covered with positively stained cells perimage at a magnification of times400

211 Enzyme-Linked Immunosorbant Assay Sera from themice on day 22 of arthritis and conditioned media fromHFLS cultured in the presence of triptolide (028 28 and28 nmolL resp) for 3 d 7 d 10 d 14 d 17 d and 21 dwas obtained and stored at minus80∘C until use The amounts ofRANKL and OPG in serum and OPG in supertant of HFLSwere detected by ELISA assay (RampD system MinneapolisMN USA) according to the manufacturerrsquos protocol andabsorbance was measured at 450 nm All experiments weredone in triplicate

212 Western Blot Analysis Isolated PBMCs(3 times 106 cellswell) or HFLS (105 cellswell) were cultured in6-well plates for 3 weeks in the presence of triptolide (02828 and 28 nmolL resp) The cells were washed twice withPBS and treated with lysis buffer (10mM HEPES-KOH [pH78] 15mM KCl 2MmMgCl

2 1 Igepal 2120583gmL leupeptin

and 1mM phenylmethylsulfonyl fluoride) The protein wasobstained to detect the levels of RNAKL in HFLS and RNAKin PBMCs by Western blot The Western blot protocol andsemiquantitative analysis were carried out following theprotocol of our previous study [23] The following antibodieswere used RANKL antibody (goat polyclonal antibody

dilution 1 200 Santa Cruz Biotechnology Inc Santa CruzCA USA) RANK antibody (goat polyclonal antibodydilution 1 200 Santa Cruz Biotechnology Inc SantaCruz CA USA) and GAPDH antibody (internal controlrabbit polyclonal antibody dilution 1 200 Santa CruzBiotechnology Inc Santa Cruz CA USA) All experimentswere done in triplicate

213 Statistical Analysis The software of SPSS version 130for Windows (SPSS Inc Chicago IL USA) and SAS 91(SAS Institute Cary NC) was used for statistical analysisContinuous variables were expressed as 119883 plusmn 119904 Arthritisincidence and percentage of arthritic limbs were analyzedby a chi-square test Arthritis index pathological scoresand number of TRAP positive cells were analyzed withnonparametric statistics (Kruskal-Wallis test) Other datawere analyzed by one-way ANOVA followed by LSD testDifferences were considered statistically significant when 119875was less than 005

3 Results

31 Effects of Triptolide on Severity of Arthritis and ArthritisProgression To investigate the effect of triptolide on arthritisthe CIAmodel inDBA1mice was used Although the diseasemanifested itself on different days after immunization wedid not observe a relation between clinical response andtime of onset of disease Consistent with the previous studies[22 27] oral administration of triptolide once a day startedat the beginning of the fist rheumatoid arthritis signs such aserythema andor oedema in joint(s) of CIAmice and continuedfor 21 days As shown in Figure 1(a) macroscopic evidence ofarthritis such as erythema or swelling wasmarkedly observedin vehicle-treated CIA mice while a dose of 32 120583g(kgsdotday)triptolide significantly attenuated arthritis severity in CIAmice Additionally the mean arthritis index (all 119875 lt 005Figure 1(b)) arthritis incidence (all119875 lt 005 Figure 1(c)) andthe percentage of arthritis limbs (all 119875 lt 005 Figure 1(d))in triptolide-treated mice were significantly lower than thosein methotrexate-treated and vehicle-treated CIA mice with adose-dependent manner In the groups receiving triptolidewith doses of 16sim32 120583g(kgsdotday) the rate of incidence andthe percentage of arthritis limbs weremarkedly reduced fromday 7 of arthritis Remarkably triptolide treatment couldeffectively suppress the loss of body weight of CIA mice (forTrip 16 group versus Vehicle 119875 lt 005 for Trip 32 groupversus Vehicle 119875 lt 005 Figure 1(e)) but methotrexatetreatment did not have this effect (Figure 1(e))

32 Triptolide Prevents Bone Destruction in CIAMice Micro-CT scan was performed to validate the efficiency of triptolidein CIA mice Figures 2(a) 2(b) and 3(a) respectivelyshowed the three-dimensional reconstructed bones of kneejoints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday)triptolide markedly reduced the extent of joint destructionin triptolide-treated CIA mice In addition four parametersincluding bone volume BMD trabecular thickness and

Evidence-Based Complementary and Alternative Medicine 5

Vehicle Trip 32 120583gkg

(a)

lowastlowast

lowastlowast

0

2

4

6

8

10

12

14

16

7 10 13 16 19 22

Art

hriti

s ind

ex

Day of arthritis

lowast

+ +

+

+

+lowast

lowast

1 4

lowast + ⋆

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(b)

lowast lowast

lowast

lowast

lowastlowast

lowast

++

lowast

lowast

lowast

lowast

++

⋆

lowast

lowast

lowast0

20

40

60

80

100

120

1 4 7 10 13 16 19 22

Art

hriti

s inc

iden

ce (

)

Day of arthritis

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(c)

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

lowast

lowastlowast lowast

+

lowast

lowast

lowast

lowastlowast

+ + ++

⋆

lowast

lowast

lowast

0

20

40

60

80

100

1 4 7 10 13 16 19 22

Art

hriti

s lim

b (

)

Day of arthritis

(d)

Normal

Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

minus15

minus10

minus5

0

5

10

15

20

Chan

ge in

bod

ywei

ght (

)

lowast

$lowast$

(e)

Figure 1 Effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice Mice were orallyadministered triptolide (Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onsetof the clinical symptoms of arthritis At the end of the experiment the arthritis index arthritis incidence the percentage of arthritic limbsand changes in bodyweight were evaluated Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 005 in comparison with the normalcontrol lowast119875 lt 005 +119875 lt 001 and lowast119875 lt 0001 in comparison with the vehicle control $119875 lt 005 in comparison with methotrexate-treatedCIA mice

6 Evidence-Based Complementary and Alternative Medicine

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

(b)

Figure 2 Micro-CT scan validates efficiency of triptolide in collagen-induced arthritis (CIA) mice Mice were orally administered triptolide(Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptomsof arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice (a) and (b)respectively showed the three-dimensional reconstructed bones of ankle and knee joints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treatedCIA mice

trabecular separation of inflamed joints in different groupswere detected to quantify the extent of joint destructionAs shown in Figure 3(b) compared with vehicle-treatedCIA mice doses of 8sim32120583g(kgsdotday) triptolide significantlyincreased BMD (all 119875 lt 005) bone volume fraction (all119875 lt 001) and trabecular thickness of inflamed joints (all119875 lt 005) and decreased trabecular separation (for Trip 16group versus Vehicle 119875 lt 005 for Trip 32 group versusVehicle 119875 lt 0001) suggesting a protective role of triptolideon volume and quality of preserved trabecular bone despitejoint inflammation Notably dose of 16 or 32 120583g(kgsdotday)triptolide could alsomore effectively change these parametersthan 01mgkgmethotrexate could (all119875 lt 005 Figure 3(b))

Histopathological evaluation of knee joint sections ofvehicle-treated CIA mice revealed inflammatory cell infil-tration synovial hyperplasia and partial bone destructionIn contrast oral administration of triptolide could distinctlyreduce the extent of inflammatory cell infiltration and bonedestruction (Figure 4(a)) To elucidate the effects of trip-tolide treatment on bone destruction at the histologic levelinflamed joints were scored with semiquantitative gradingscales As shown in Figure 4(b) the bone destruction scores

in triptolide-treated CIA mice were significantly decreasedwith a dose-dependent tendency in comparison with vehicle-treated CIA mice (for Trip 16 group versus vehicle group119875 lt 005 for Trip 32 group versus vehicle group 119875 lt 001)MTX also reduced significantly the bone destruction scoresof inflamed joints compared with vehicle-treated CIA mice(119875 lt 005 Figure 4(b)) although this value remained higherthan those for triptolide-treated groups

To confirm the effect of triptolide on the number ofosteoclasts knee joint sections were stained with TRAPOnly TRAP-positive multinucleated cells located at the bonesurfacewithin the bone destructionwere considered as osteo-clasts (Figure 4(a)) Comparedwith vehicle-treatedCIAmicethe number of osteoclasts in the areas of bone destructionwassignificantly decreased in triptolide-treatedmice with a dose-dependent tendency (all 119875 lt 001 Figure 4(c)) Methotrexatealso reduced significantly the number of osteoclasts in theareas of bone destruction compared with vehicle control(119875 lt 005 Figure 4(c)) although this value remained higherthan those for triptolide-treated groups (all 119875 lt 0001Figure 4(c))

Evidence-Based Complementary and Alternative Medicine 7

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

lowast

+lowast

++

0100200300400500600700

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

BMD

(mg

cc)

120583gkg

+

01020304050607

BVF

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

0

lowast

+++

lowast

0001002003004005006007

TbTh

(m

m)

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowast + + +

0

004

008

012

016

TbS

p (m

m)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowast

(b)

Figure 3 Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice Mice wereorally administered triptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of theonset of the clinical symptoms of arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolidein CIAmice (a)The two-dimensional reconstructed bones of knee joints in different groups Compared with vehicle-treated CIAmice doseof 32120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIAmice (b)The values of four parametersincluding bone volume bone mineral density (BMD) trabecular thickness (TbTh) and trabecular separation (TbSp) of proximal end of thetibia facing the articular cavity in different groups Compared with vehicle-treated andmethotrexate-treated CIAmice triptolide significantlyincreased BMD bone volume fraction (BVF) and TbTh of inflamed joints and decreased TbSp of inflamed joints Data are represented asthe mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normal control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison withthe vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 in comparison with methotrexate-treated CIA mice

33 Triptolide Inhibits Osteoclast Differentiation by TargetingRANKLRANKOPG Signal Pathway To obtain insights intothemechanisms of the inhibitory effects of triptolide on bonedestruction in inflamed joints of CIA mice the expressionof RANKL RANK and OPG at mRNA and protein levelsin inflamed joints were respectively detected by quantitativereal-time RT-PCR (Figure 5(a)) and immunohistochemistry(Figure 5(b)) and the serum levels of RANKL and OPG pro-teins were detected by ELISA assay (Figure 5(c)) Comparedwith vehicle-treated CIA mice doses of 8sim32 120583g(kgsdotday)

triptolide significantly reduced the expression of RANKL (all119875 lt 0001 only except for Trip 8 group at protein level injoints Figure 5) and RANK (all 119875 lt 005 Figure 5(a)) andenhanced the expression of OPG (all 119875 lt 0001 only exceptfor Trip 8 group at protein level in joints and sera Figure 5)More interestingly triptolide treatments markedly reducedthe ratio of RANKL to OPG in the sera and inflamed jointsof CIA mice with a dose-dependent tendency (all 119875 lt 001Figure 5) Methotrexate also reduced significantly the ratioof RANKL to OPG in the sera and inflamed joints of CIA

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

50 120583m

5 120583m

(a)

lowastlowast

0

02

04

06

08

1

12

14

Bone

des

truc

tion

scor

e

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

(b)

+ ++

lowast

0

10

20

30

40

50

Num

ber o

f oste

ocla

st

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

lowastlowastlowast lowastlowastlowast

(c)

Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Vehicle Trip 32 120583gkg

(a)

lowastlowast

lowastlowast

0

2

4

6

8

10

12

14

16

7 10 13 16 19 22

Art

hriti

s ind

ex

Day of arthritis

lowast

+ +

+

+

+lowast

lowast

1 4

lowast + ⋆

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(b)

lowast lowast

lowast

lowast

lowastlowast

lowast

++

lowast

lowast

lowast

lowast

++

⋆

lowast

lowast

lowast0

20

40

60

80

100

120

1 4 7 10 13 16 19 22

Art

hriti

s inc

iden

ce (

)

Day of arthritis

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

(c)

Normal VehicleTrip 8 120583gkg Trip 16 120583gkgTrip 32 120583gkg MTX 01 mgkg

lowast

lowastlowast lowast

+

lowast

lowast

lowast

lowastlowast

+ + ++

⋆

lowast

lowast

lowast

0

20

40

60

80

100

1 4 7 10 13 16 19 22

Art

hriti

s lim

b (

)

Day of arthritis

(d)

Normal

Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

minus15

minus10

minus5

0

5

10

15

20

Chan

ge in

bod

ywei

ght (

)

lowast

$lowast$

(e)

Figure 1 Effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice Mice were orallyadministered triptolide (Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onsetof the clinical symptoms of arthritis At the end of the experiment the arthritis index arthritis incidence the percentage of arthritic limbsand changes in bodyweight were evaluated Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 005 in comparison with the normalcontrol lowast119875 lt 005 +119875 lt 001 and lowast119875 lt 0001 in comparison with the vehicle control $119875 lt 005 in comparison with methotrexate-treatedCIA mice

6 Evidence-Based Complementary and Alternative Medicine

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

(b)

Figure 2 Micro-CT scan validates efficiency of triptolide in collagen-induced arthritis (CIA) mice Mice were orally administered triptolide(Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptomsof arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice (a) and (b)respectively showed the three-dimensional reconstructed bones of ankle and knee joints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treatedCIA mice

trabecular separation of inflamed joints in different groupswere detected to quantify the extent of joint destructionAs shown in Figure 3(b) compared with vehicle-treatedCIA mice doses of 8sim32120583g(kgsdotday) triptolide significantlyincreased BMD (all 119875 lt 005) bone volume fraction (all119875 lt 001) and trabecular thickness of inflamed joints (all119875 lt 005) and decreased trabecular separation (for Trip 16group versus Vehicle 119875 lt 005 for Trip 32 group versusVehicle 119875 lt 0001) suggesting a protective role of triptolideon volume and quality of preserved trabecular bone despitejoint inflammation Notably dose of 16 or 32 120583g(kgsdotday)triptolide could alsomore effectively change these parametersthan 01mgkgmethotrexate could (all119875 lt 005 Figure 3(b))

Histopathological evaluation of knee joint sections ofvehicle-treated CIA mice revealed inflammatory cell infil-tration synovial hyperplasia and partial bone destructionIn contrast oral administration of triptolide could distinctlyreduce the extent of inflammatory cell infiltration and bonedestruction (Figure 4(a)) To elucidate the effects of trip-tolide treatment on bone destruction at the histologic levelinflamed joints were scored with semiquantitative gradingscales As shown in Figure 4(b) the bone destruction scores

in triptolide-treated CIA mice were significantly decreasedwith a dose-dependent tendency in comparison with vehicle-treated CIA mice (for Trip 16 group versus vehicle group119875 lt 005 for Trip 32 group versus vehicle group 119875 lt 001)MTX also reduced significantly the bone destruction scoresof inflamed joints compared with vehicle-treated CIA mice(119875 lt 005 Figure 4(b)) although this value remained higherthan those for triptolide-treated groups

To confirm the effect of triptolide on the number ofosteoclasts knee joint sections were stained with TRAPOnly TRAP-positive multinucleated cells located at the bonesurfacewithin the bone destructionwere considered as osteo-clasts (Figure 4(a)) Comparedwith vehicle-treatedCIAmicethe number of osteoclasts in the areas of bone destructionwassignificantly decreased in triptolide-treatedmice with a dose-dependent tendency (all 119875 lt 001 Figure 4(c)) Methotrexatealso reduced significantly the number of osteoclasts in theareas of bone destruction compared with vehicle control(119875 lt 005 Figure 4(c)) although this value remained higherthan those for triptolide-treated groups (all 119875 lt 0001Figure 4(c))

Evidence-Based Complementary and Alternative Medicine 7

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

lowast

+lowast

++

0100200300400500600700

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

BMD

(mg

cc)

120583gkg

+

01020304050607

BVF

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

0

lowast

+++

lowast

0001002003004005006007

TbTh

(m

m)

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowast + + +

0

004

008

012

016

TbS

p (m

m)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowast

(b)

Figure 3 Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice Mice wereorally administered triptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of theonset of the clinical symptoms of arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolidein CIAmice (a)The two-dimensional reconstructed bones of knee joints in different groups Compared with vehicle-treated CIAmice doseof 32120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIAmice (b)The values of four parametersincluding bone volume bone mineral density (BMD) trabecular thickness (TbTh) and trabecular separation (TbSp) of proximal end of thetibia facing the articular cavity in different groups Compared with vehicle-treated andmethotrexate-treated CIAmice triptolide significantlyincreased BMD bone volume fraction (BVF) and TbTh of inflamed joints and decreased TbSp of inflamed joints Data are represented asthe mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normal control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison withthe vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 in comparison with methotrexate-treated CIA mice

33 Triptolide Inhibits Osteoclast Differentiation by TargetingRANKLRANKOPG Signal Pathway To obtain insights intothemechanisms of the inhibitory effects of triptolide on bonedestruction in inflamed joints of CIA mice the expressionof RANKL RANK and OPG at mRNA and protein levelsin inflamed joints were respectively detected by quantitativereal-time RT-PCR (Figure 5(a)) and immunohistochemistry(Figure 5(b)) and the serum levels of RANKL and OPG pro-teins were detected by ELISA assay (Figure 5(c)) Comparedwith vehicle-treated CIA mice doses of 8sim32 120583g(kgsdotday)

triptolide significantly reduced the expression of RANKL (all119875 lt 0001 only except for Trip 8 group at protein level injoints Figure 5) and RANK (all 119875 lt 005 Figure 5(a)) andenhanced the expression of OPG (all 119875 lt 0001 only exceptfor Trip 8 group at protein level in joints and sera Figure 5)More interestingly triptolide treatments markedly reducedthe ratio of RANKL to OPG in the sera and inflamed jointsof CIA mice with a dose-dependent tendency (all 119875 lt 001Figure 5) Methotrexate also reduced significantly the ratioof RANKL to OPG in the sera and inflamed joints of CIA

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

50 120583m

5 120583m

(a)

lowastlowast

0

02

04

06

08

1

12

14

Bone

des

truc

tion

scor

e

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

(b)

+ ++

lowast

0

10

20

30

40

50

Num

ber o

f oste

ocla

st

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

lowastlowastlowast lowastlowastlowast

(c)

Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

(b)

Figure 2 Micro-CT scan validates efficiency of triptolide in collagen-induced arthritis (CIA) mice Mice were orally administered triptolide(Trip 8 16 and 32120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptomsof arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolide in CIA mice (a) and (b)respectively showed the three-dimensional reconstructed bones of ankle and knee joints in different groups Compared with vehicle-treatedand methotrexate-treated CIA mice dose of 32 120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treatedCIA mice

trabecular separation of inflamed joints in different groupswere detected to quantify the extent of joint destructionAs shown in Figure 3(b) compared with vehicle-treatedCIA mice doses of 8sim32120583g(kgsdotday) triptolide significantlyincreased BMD (all 119875 lt 005) bone volume fraction (all119875 lt 001) and trabecular thickness of inflamed joints (all119875 lt 005) and decreased trabecular separation (for Trip 16group versus Vehicle 119875 lt 005 for Trip 32 group versusVehicle 119875 lt 0001) suggesting a protective role of triptolideon volume and quality of preserved trabecular bone despitejoint inflammation Notably dose of 16 or 32 120583g(kgsdotday)triptolide could alsomore effectively change these parametersthan 01mgkgmethotrexate could (all119875 lt 005 Figure 3(b))

Histopathological evaluation of knee joint sections ofvehicle-treated CIA mice revealed inflammatory cell infil-tration synovial hyperplasia and partial bone destructionIn contrast oral administration of triptolide could distinctlyreduce the extent of inflammatory cell infiltration and bonedestruction (Figure 4(a)) To elucidate the effects of trip-tolide treatment on bone destruction at the histologic levelinflamed joints were scored with semiquantitative gradingscales As shown in Figure 4(b) the bone destruction scores

in triptolide-treated CIA mice were significantly decreasedwith a dose-dependent tendency in comparison with vehicle-treated CIA mice (for Trip 16 group versus vehicle group119875 lt 005 for Trip 32 group versus vehicle group 119875 lt 001)MTX also reduced significantly the bone destruction scoresof inflamed joints compared with vehicle-treated CIA mice(119875 lt 005 Figure 4(b)) although this value remained higherthan those for triptolide-treated groups

To confirm the effect of triptolide on the number ofosteoclasts knee joint sections were stained with TRAPOnly TRAP-positive multinucleated cells located at the bonesurfacewithin the bone destructionwere considered as osteo-clasts (Figure 4(a)) Comparedwith vehicle-treatedCIAmicethe number of osteoclasts in the areas of bone destructionwassignificantly decreased in triptolide-treatedmice with a dose-dependent tendency (all 119875 lt 001 Figure 4(c)) Methotrexatealso reduced significantly the number of osteoclasts in theareas of bone destruction compared with vehicle control(119875 lt 005 Figure 4(c)) although this value remained higherthan those for triptolide-treated groups (all 119875 lt 0001Figure 4(c))

Evidence-Based Complementary and Alternative Medicine 7

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

lowast

+lowast

++

0100200300400500600700

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

BMD

(mg

cc)

120583gkg

+

01020304050607

BVF

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

0

lowast

+++

lowast

0001002003004005006007

TbTh

(m

m)

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowast + + +

0

004

008

012

016

TbS

p (m

m)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowast

(b)

Figure 3 Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice Mice wereorally administered triptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of theonset of the clinical symptoms of arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolidein CIAmice (a)The two-dimensional reconstructed bones of knee joints in different groups Compared with vehicle-treated CIAmice doseof 32120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIAmice (b)The values of four parametersincluding bone volume bone mineral density (BMD) trabecular thickness (TbTh) and trabecular separation (TbSp) of proximal end of thetibia facing the articular cavity in different groups Compared with vehicle-treated andmethotrexate-treated CIAmice triptolide significantlyincreased BMD bone volume fraction (BVF) and TbTh of inflamed joints and decreased TbSp of inflamed joints Data are represented asthe mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normal control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison withthe vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 in comparison with methotrexate-treated CIA mice

33 Triptolide Inhibits Osteoclast Differentiation by TargetingRANKLRANKOPG Signal Pathway To obtain insights intothemechanisms of the inhibitory effects of triptolide on bonedestruction in inflamed joints of CIA mice the expressionof RANKL RANK and OPG at mRNA and protein levelsin inflamed joints were respectively detected by quantitativereal-time RT-PCR (Figure 5(a)) and immunohistochemistry(Figure 5(b)) and the serum levels of RANKL and OPG pro-teins were detected by ELISA assay (Figure 5(c)) Comparedwith vehicle-treated CIA mice doses of 8sim32 120583g(kgsdotday)

triptolide significantly reduced the expression of RANKL (all119875 lt 0001 only except for Trip 8 group at protein level injoints Figure 5) and RANK (all 119875 lt 005 Figure 5(a)) andenhanced the expression of OPG (all 119875 lt 0001 only exceptfor Trip 8 group at protein level in joints and sera Figure 5)More interestingly triptolide treatments markedly reducedthe ratio of RANKL to OPG in the sera and inflamed jointsof CIA mice with a dose-dependent tendency (all 119875 lt 001Figure 5) Methotrexate also reduced significantly the ratioof RANKL to OPG in the sera and inflamed joints of CIA

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

50 120583m

5 120583m

(a)

lowastlowast

0

02

04

06

08

1

12

14

Bone

des

truc

tion

scor

e

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

(b)

+ ++

lowast

0

10

20

30

40

50

Num

ber o

f oste

ocla

st

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

lowastlowastlowast lowastlowastlowast

(c)

Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Normal Vehicle Trip 32 120583gkg MTX 01 mgkg

(a)

lowast

+lowast

++

0100200300400500600700

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

BMD

(mg

cc)

120583gkg

+

01020304050607

BVF

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

0

lowast

+++

lowast

0001002003004005006007

TbTh

(m

m)

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

Normal Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowast + + +

0

004

008

012

016

TbS

p (m

m)

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast lowastlowast

(b)

Figure 3 Inhibitory effects of triptolide in periarticular bone erosion in knee joints of collagen-induced arthritis (CIA) mice Mice wereorally administered triptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of theonset of the clinical symptoms of arthritis At the end of the experiment Micro-CT scan was performed to validate the efficiency of triptolidein CIAmice (a)The two-dimensional reconstructed bones of knee joints in different groups Compared with vehicle-treated CIAmice doseof 32120583g(kgsdotday) triptolide markedly reduced the extent of joint destruction in triptolide-treated CIAmice (b)The values of four parametersincluding bone volume bone mineral density (BMD) trabecular thickness (TbTh) and trabecular separation (TbSp) of proximal end of thetibia facing the articular cavity in different groups Compared with vehicle-treated andmethotrexate-treated CIAmice triptolide significantlyincreased BMD bone volume fraction (BVF) and TbTh of inflamed joints and decreased TbSp of inflamed joints Data are represented asthe mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normal control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison withthe vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 in comparison with methotrexate-treated CIA mice

33 Triptolide Inhibits Osteoclast Differentiation by TargetingRANKLRANKOPG Signal Pathway To obtain insights intothemechanisms of the inhibitory effects of triptolide on bonedestruction in inflamed joints of CIA mice the expressionof RANKL RANK and OPG at mRNA and protein levelsin inflamed joints were respectively detected by quantitativereal-time RT-PCR (Figure 5(a)) and immunohistochemistry(Figure 5(b)) and the serum levels of RANKL and OPG pro-teins were detected by ELISA assay (Figure 5(c)) Comparedwith vehicle-treated CIA mice doses of 8sim32 120583g(kgsdotday)

triptolide significantly reduced the expression of RANKL (all119875 lt 0001 only except for Trip 8 group at protein level injoints Figure 5) and RANK (all 119875 lt 005 Figure 5(a)) andenhanced the expression of OPG (all 119875 lt 0001 only exceptfor Trip 8 group at protein level in joints and sera Figure 5)More interestingly triptolide treatments markedly reducedthe ratio of RANKL to OPG in the sera and inflamed jointsof CIA mice with a dose-dependent tendency (all 119875 lt 001Figure 5) Methotrexate also reduced significantly the ratioof RANKL to OPG in the sera and inflamed joints of CIA

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

50 120583m

5 120583m

(a)

lowastlowast

0

02

04

06

08

1

12

14

Bone

des

truc

tion

scor

e

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

(b)

+ ++

lowast

0

10

20

30

40

50

Num

ber o

f oste

ocla

st

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

lowastlowastlowast lowastlowastlowast

(c)

Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Vehicle Trip 32 120583gkg MTX 01 mgkg

50 120583m

5 120583m

(a)

lowastlowast

0

02

04

06

08

1

12

14

Bone

des

truc

tion

scor

e

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

(b)

+ ++

lowast

0

10

20

30

40

50

Num

ber o

f oste

ocla

st

Vehicle Trip 8 Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg120583gkg

lowastlowast

lowastlowastlowast lowastlowastlowast

(c)

Figure 4 Triptolide inhibits osteoclast differentiation in CIAmice Mice were orally administered triptolide (Trip 8 16 and 32 120583gkg resp)methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinical symptoms of arthritis At the end of theexperiment the osteoclast differentiation was evaluated (a) Tartrate-resistant acid phosphatase (TRAP) stained section from knee joints ofvehicle-treated triptolide-treated and methotrexate-treated CIAmiceThe under graphs is the magnification of the yellow pane in the uppergraphs respectively (b) The bone destruction score in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice(c) The number of osteoclasts (multinucleated TRAP positive cells) in knee joints of vehicle-treated triptolide-treated and methotrexate-treated CIA mice Data are represented as the mean plusmn SD (119899 = 12) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehiclecontrol +119875 lt 005 and ++119875 lt 001 in comparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

mice compared with vehicle controls (all 119875 lt 001 Figure 5)although this value remained higher than those for triptolide-treated groups (119875 lt 005 only except for Trip 8 group atprotein level in joints Figure 5)

To further validate the above mechanisms we alsoassessed the effects of triptolide on osteoclastogenesis in thecoculture system of HFLS and PBMCs Numerous TRAP-positive multinucleated cells considered as osteoclasts wereformed (Figure 6(a)) but few TRAP-positive multinucleatedcells were formedwhen concentration of 28 nmolL triptolidewere added into this coculture system (Figure 6(a)) Thenumber of osteoclasts per area counted under a light micro-scope was significantly decreased in the coculture systemwith the addition of 28sim28 nmolL triptolide compared thatwithout (119875 lt 001 Figure 6(b)) Then the soluble OPGconcentration in HFLS measured by ELISA was increasedand reached the peak at day 14 in the culture period (Fig-ure 6(c)) Moreover the expression level of RANKL proteinin HFLS and that of RANK protein in PBMCs were alsoreduced significantly after the treatment of triptolide in adose-dependent manner (all 119875 lt 005 Figure 6(c)) Theseresults suggested that triptolide play a pivotal role in theosteoclastogenesis through the downregulation of RANKLand RANK and the upregulation of OPG in the coculturesystem of HFLS and PBMCs

4 Discussion

RA is a chronic and progressive inflammatory disease withmultiple underlying pathogenic mechanisms caused by var-ious risk factors The disease progression of RA is associ-ated with chronic soft tissue inflammation which is oftenfollowed by bone and cartilage destruction of inflamed joints[28] Therefore to prevent bone and cartilage destructionis the most important issue in the treatment of RA Thetherapeutic mechanisms of triptolide a common medicinefor RA therapy have not been fully elucidated Recentstudies have mainly explained its suppressive effects oninflammation of RA The previous study of our group hasalready demonstrated that triptolide can potently suppressthe inflammatory responses and cartilage destruction inCIA mice [22] Thus this research is focus on the effectsof triptolide on bone destruction in CIA mice The mainfindings of our study are as the following two points (1)triptolide attenuates RA partially by preventing the bonedestruction (2) triptolide inhibits osteoclast formation byregulating RANKLRANKOPG signal pathway

In order to determine the therapeutic efficiency of trip-tolide for the treatment of RA methotrexate which is thefirst-line therapy for this disease was used as the controldrug Methotrexate is not only effective against inflammatorysymptoms but also in the prevention of bone destructionHowever accumulating reports have indicated that highdosage or the prolonged administration of low dosage ofmethotrexate may cause distinctive osteopenia by inhibitingthe osteoblast activity and stimulating the osteoclast recruit-ment [29 30] Thus it is of great clinical significance to finda novel drug which can effectively prevent both the joint

damage and the systemic bone mass loss of RA patients Inline with the data of our previous study [22] we here firstfound that triptolide aswell asmethotrexate efficiently atten-uated the severity of arthritis in CIA mice by reducing themean arthritis index and the percentage of arthritic limbs butwithout the subsequent loss of body weight Then triptolideimproved histological findings in a dose-dependent mannerby decreasing the extent of inflammatory cell infiltration andbone destruction in inflamed joints of CIA mice more effec-tively than methotrexate These results were also confirmedby three-dimensional micro-CT which showed that bonedestruction and osteopenia were the common alterationsof the bone in the affected joints in RA Four parametersincluding bone volume BMD trabecular thickness andtrabecular separation of inflamed joints of three-dimensionalmicro-CT were detected to quantify these alterations in allCIA mice of different groups Our data further suggest thatoral administration of triptolidemay effectively preserve bothbone density and trabecular thickness of inflamed joints

Recent studies have identified osteoclasts as the principalcell type responsible for bone destruction in RA [31 32]Thus we hypothesized that triptolide would play a rolein osteoclastogenesis in rheumatoid synovium Consistentwith this hypothesis we found that the administration oftriptolide significantly reduced the number of osteoclastsin the areas of bone destruction with a dose-dependenttendency by down-regulating the expression of RANKL andRANK up-regulating the expression of OPG and reducingthe ratio of RANKL to OPG in CIA mice Additionallywe also investigated the effects of triptolide on osteoclastformation in the coculture system of HFLS and PBMCsbecause HFLS can efficiently induce the formation of TRAP-positive multinucleated cells when cocultured with PBMCs[26 33] According to the results of in vitro study weconfirmed the inhibitive effects of triptolide on osteoclastformation which was found in vivo study

In conclusion our data offer the convincing evidencefor the first time that triptolide may attenuate RA partiallyby preventing the bone destruction Triptolide may inhibitosteoclast formation by regulating RANKLRANKOPG sig-nal pathway targeting which with triptolide may thereforebe an important therapeutic strategy for preventing bonedestruction in RA

Authorsrsquo Contribution

C Liu and Y Zhang contributed equally to this paper

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (no 30672647) the Beijing NaturalScience Foundation (no 7082069) and the Creation for

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

++++

0

5

10

15

20

25

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

RAN

KL m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast +lowast

005

115

225

335

4

RAN

K m

RNA

in jo

ints

(rel

ativ

e exp

ress

ion)

+ + +

+ + +

+ + +

lowast

0

5

10

15

20

25

OPG

mRN

A in

join

ts(r

elat

ive e

xpre

ssio

n)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg Normal Vehicle Trip 8

120583gkgTrip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

+ + +

+ + + + + +

0123456789

10

RAN

KL m

RNA

OPG

mRN

Ain

join

ts

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

(a)

+ + +

lowast

0

2

4

6

8

10

12

Posit

ive e

xpre

ssio

n of

RAN

KL p

rote

in in

join

ts (

)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++ + + +

lowast

012345678

Posit

ive e

xpre

ssio

n of

OPG

pro

tein

in jo

ints

()

+

++

lowastlowast

0

1

2

3

4

5

RAN

KL p

rote

inO

PG p

rote

inin

join

ts

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(b)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0102030405060

RAN

KL p

rote

in in

sera

(pg

mL)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowast

010203040506070

OPG

pro

tein

in se

ra (n

gm

L)

Normal Vehicle Trip 8120583gkg

Trip 16120583gkg

Trip 32120583gkg

MTX 01mgkg

lowastlowast

++

0

3

6

9

12

15

RAN

KL p

rote

inO

PG p

rote

inin

sera

(times10minus4)lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 5 Triptolide inhibits osteoclast differentiation by targeting RANKLRANKOPG signal pathway Mice were orally administeredtriptolide (Trip 8 16 and 32 120583gkg resp) methotrexate (MTX 01mgkg) or vehicle for 21 days from the first day of the onset of the clinicalsymptoms of arthritis At the end of the experiment the changes in RANKL RANK and OPG expression at both mRNA and protein levelsin the ankle joint and serum were respectively detected by real-time PCR immunohistochemistry and ELISA assay Triptolide reduces theexpression of RANKL and RANK and enhances the expression of OPG and the ratio of RANKL to OPG in the ankle joint at mRNA (a) andprotein (b) levels and in serum (c) of CIA mice Data are represented as the mean plusmn SD (119899 = 12) 119875 lt 0001 in comparison with the normalcontrol lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 in comparison with the vehicle control +119875 lt 005 ++119875 lt 001 and +++119875 lt 0001 incomparison with methotrexate-treated CIA mice

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

Vehicle Trip 28 nmolL

(a)

lowastlowast

lowastlowast

0

5

10

15

20

25

30

35

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

Num

ber o

f oste

ocla

sts p

er ar

ea

(b)

lowastlowastlowast

lowastlowastlowast

002040608

112

Relat

ive e

xpre

ssio

n of

RAN

KL p

rote

in

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

002040608

112

Relat

ive e

xpre

ssio

nof

RA

NK

prot

ein

Vehicle Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

050

100150200250300350

3 7 10 14 17 21OPG

conc

entr

atio

n (p

gm

L)

Days

VehicleTrip 028 nmolL

Trip 28 nmolLTrip 28 nmolL

lowastlowast lowastlowastlowastlowastlowast

VehicleTrip 028

nmolLTrip 28nmolL

Trip 28nmolL Vehicle

Trip 028nmolL

Trip 28nmolL

Trip 28nmolL

RANKL

GAPDH

RANK

GAPDH

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 6 Triptolide inhibits osteoclastogenesis in the coculture system of human fibroblast-like synovial cells (HFLS) and peripheralblood mononuclear cells (PBMCs) (a) Numerous tartrate-resistant acid phosphatase- (TRAPs) positive multinucleated cells considered asosteoclasts were formed in the coculture system of HFLS and PBMCs while few TRAP-positive multinucleated cells were formed whenconcentration of 28 nmolL triptolide were added into this coculture system (b) The number of osteoclasts per area counted under a lightmicroscope (c) Expression levels of RANKL and RANKproteins in PBMCs andHFLSmeasured byWestern blot Soluble OPG concentrationin the culture supernatant of HFLS was measured by ELISA Data are represented as the mean plusmn SD (119899 = 3) lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 in comparison with the vehicle control

Significant New Drugs Project of China (no 2009ZX09301-005-007 2009ZX090502-019)

References

[1] C Bohler H Radner M Ernst et al ldquoRheumatoid arthritis andfalls the influence of disease activityrdquo Rheumatology vol 51 pp2051ndash2057 2012

[2] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 no 2 pp ii84ndashii86 2002

[3] Y Nanke T Yago and S Kotake ldquoThe effects of diseasemodifying anti-rheumatic drugs on osteoclastogenesis andbone destruction in rheumatoid arthritisrdquoNihon RinshoMenekiGakkai Kaishi vol 34 pp 493ndash500 2011

[4] M Stolina G Schett D Dwyer et al ldquoRANKL inhibition byosteoprotegerin prevents bone loss without affecting local or

systemic inflammation parameters in two rat arthritis modelscomparison with anti-TNFalpha or anti-IL-1 therapiesrdquo Arthri-tis Research ampTherapy vol 11 article R187 2009

[5] Y D Bai F S Yang K Xuan Y X Bai and B LWu ldquoInhibitionof RANKRANKL signal transduction pathway a promisingapproach for osteoporosis treatmentrdquo Medical Hypotheses vol71 no 2 pp 256ndash258 2008

[6] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[7] P Geusens ldquoThe role of RANK ligandosteoprotegerin inrheumatoid arthritisrdquo Therapeutic Advances in MusculoskeletalDisease vol 4 pp 225ndash233 2012

[8] T Y Ho K Santora J C Chen A L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) and

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

osteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[9] Y Saeki T Matsui K Saisho and S Tohma ldquoCurrent treat-ments of rheumatoid arthritis from the ldquoNinJardquo registryrdquoExpertReview of Clinical Immunology vol 8 pp 455ndash465 2012

[10] C Xiao J Zhou Y He et al ldquoEffects of triptolide from RadixTripterygium wilfordii (Leigongteng) on cartilage cytokines andtranscription factor NF-120581B a study on induced arthritis in ratsrdquoChinese Medicine vol 4 article 13 2009

[11] J Wang A Wang H Zeng et al ldquoEffect of triptolide on T-cell receptor beta variable gene mRNA expression in rats withcollagen-induced arthritisrdquoTheAnatomical Record vol 295 pp922ndash927 2012

[12] M Xue Z Z Jiang J P Liu et al ldquoComparative study on theanti-inflammatory and immune suppressive effect ofWilforlideArdquo Fitoterapia vol 81 no 8 pp 1109ndash1112 2010

[13] R Goldbach-Mansky M Wilson R Fleischmann et al ldquoCom-parison of Tripterygium wilfordii Hook F versus sulfasalazinein the treatment of rheumatoid arthritis a randomized trialrdquoAnnals of Internal Medicine vol 151 no 4 pp 229ndash240 W49ndashW51 2009

[14] R Matta X Wang H Ge W Ray L D Nelin and YLiu ldquoTriptolide induces anti-inflammatory cellular responsesrdquoAmerican Journal of Translational Research vol 1 no 3 pp 267ndash282 2009

[15] X D Pan and X C Chen ldquoAdvances in the study ofimmunopharmacological effects and mechanisms of extractsof Tripterygium wilfordii Hook f in neuroimmunologic disor-dersrdquo Yaoxue Xuebao vol 43 no 12 pp 1179ndash1185 2008

[16] Z K Chen N Wang and H S Lu ldquoOverexpression ofprogrammed cell death 5 factor enhances triptolides-inducedfibroblast-like synoviocytes apoptosis in rheumatoid arthritisrdquoBeijing Da Xue Xue Bao vol 40 no 6 pp 567ndash571 2008

[17] Y Wang L Jia and C Y Wu ldquoTriptolide inhibits the differen-tiation of Th17 cells and suppresses collagen-induced arthritisrdquoScandinavian Journal of Immunology vol 68 no 4 pp 383ndash3902008

[18] Y Lu W J Wang J H Leng L F Cheng L Feng andH P Yao ldquoInhibitory effect of triptolide on interleukin-18and its receptor in rheumatoid arthritis synovial fibroblastsrdquoInflammation Research vol 57 no 6 pp 260ndash265 2008

[19] W Yifan W Dengming L Zheng L Yanping and S JunkanldquoTriptolide inhibits CCR5 expressed in synovial tissue of ratadjuvant-induced arthritisrdquo Pharmacological Reports vol 59no 6 pp 795ndash799 2007

[20] Y Hu W Cheng W Cai Y Yue J Li and P Zhang ldquoAdvancesin research on animal models of rheumatoid arthritisrdquo ClinicalRheumatology vol 32 no 2 pp 161ndash165 2012

[21] L K Myers E F Rosloniec M A Cremer and A H KangldquoCollageninduced arthritis an animal model of autoimmunityrdquoLife sciences vol 61 pp 1861ndash1872 1878

[22] N Lin C Liu C Xiao et al ldquoTriptolide a diterpenoidtriepoxide suppresses inflammation and cartilage destructionin collagen-induced arthritis micerdquo Biochemical Pharmacologyvol 73 no 1 pp 136ndash146 2007

[23] N Lin T Sato and A Ito ldquoTriptolide a novel diterpenoidtriepoxide from Tripterygium wilfordii Hook f suppressesthe production and gene expression of pro-matrix metallo-proteinases 1 and 3 and augments those of tissue inhibitorsof metalloproteinases 1 and 2 in human synovial fibroblastsrdquoArthritis amp Rheumatism vol 44 pp 2193ndash2200 2001

[24] L C Rall and R Roubenoff ldquoRheumatoid cachexia metabolicabnormalities mechanisms and interventionsrdquo Rheumatologyvol 43 no 10 pp 1219ndash1223 2004

[25] I M Jou A L Shiau S Y Chen et al ldquoThrombospondin 1as an effective gene therapeutic strategy in collagen-inducedarthritisrdquo Arthritis amp Rheumatism vol 52 no 1 pp 339ndash3442005

[26] K Nakano Y Okada K Saito and Y Tanaka ldquoInduction ofRANKL expression and osteoclast maturation by the bindingof fibroblast growth factor 2 to heparan sulfate proteoglycan onrheumatoid synovial fibroblastsrdquo Arthritis amp Rheumatism vol50 no 8 pp 2450ndash2458 2004

[27] J van Holten K Reedquist P Sattonet-Roche et al ldquoTreatmentwith recombinant interferon-beta reduces inflammation andslows cartilage destruction in the collagen-induced arthritismodel of rheumatoid arthritisrdquo Arthritis Research amp Therapyvol 6 no 3 pp R239ndashR249 2004

[28] J Anderson L Caplan J Yazdany et al ldquoRheumatoid arthritisdisease activity measures American College of Rheumatologyrecommendations for use in clinical practicerdquo Arthritis Care ampResearch vol 64 pp 640ndash647 2012

[29] V Strand S Cohen M Schiff et al ldquoTreatment of activerheumatoid arthritis with leflunomide compared with placeboandmethotrexaterdquoArchives of InternalMedicine vol 159 no 21pp 2542ndash2550 1999

[30] B Le Goff E Soltner C Charrier et al ldquoA combination ofmethotrexate and zoledronic acid prevents bone erosions andsystemic bone mass loss in collagen induced arthritisrdquo ArthritisResearch ampTherapy vol 11 no 6 article R185 2009

[31] T Braun and J Zwerina ldquoPositive regulators of osteoclastoge-nesis and bone resorption in rheumatoid arthritisrdquo ArthritisResearch ampTherapy vol 13 article 235 2011

[32] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[33] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis amp Rheumatism vol 43 pp 259ndash269 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

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Stem CellsInternational

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MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom