Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 809579 8 pageshttpdxdoiorg1011552013809579

Research ArticleTetrandrine Inhibits the Wnt120573-Catenin Signalling Pathway andAlleviates Osteoarthritis An In Vitro and In Vivo Study

Xindie Zhou Weijun Li Lifeng Jiang Jiapeng Bao Lijiang Tao Jin Li and Lidong Wu

Department of Orthopedics Surgery The Second Affiliated Hospital of Medical College Zhejiang University Hangzhou 310000 China

Correspondence should be addressed to Lidong Wu ldwuyahoocom

Received 27 November 2012 Accepted 3 February 2013

Academic Editor Mei Tian

Copyright copy 2013 Xindie Zhou 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

There is currently no effective drug treatment for the early phase of osteoarthritis (OA) one of the most common senile diseasesThe goal of this study was to investigate the protective effect of the tetrandrine (Tet) on OA in vitro and in vivo In an in vitroexperiment quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate changes in gene expression uponthe addition of Tet in chondrocytes processed with IL-1120573 changes in protein profiles were assessed by Western blotting In vivo todetermine whether Tet has the protective effects on articular cartilage a rabbit anterior cruciate ligament transaction model of OAwas established Expression of matrix metalloproteinase and 120573-catenin genes increased significantly while that of tissue inhibitor ofmetalloproteinase-1 decreased significantly in the OA group both in vivo and in chondrocytes However the changes of expressionwere reversed by Tet and there was less cartilage degradation in vivo compared with the OA group as assessed by histological andmacroscopic observations Thus Tet may play a useful role in the treatment of OA through the Wnt120573-catenin signalling pathwayand has potential for the treatment of OA

1 Introduction

Osteoarthritis (OA) is the most common joint disorder andis a major cause of pain disability and loss of quality oflife It is regarded as a degenerative disease and is associatedwith joint marginal osteophyte formation It is believed thatobesity bonemass joint injury and instability developmentaldiseases trauma joint deformity and age are commonfactors in OA especially of the hip and knee joints [1]Although OA is regarded primarily as a noninflammatoryarthropathy symptoms of local inflammation as well assynovitis are present in many patients and animal modelsof OA during cartilage destruction [2] Interleukin-1 (IL-1) originally known as a lymphokine was first describedin the context of cellular interactions in articular tissues asa monocytemacrophage product that induced collagenaseand prostaglandin production in synovial fibroblast cultures[3] A previous study showed that IL-1 mediated markeddownregulation of the matrix metalloproteinases (MMPs) inchondrocytes [4]

It is now generally accepted that chondrocyte senescenceand destruction are the final results of abnormal biome-chanical factors and that biochemical and genetic factors

also play important roles in the normal functional activitiesof these cells MMPs have received much attention becausethey specifically degrade native collagens and proteoglycansLevels of MMPs are increased in the cartilage of OA patientsand there is a decrease in levels of tissue inhibitor ofmetalloproteinase-1 (TIMP-1) [5] Among these enzymesMMP-1 and MMP-13 play major roles in degrading thecomponents of the cartilage matrix especially the aggrecansand collagens Active stromelysin (MMP-3) also serves asan activator of latent collagenases and digests proteoglycanaggregates in human articular cartilage [6] TIMPs act asinhibitors ofMMPs TIMP levels are elevated inOA cartilagepossibly reflecting an endogenous adaptive response to theincreased levels of active proteinase activity [7]

Nonsteroidal anti-inflammatory drugs (NSAIDs) corti-costeroids and hyaluronan have been clinically used for thetreatment of OA in the clinic However they fail to reversecartilage damage and a proportion of patients still progressand ultimately require surgery Thus there is a continuingneed for better agents with which to treat OA [8]

Here we investigated the activity of tetrandrine (Tet)in the protection of articular cartilage Tet (International

2 Evidence-Based Complementary and Alternative Medicine

Union of Pure and Applied Chemistry name 6610158407 12-tetramethoxy-221015840-dimethyl-1120573-berbaman Chemical Ab-stracts Service number 518-34-3 C

38H42N2O6 molecular

weight 62274988) is a bisbenzylisoquinoline alkaloidpurified from the root of Stephania tetrandrine of the Men-ispermaceae family It has been used as an antihypertensiveand antiarrhythmic agent as well as an operative agentin cardiovascular disease because of its calcium channel-blocking effects and it has been shown to exhibit antifibroticactivity in silicosis [9 10] Early findings showed that Tetmay have value in the therapy of chronic inflammatorydiseases in which IL-1 or TNF play a role in pathogenesis[11 12] Recent studies have shown that Wnt120573-cateninsignalling also participates in the response to mechanicalinjury to cartilage and that Tet may have an effect on OAthrough Wnt120573-catenin signalling from a study of theeffect of Tet on tumour growth in human colorectal cancer[13 14] However little is known about its possible use inthe treatment of OA In this study we evaluated the effectsof Tet on cartilage degradation following intra-articularinjection in an experimental OA model and at 20mgL in anarticular chondrocyte experiment at this concentration theeffectiveness of Tet in vitro has been confirmed

2 Materials and Methods

21 Reagents Tet (Sigma St Louis MO USA) was dissolvedin 001molL HCl (1mg Tet dissolved in 10 uL 001molLHCl to mother liquor) and diluted to 20mgL by Dulbeccorsquosmodified Eaglersquos medium (DMEM) for the in vivo test and byphosphate-buffered saline (PBS) for the in vitro test

22 MTT Assays Cells were cultured in a 96-well plate(8000well) After incubationwith various concentrations (5ndash100mgL) of Tet for 24 h in a serum-free medium MTT(5mgmL)was added (20 uLwell) Cells were then incubatedwith MTT for 4 h and culture medium was removed andDMSO was added (150mLwell) Absorbance was measuredat 570 nm This step was repeated for three times to getaverage results to reduce errors

23 Primary Cell Culture and Treatment The study wasapproved by the Institutional Animal Care and Use Com-mittee of Zhejiang University (Hangzhou China) Four-week-old New Zealand white rabbits were sacrificed by airembolism Immediately cartilage harvested from the kneejoints of rabbits under sterile conditions was digested with025 pancreatic enzymes for 30min to remove other tissuesand cells then digested with 02 collagenase II at 37∘Cfor 4 h as described previously with minor modificationsCells were grown to confluence in DMEM supplementedwith 10 fetal bovine serum (FBS) 100UmL penicillin and100mgmL streptomycin at 37∘Cwith 5CO

2 Cells from the

third passage were used Experiments were performed aftersubconfluent cells were serum-starved overnight Cells wereseeded in six-well plates (1times105well) and subconfluent cellswere preincubated with three concentrations of Tet (derivedfrom preliminary tests) for 1 h followed by stimulation with

IL-1120573 (10 ngmL) for 24 h Cells were then harvested andsubjected to determination ofMMP-1 MMP-3MMP-13 andTIMP-1 mRNA expression levels to assess the optimum Tetconcentration which was then used in subsequent experi-ments

24 Gene Expression Analysis Total RNAwas extracted fromchondrocytes treated with various Tet concentrations andcartilage harvested from the in vitro test using the TRIzolreagent (Invitrogen Carlsbad CA USA) according to themanufacturerrsquos protocol Total RNA (600 120583g) 1 120583L of primermix and 1-120583L dNTPs (10mM) were added to a 200-120583LRNase-free centrifuge tube DEPC-treated water was added(15 120583L) and the centrifuge tube was incubated on ice Thetube was then incubated at 70∘C for 5min Next 4 120583L of5 times first-strand buffer 2 120583L of 01M DTT 25 units of RNaseinhibitor and 200 units of Superscript II reverse transcriptase(Invitrogen) were added RNA was reverse-transcribed intocDNA MMP-1 MMP-3 MMP-13 and TIMP-1 expressionlevels were quantified by quantitative real-time polymerasechain reaction (qRT-PCR) using the iCycler system (Bio-Rad Hercules CA USA) and iQ SYBRGreen Supermix PCRkit (Bio-Rad) based on sequence information (Table 1) Aparallel amplification with rabbit 18S primers was carriedout to normalise the expression data of the targeted genetranscripts The relative levels of targeted gene expressionswere calculated following the formula

2minus(Δ119888119905 target gene minusΔ119888119905 18s rRNA) (1)

25 Western Blot Analysis Cells were rinsed with icedPBS and total protein was extracted from three samplesusing a total protein extraction kit then quantified with aBCA quantification kit Proteins were resolved by sodiumdodecyl sulphate polyacrylamide gel electrophoresis andthen transferred onto polyvinylidene fluoride membranesAfter blocking for 1 h with 5 milk in Tris-buffered saline-Tween membranes were incubated with antibodies againstMMP-3 (anti-MMP-3 antibody Santa Cruz sc-6839 USA)TIMP-1 (anti-TIMP1 antibody Abcam ab126847 USA) 120573-catenin (anti-beta catenin antibody Abcam ab83295 USA)and 120573-actin (Santa Cruz Biotechnology) overnight at 4∘CMembranes were incubated with goat anti-mouse IgG-HRPand goat anti-rabbit secondary antibody at room tempera-ture for 1 h and signals were detected using an EnhancedChemiluminescence kit (GE Healthcare Shanghai China)with exposure to X-ray film (Kodak Hangzhou China)

26 Induction of OA in Rabbits Fifteen New Zealand whiterabbits weighing 20 kg were used (Animal Centre of Zhe-jiang University) All experiments were conducted withthe approval of Zhejiang University Animal Care and UseCommittee

Ten of the 15 rabbits underwent bilateral anterior cruciateligament transections (ACLTs) on the knee joints to induceOA and were divided into two groups randomly The otherfive rabbits (control group) received sham operations which

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers of targeted genes

Gene Genbank accession Primer sequences(51015840 to 31015840) Size (bp) Annealing (∘C)

Rabbit MMP-1 M25663 F CAGGAGCCTTCCCAAGAGGAA 79 62R CTTGTCTCTTGCATATCAGGATGATG

Rabbit MMP-3 NM 001082280 F ACACCGGATCTGCCAAGAGA 89 63R CTGGAGAACGTGAGTGGAGTCA

Rabbit MMP-13 NM 001082037 F CAGATGGGCATATCCCTCTAAGAA 88 63R CCATGACCAAATCTACAGTCCTCAC

Rabbit TIMP-1 AY829730 F CAACTGCGGAACGGGCTCTTG 102 63R CGGCAGCGTAGGTCTTGGTGAA

Rabbit 120573-catenin DQ786777 F CGTACGCACCATGCAGAACACAAA 154 63R ATCCACTGGTGAACCGAGCATCTT

Rabbit 18S EU236696 F GACGGACCAGAGCGAAAGC 119 62R CGCCAGTCGGCATCGTTTATG

involved opening the articular cavity and resuturing it with-out cutting the short anterior cruciate ligament After surgeryall animals were returned to their cages the limbs were notimmobilised At 1 month after surgery the Tet group wasgiven intra-articular injections of 03mL Tet (20mgL) inboth knees once per week for 6 weeks The OA group wasinjected with 03mL solvent alone in both knees under thesame conditions (solvent 10120583L of 001molL HCl dissolvedin 50mL of PBS) In the sham-operation group no otherprocedures were conducted Rabbits were sacrificed 7 daysafter the last injection

27 Histological Examination Five samples from each groupwere fixed in 4 paraformaldehyde [15] decalcified with 10formic acid buffered at pH 74 dehydrated through a seriesof ethanol solutions embedded in paraffin cut into 3-120583msections and stained with safranin O-fast greenThe sampleswere scored for the degree of histological change using theMankin score system [16] Two independent researchersassessed the extent of histological cartilage damage in ablinded manner

28 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) Statistical analysis of MTT assaydata was performed by an unpaired t-test while histologicaland gene expression data were analyzed by a paired t-testDifferences were considered significant at 119875 lt 005

3 Results

31 Effects of Tet on Viability The chondrocyte toxicities of100 50 20 10 and 5mgL Tet were assessed by MTT assayStatistical analysis of MTT assay data was conducted usingan unpaired t-test (Figure 1) Concentrations of gt20mgLhad toxic effects on chondrocytes As a result 20mgL wasselected as the optimum concentration and was used insubsequent experiments

32 Effects of Tet on the Expression of MMP-1 MMP-3MMP-13 TIMP-1 and 120573-catenin in Rabbit Chondrocytes

0 5 10 20 50 1000

05

1

15

2

Tet (mgL)

OD

lowast

lowast

lowast

lowast

lowast

Figure 1 The effects of Tetrandrine (Tet) on chondrocyte prolifer-ation as determined by the MTT assay Chondrocytes (8000) wereinitially plated in each well of a 96-well plate and treated with Tetat 0 5 10 20 50 100mgL At 24 h the number of viable cells wasmeasured byMTT assay Values aremeanplusmn SD of three independentexperiments lowastStatistical significance in viable cell numbers (119875 lt005)

and Cartilage qRT-PCR was performed to determine theexpression levels of MMPs in chondrocytes To investigatethe effects of Tet on gene expression chondrocytes werepreincubated with Tet for 1 h prior to stimulation with IL-1120573 for 24 h Chondrocytes stimulated with IL-1120573 showedinduction of MMP-1 MMP-3 and MMP-13 gene expressionbut downregulation of TIMP-1 expression (Figure 2(a)) Tetinhibited the IL-1120573-mediated induction of MMP-1 MMP-3and MMP-13 gene expression and induced the expression ofTIMP-1 We next examined the effects of IL-1120573 and Tet onprotein expression of MMP-1 MMP-3 MMP-13 and TIMP-1 in chondrocytes The optimum Tet concentration was used(Figures 2(b) and 2(c)) Treatment with IL-1120573 resulted in theupregulation ofMMP-1MMP-3 andMMP-13 and the down-regulation of TIMP-1 at the protein level These effects wereblocked by Tet We identified similar changes in the analysisof the expression of the MMP TIMP-1 and 120573-catenin genesin vivo (Figure 3(a))

4 Evidence-Based Complementary and Alternative Medicine

minus

minus minus

+ +5

+ +10 20

0

200

400

600

800

Gen

e exp

ress

ion

ofM

MP-

3

0

20

40

60

80

Gen

e exp

ress

ion

of

MM

P-13

01020304050

Gen

e exp

ress

ion

ofTI

MP-

1

0

5

10

15

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowast

lowast

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

0

20

40

60

80

Gen

e exp

ress

ion

ofM

MP-

1lowast

lowast

lowastlowast

Gen

e exp

ress

ion

of120573

-cat

enin

(a)

0

20

40

60

80

minus

minus

minus

+ ++

MM

P-3

0

20

40

60

80

TIM

P-1

0

20

40

60

IL-1120573 (10 ngmL)Tet (mgL) minus

minus

minus

+ ++

IL-1120573 (10 ngmL)Tet (mgL) minus

minus

minus

+ ++

IL-1120573 (10 ngmL)Tet (mgL)

lowast

lowast

lowast

lowastlowast

lowast

120573-c

aten

in

(b)

minus

minus

1

+ +

2 320minus

120573-catenin

TIMP-1

MMP-3

120573-actin

IL-1120573 (10 ngmL)Tet (mgL)

(c)

Figure 2 Effects of Tet on the expression of matrix metalloproteinase-1 (MMP-1) MMP-3 MMP-13 tissue inhibitors of metalloproteinase-1 (TIMP-1) and 120573-catenin in chondrocytes Cells were pretreated with Tet (0mgL 5mgL 10mgL 20mgL) for one hour followedby stimulation with interleukin-1120573 (IL-1120573) (10 ngmL) for 24 h qRT-PCR was performed to determine the expression levels of MMPs inchondrocytes in (a) The protein concentrations of MMP-3 TIMP-1 and 120573-catenin were detected by Western blot analysis with the optimalconcentration (b) The protein concentrations of MMP-3 MMP-13 and TIMP-1 were quantitated (c) Data are expressed as mean plusmn SDlowast119875 lt 005 compared with cells stimulated with IL-1120573 alone

33 Macroscopic Observations In the sham-operation (con-trol) group the cartilage on the femoral condyles was macro-scopically normal with a smooth glistening surface andno cartilage defect or osteophyte was observed In the OAgroup general characteristics of OA including erosion andosteophyte formation were seen on the side of the femoralcondyles after surgery The Tet group showed less bonewear than the OA group as determined by gross appearance(Figure 3(b))

34 Histopathological Changes in Articular Cartilage Histo-pathological changes in the rabbits after surgery centredmainly on the thinner cartilage layer abraded surface andreduced safranin O-fast green staining in the cartilage Tetinhibited the cartilage degradation which developed as OAprogressed However osteophyte proliferation could not bereversed (Figure 3(c)) Consistent with these findings theMankin score was reduced in the Tet group compared withthe OA group (Table 2 and Figure 4)

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

1

0

10

20

30

40

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

3

0

50

100

150

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

13

0

05

1

15

Normal OA Tet

Gen

e exp

ress

ion

ofTI

MP-

1

0

10

20

30

Normal OA Tet

lowast lowast

lowast

lowast

lowast

Gen

e exp

ress

ion

of120573

-cat

enin

(a)

(b)

Normal OA Tet

(c)

Figure 3 Effects of Tet on the expression of MMP-1 MMP-3 MMP-13 TIMP-1 and 120573-catenin in vivo experiment on cartilage in (a)The Tetand OA groups were treated with ACLT and only the Tet groupwas given treatment of Tet (20mgL) for 6 weeks after surgery Rabbits weresacrificed after last injection in articular cavity Rabbits receive the sham-operation regarding as the Normal group Representative picturesof macroscopic observations in (b) and Safranin O staining are shown in (c)

4 Discussion

OA may be of unknown origin (idiopathic primary) orrelated to a known medical condition or event the majorpathological changes occur in the structure of the hyalinecartilage with a variable degree of synovial inflammationThese changes are ascribed to a complex network of biochem-ical factors including proteolytic enzymes matrix metallo-proteinases and cytokines which interact and lead to thebreakdown of cartilage macromolecules Cytokines such as

TNF-120572 and IL-1 produced by mononuclear cells activatedsynoviocytes or even articular cartilage itself significantlyupregulate MMP gene expression [17] As a result cytokinesaffect compensatory chondrocyte synthesis pathways leadingto the degradation of the extracellular matrix (ECM) Manystudies have demonstrated that IL-1 and TNF-120572 inhibitchondrocyte compensatory biosynthesis pathways whichcan further compromise cartilage repair [18] IL-1120573 is knownto play a pivotal role in cartilage degradation through theinduction of MMPs secreted by chondrocytes Chondrocytes

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Histological score of articular cartilage

Femoral condyle Normal group Tet group OA groupStructural changes 02 plusmn 040 30 plusmn 063lowast 38 plusmn 117

Cellular changes 04 plusmn 049 16 plusmn 080lowast 18 plusmn 075

Safranin staining 04 plusmn 049 18 plusmn 075lowast 22 plusmn 075

Tide mark 02 plusmn 04 04 plusmn 049lowast 06 plusmn 049

Sum of score 12 plusmn 117 68 plusmn 133lowast 84 plusmn 185

Values are the means plusmn SD 119875 lt 005 when Normal group compared with OA group lowast119875 lt 005 when Normal group compared with Tet group

0

5

10

15

Normal Tet OA

Mod

ified

Man

kin

scor

e

lowast

Figure 4 The Mankin scores are also presented 119875 lt 005 whenNormal group compared with OA group lowast119875 lt 005 when Normalgroup compared with Tet group

stimulated with IL-1120573 in vitro have been used to mimic themicroenvironment that occurs in OA [19]

In this in vitro study we used IL-1120573 to induce MMP geneexpression based on the hypothesis above then evaluated theeffect of Tet on MMP induction in rabbit chondrocytes aswell as its effects on ACLT-induced OA in the rabbit model[20] We demonstrated that Tet inhibited the IL-1120573-mediatedinduction of MMP-1 MMP-3 and MMP-13 and induced theexpression of TIMP-1 a metalloproteinase inhibitor in rabbitarticular chondrocytes these effects were manifested at boththe mRNA and protein levels The MMPs are a family ofproteinases that are involved in ECM degradation MMP-1and MMP-13 in particular are interstitial collagenases thatdegrade type II collagen in cartilage a major component ofthe ECMwhich is a committed step in the progression ofOAAs a result targetingMMPs represents a promising approachto the treatment of OA MMPs can be induced by IL-1120573and increased expression of MMPs has been identified inosteoarthritic cartilage [21] Similarly serum concentrationsof MMP-3 are elevated in patients with OA compared withnormal subjects [22]

In the present study pretreatment with 20mgL Tetresulted in significantly decreased expression levels of MMP-1 MMP-3 and MMP-13 in IL-1120573-induced chondrocytes Tetalso induced the expression of TIMP-1 without causing cyto-toxicity Because TIMPs are endogenous inhibitors of MMPswe suggest that Tet may modulate the balance between

MMP and TIMP expression to exert its antiarthritic effectsin IL-1120573-treated rabbit articular chondrocytes [23] We alsoinvestigated in vivo the effects of Tet on cartilage degradationin an ACLT-based experimental model of OA in rabbitsThismodel has been widely used to evaluate the efficacy of agentsin the treatment ofOAdue to themodelrsquosmechanical instabil-ity which leads to cartilage degradation [24] As shown in thisstudy ACLT in the rabbit resulted in cartilage degradationand injection of Tet into the articular cavity (03mL 20mgL)for 6 weeks inhibited cartilage degradation as assessed byhistological evaluationThe results were consistentwith the invitro findings These observations provide evidence that Tetpossesses chondroprotective activity both in vitro and in vivo

Several signalling pathways are involved in the inductionofMMPs by IL-1120573 in chondrocytes Among them theWnt120573-catenin signalling pathway and proteins regulate organdevelopment tumourigenesis and bone homeostasis amongother functions and are believed to play important roles inthe induction of MMP expression [25 26] Given this agentsthat interfere with theWnt120573-catenin signalling pathwaymayaffect MMP expression In the present study we investigatedwhether Tet affected activation depending on the Wnt120573-catenin signalling pathway 120573-catenin is a multifunctionalprotein involved in the formation of adhesive ldquotaperdquo byinteracting with cadherin at cell junctions Furthermorefree 120573-catenin can penetrate into the nucleus to affect geneexpression [27]

Our in vitro and in vivo findings suggest that expressionof MMP and 120573-catenin genes showed a similar trend as didthe protein levels We demonstrated that Tet effectively tar-gets the Wnt120573-catenin signalling pathway in chondrocytessuggesting that inhibition of the Wnt120573-catenin signallingpathway may at least in part account for changes in thetrends in the expression of MMPs and TIMP-1 as well ascartilage protection This study is to our knowledge the firstto report the effects of Tet on Wnt120573-catenin signalling inchondrocytes and in an ACLT-based experimental model ofOA in rabbits These findings are consistent with previousstudies of the effects of Tet on the Wnt120573-catenin signallingpathway in other cell systems [14]The discrepancies betweenour findings and these reports may be related to differencesin cell type and stimulation conditions

In conclusionmodulation of cytokines such as IL-1120573 andTNF-120572 which control MMP gene overexpression appears tobe a fertile target for drug development for the treatment ofOA Several studies have illustrated the potential importance

Evidence-Based Complementary and Alternative Medicine 7

of modulating IL-1 activity as a means to reduce the progres-sion of structural changes in OA [24] In the present study wedemonstrated that Tet possessed chondroprotective effectsin IL-1120573-induced rabbit chondrocytes and an experimentalmodel of OA The inhibition of MMPs by Tet was associatedat least in part with inhibition of theWnt120573-catenin signallingpathway Our results indicate that Tet shows promise as atherapeutic agent for the treatment of OA However furtherstudies are needed to confirm and extend these preliminaryfindings

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

X Zhou and LWu designed the research X ZhouW Li andL Jiang performed the research X Zhou and J Bao L Taoand J Li analyzed the data and X Zhou wrote the paper

Acknowledgment

This study was supported by the National Natural ScienceFoundation of China (81071492)

References

[1] D T Felson ldquoEpidemiology of hip and knee osteoarthritisrdquoEpidemiologic Reviews vol 10 pp 1ndash28 1988

[2] M B Goldring ldquoThe role of the chondrocyte in osteoarthritisrdquoArthritis amp Rheumatism vol 43 no 9 pp 1916ndash1926 2000

[3] MGowenDDWood andRGG Russell ldquoStimulation of theproliferation of human bone cells in vitro by human monocyteproducts with interleukin-1 activityrdquo Journal of Clinical Investi-gation vol 75 no 4 pp 1223ndash1229 1985

[4] J A Mengshol M P Vincenti C I Coon A Barchowsky andC E Brinckerhoff ldquoInterleukin-1 induction of collagenase 3(matrix metalloproteinase 13) gene expression in chondrocytesrequires p38 c-Jun N-terminal kinase and nuclear factorkappaB differential regulation of collagenase 1 and collagenase3rdquo Arthritis amp Rheumatism vol 43 no 4 pp 801ndash811 2000

[5] N Ishiguro T Ito H Ito et al ldquoRelationship of matrix metal-loproteinases and their inhibitors to cartilage proteoglycan andcollagen turnover analyses of synovial fluid from patients withosteoarthritisrdquo Arthritis amp Rheumatism vol 42 no 1 pp 129ndash136 2001

[6] Y Okada M Shinmei O Tanaka et al ldquoLocalization of matrixmetalloproteinase 3 (stromelysin) in osteoarthritic cartilage andsynoviumrdquo Laboratory Investigation vol 66 no 6 pp 680ndash6901992

[7] R M Hembry M R Bagga J J Reynolds and D L HamblenldquoImmunolocalisation studies on six matrix metalloproteinasesand their inhibitors TIMP-1 and TIMP-2 in synovia frompatients with osteo- and rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 54 no 1 pp 25ndash32 1995

[8] I Uthman J P Raynauld and B Haraoui ldquoIntra-articulartherapy in osteoarthritisrdquo Postgraduate Medical Journal vol 79no 934 pp 449ndash453 2003

[9] N H Mashour G I Lin and W H Frishman ldquoHerbalmedicine for the treatment of cardiovascular disease clinicalconsiderationsrdquo Archives of Internal Medicine vol 158 no 20pp 2225ndash2234 1998

[10] X F Yu C Q Zou and M B Lin ldquoObservation of the effect oftetrandrine on experimental silicosis of ratsrdquo Ecotoxicology andEnvironmental Safety vol 7 no 3 pp 306ndash312 1983

[11] L J Ho and J H Lai ldquoChinese herbs as immunomodulators andpotential disease-modifying antirheumatic drugs in autoim-mune disordersrdquoCurrent DrugMetabolism vol 5 no 2 pp 181ndash192 2004

[12] H S Kim YH Zhang andY P Yun ldquoEffects of tetrandrine andfangchinoline on experimental thrombosis in mice and humanplatelet aggregationrdquo Planta Medica vol 65 no 2 pp 135ndash1381999

[13] F P Luyten P Tylzanowski and R J Lories ldquoWnt signaling andosteoarthritisrdquo Bone vol 44 no 4 pp 522ndash527 2009

[14] B C He J L Gao B Q Zhang et al ldquoTetrandrine inhibitsWntbeta-catenin signaling and suppresses tumor growth ofhuman colorectal cancerrdquoMolecular Pharmacology vol 79 no2 pp 211ndash219 2011

[15] L J Brunet J AMcMahonA PMcMahon andRMHarlandldquoNoggin cartilage morphogenesis and joint formation in themammalian skeletonrdquo Science vol 280 no 5368 pp 1455ndash14571998

[16] H J Mankin M E Johnson and L Lippiello ldquoBiochem-ical and metabolic abnormalities in articular cartilage fromosteoarthritic human hips III Distribution and metabolism ofamino sugar-containing macromoleculesrdquo Journal of Bone andJoint Surgery A vol 63 no 1 pp 131ndash139 1981

[17] J C Fernandes J Martel-Pelletier and J P Pelletier ldquoThe roleof cytokines in osteoarthritis pathophysiologyrdquoBiorheology vol39 no 1-2 pp 237ndash246 2002

[18] M Lotz ldquoCytokines in cartilage injury and repairrdquo ClinicalOrthopaedics and Related Research supplement 391 pp S108ndashS115 2001

[19] F Moldovan J P Pelletier F C Jolicoeur J M Cloutier and JMartel-Pelletier ldquoDiacerhein and rhein reduce the ICE-inducedIL-1120573 and IL-18 activation in human osteoarthritic cartilagerdquoOsteoarthritis and Cartilage vol 8 no 3 pp 186ndash196 2000

[20] P F Hu Y Chen P F Cai L F Jiang and L D WuldquoSphingosine-1-phosphate a potential therapeutic target forrheumatoid arthritisrdquoMolecular Biology Reports vol 38 no 6pp 4225ndash4230 2011

[21] M P Vincenti and C E Brinckerhoff ldquoTranscriptional reg-ulation of collagenase (MMP-1 MMP-13) genes in arthritisintegration of complex signaling pathways for the recruitmentof gene-specific transcription factorsrdquo Arthritis Research vol 4no 3 pp 157ndash164 2002

[22] L S Lohmander P J Neame and J D Sandy ldquoThe structureof aggrecan fragments in human synovial fluid evidence thataggrecanase mediates cartilage degradation in inflammatoryjoint disease joint injury and osteoarthritisrdquo Arthritis andRheumatism vol 36 no 9 pp 1214ndash1222 1993

[23] D D Dean J Martel-Pelletier J P Pelletier D S Howelland J F Woessner ldquoEvidence for metalloproteinase inhibitorimbalance in human osteoarthritic cartilagerdquo Journal of ClinicalInvestigation vol 84 no 2 pp 678ndash685 1989

[24] W P Chen P F Hu J P Bao and L D Wu ldquoMorin exertsantiosteoarthritic properties an in vitro and in vivo studyrdquoExperimental Biology andMedicine vol 237 no 4 pp 380ndash3862012

8 Evidence-Based Complementary and Alternative Medicine

[25] G Schett J Zwerina and J P David ldquoThe role of Wnt proteinsin arthritisrdquoNature Clinical Practice Rheumatology vol 4 no 9pp 473ndash480 2008

[26] T Yuasa T Otani T Koike M Iwamoto and M Enomoto-Iwamoto ldquoWnt120573-catenin signaling stimulates matrix catabolicgenes and activity in articular chondrocytes is possible role injoint degenerationrdquo Laboratory Investigation vol 88 no 3 pp264ndash274 2008

[27] M Corr ldquoWnt-120573-catenin signaling in the pathogenesis ofosteoarthritisrdquo Nature Clinical Practice Rheumatology vol 4no 10 pp 550ndash556 2008

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers of targeted genes

Gene Genbank accession Primer sequences(51015840 to 31015840) Size (bp) Annealing (∘C)

Rabbit MMP-1 M25663 F CAGGAGCCTTCCCAAGAGGAA 79 62R CTTGTCTCTTGCATATCAGGATGATG

Rabbit MMP-3 NM 001082280 F ACACCGGATCTGCCAAGAGA 89 63R CTGGAGAACGTGAGTGGAGTCA

Rabbit MMP-13 NM 001082037 F CAGATGGGCATATCCCTCTAAGAA 88 63R CCATGACCAAATCTACAGTCCTCAC

Rabbit TIMP-1 AY829730 F CAACTGCGGAACGGGCTCTTG 102 63R CGGCAGCGTAGGTCTTGGTGAA

Rabbit 120573-catenin DQ786777 F CGTACGCACCATGCAGAACACAAA 154 63R ATCCACTGGTGAACCGAGCATCTT

Rabbit 18S EU236696 F GACGGACCAGAGCGAAAGC 119 62R CGCCAGTCGGCATCGTTTATG

involved opening the articular cavity and resuturing it with-out cutting the short anterior cruciate ligament After surgeryall animals were returned to their cages the limbs were notimmobilised At 1 month after surgery the Tet group wasgiven intra-articular injections of 03mL Tet (20mgL) inboth knees once per week for 6 weeks The OA group wasinjected with 03mL solvent alone in both knees under thesame conditions (solvent 10120583L of 001molL HCl dissolvedin 50mL of PBS) In the sham-operation group no otherprocedures were conducted Rabbits were sacrificed 7 daysafter the last injection

27 Histological Examination Five samples from each groupwere fixed in 4 paraformaldehyde [15] decalcified with 10formic acid buffered at pH 74 dehydrated through a seriesof ethanol solutions embedded in paraffin cut into 3-120583msections and stained with safranin O-fast greenThe sampleswere scored for the degree of histological change using theMankin score system [16] Two independent researchersassessed the extent of histological cartilage damage in ablinded manner

28 Statistical Analysis All data were expressed as mean plusmnstandard deviation (SD) Statistical analysis of MTT assaydata was performed by an unpaired t-test while histologicaland gene expression data were analyzed by a paired t-testDifferences were considered significant at 119875 lt 005

3 Results

31 Effects of Tet on Viability The chondrocyte toxicities of100 50 20 10 and 5mgL Tet were assessed by MTT assayStatistical analysis of MTT assay data was conducted usingan unpaired t-test (Figure 1) Concentrations of gt20mgLhad toxic effects on chondrocytes As a result 20mgL wasselected as the optimum concentration and was used insubsequent experiments

32 Effects of Tet on the Expression of MMP-1 MMP-3MMP-13 TIMP-1 and 120573-catenin in Rabbit Chondrocytes

0 5 10 20 50 1000

05

1

15

2

Tet (mgL)

OD

lowast

lowast

lowast

lowast

lowast

Figure 1 The effects of Tetrandrine (Tet) on chondrocyte prolifer-ation as determined by the MTT assay Chondrocytes (8000) wereinitially plated in each well of a 96-well plate and treated with Tetat 0 5 10 20 50 100mgL At 24 h the number of viable cells wasmeasured byMTT assay Values aremeanplusmn SD of three independentexperiments lowastStatistical significance in viable cell numbers (119875 lt005)

and Cartilage qRT-PCR was performed to determine theexpression levels of MMPs in chondrocytes To investigatethe effects of Tet on gene expression chondrocytes werepreincubated with Tet for 1 h prior to stimulation with IL-1120573 for 24 h Chondrocytes stimulated with IL-1120573 showedinduction of MMP-1 MMP-3 and MMP-13 gene expressionbut downregulation of TIMP-1 expression (Figure 2(a)) Tetinhibited the IL-1120573-mediated induction of MMP-1 MMP-3and MMP-13 gene expression and induced the expression ofTIMP-1 We next examined the effects of IL-1120573 and Tet onprotein expression of MMP-1 MMP-3 MMP-13 and TIMP-1 in chondrocytes The optimum Tet concentration was used(Figures 2(b) and 2(c)) Treatment with IL-1120573 resulted in theupregulation ofMMP-1MMP-3 andMMP-13 and the down-regulation of TIMP-1 at the protein level These effects wereblocked by Tet We identified similar changes in the analysisof the expression of the MMP TIMP-1 and 120573-catenin genesin vivo (Figure 3(a))

4 Evidence-Based Complementary and Alternative Medicine

minus

minus minus

+ +5

+ +10 20

0

200

400

600

800

Gen

e exp

ress

ion

ofM

MP-

3

0

20

40

60

80

Gen

e exp

ress

ion

of

MM

P-13

01020304050

Gen

e exp

ress

ion

ofTI

MP-

1

0

5

10

15

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowast

lowast

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

0

20

40

60

80

Gen

e exp

ress

ion

ofM

MP-

1lowast

lowast

lowastlowast

Gen

e exp

ress

ion

of120573

-cat

enin

(a)

0

20

40

60

80

minus

minus

minus

+ ++

MM

P-3

0

20

40

60

80

TIM

P-1

0

20

40

60

IL-1120573 (10 ngmL)Tet (mgL) minus

minus

minus

+ ++

IL-1120573 (10 ngmL)Tet (mgL) minus

minus

minus

+ ++

IL-1120573 (10 ngmL)Tet (mgL)

lowast

lowast

lowast

lowastlowast

lowast

120573-c

aten

in

(b)

minus

minus

1

+ +

2 320minus

120573-catenin

TIMP-1

MMP-3

120573-actin

IL-1120573 (10 ngmL)Tet (mgL)

(c)

Figure 2 Effects of Tet on the expression of matrix metalloproteinase-1 (MMP-1) MMP-3 MMP-13 tissue inhibitors of metalloproteinase-1 (TIMP-1) and 120573-catenin in chondrocytes Cells were pretreated with Tet (0mgL 5mgL 10mgL 20mgL) for one hour followedby stimulation with interleukin-1120573 (IL-1120573) (10 ngmL) for 24 h qRT-PCR was performed to determine the expression levels of MMPs inchondrocytes in (a) The protein concentrations of MMP-3 TIMP-1 and 120573-catenin were detected by Western blot analysis with the optimalconcentration (b) The protein concentrations of MMP-3 MMP-13 and TIMP-1 were quantitated (c) Data are expressed as mean plusmn SDlowast119875 lt 005 compared with cells stimulated with IL-1120573 alone

33 Macroscopic Observations In the sham-operation (con-trol) group the cartilage on the femoral condyles was macro-scopically normal with a smooth glistening surface andno cartilage defect or osteophyte was observed In the OAgroup general characteristics of OA including erosion andosteophyte formation were seen on the side of the femoralcondyles after surgery The Tet group showed less bonewear than the OA group as determined by gross appearance(Figure 3(b))

34 Histopathological Changes in Articular Cartilage Histo-pathological changes in the rabbits after surgery centredmainly on the thinner cartilage layer abraded surface andreduced safranin O-fast green staining in the cartilage Tetinhibited the cartilage degradation which developed as OAprogressed However osteophyte proliferation could not bereversed (Figure 3(c)) Consistent with these findings theMankin score was reduced in the Tet group compared withthe OA group (Table 2 and Figure 4)

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

1

0

10

20

30

40

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

3

0

50

100

150

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

13

0

05

1

15

Normal OA Tet

Gen

e exp

ress

ion

ofTI

MP-

1

0

10

20

30

Normal OA Tet

lowast lowast

lowast

lowast

lowast

Gen

e exp

ress

ion

of120573

-cat

enin

(a)

(b)

Normal OA Tet

(c)

Figure 3 Effects of Tet on the expression of MMP-1 MMP-3 MMP-13 TIMP-1 and 120573-catenin in vivo experiment on cartilage in (a)The Tetand OA groups were treated with ACLT and only the Tet groupwas given treatment of Tet (20mgL) for 6 weeks after surgery Rabbits weresacrificed after last injection in articular cavity Rabbits receive the sham-operation regarding as the Normal group Representative picturesof macroscopic observations in (b) and Safranin O staining are shown in (c)

4 Discussion

OA may be of unknown origin (idiopathic primary) orrelated to a known medical condition or event the majorpathological changes occur in the structure of the hyalinecartilage with a variable degree of synovial inflammationThese changes are ascribed to a complex network of biochem-ical factors including proteolytic enzymes matrix metallo-proteinases and cytokines which interact and lead to thebreakdown of cartilage macromolecules Cytokines such as

TNF-120572 and IL-1 produced by mononuclear cells activatedsynoviocytes or even articular cartilage itself significantlyupregulate MMP gene expression [17] As a result cytokinesaffect compensatory chondrocyte synthesis pathways leadingto the degradation of the extracellular matrix (ECM) Manystudies have demonstrated that IL-1 and TNF-120572 inhibitchondrocyte compensatory biosynthesis pathways whichcan further compromise cartilage repair [18] IL-1120573 is knownto play a pivotal role in cartilage degradation through theinduction of MMPs secreted by chondrocytes Chondrocytes

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Histological score of articular cartilage

Femoral condyle Normal group Tet group OA groupStructural changes 02 plusmn 040 30 plusmn 063lowast 38 plusmn 117

Cellular changes 04 plusmn 049 16 plusmn 080lowast 18 plusmn 075

Safranin staining 04 plusmn 049 18 plusmn 075lowast 22 plusmn 075

Tide mark 02 plusmn 04 04 plusmn 049lowast 06 plusmn 049

Sum of score 12 plusmn 117 68 plusmn 133lowast 84 plusmn 185

Values are the means plusmn SD 119875 lt 005 when Normal group compared with OA group lowast119875 lt 005 when Normal group compared with Tet group

0

5

10

15

Normal Tet OA

Mod

ified

Man

kin

scor

e

lowast

Figure 4 The Mankin scores are also presented 119875 lt 005 whenNormal group compared with OA group lowast119875 lt 005 when Normalgroup compared with Tet group

stimulated with IL-1120573 in vitro have been used to mimic themicroenvironment that occurs in OA [19]

In this in vitro study we used IL-1120573 to induce MMP geneexpression based on the hypothesis above then evaluated theeffect of Tet on MMP induction in rabbit chondrocytes aswell as its effects on ACLT-induced OA in the rabbit model[20] We demonstrated that Tet inhibited the IL-1120573-mediatedinduction of MMP-1 MMP-3 and MMP-13 and induced theexpression of TIMP-1 a metalloproteinase inhibitor in rabbitarticular chondrocytes these effects were manifested at boththe mRNA and protein levels The MMPs are a family ofproteinases that are involved in ECM degradation MMP-1and MMP-13 in particular are interstitial collagenases thatdegrade type II collagen in cartilage a major component ofthe ECMwhich is a committed step in the progression ofOAAs a result targetingMMPs represents a promising approachto the treatment of OA MMPs can be induced by IL-1120573and increased expression of MMPs has been identified inosteoarthritic cartilage [21] Similarly serum concentrationsof MMP-3 are elevated in patients with OA compared withnormal subjects [22]

In the present study pretreatment with 20mgL Tetresulted in significantly decreased expression levels of MMP-1 MMP-3 and MMP-13 in IL-1120573-induced chondrocytes Tetalso induced the expression of TIMP-1 without causing cyto-toxicity Because TIMPs are endogenous inhibitors of MMPswe suggest that Tet may modulate the balance between

MMP and TIMP expression to exert its antiarthritic effectsin IL-1120573-treated rabbit articular chondrocytes [23] We alsoinvestigated in vivo the effects of Tet on cartilage degradationin an ACLT-based experimental model of OA in rabbitsThismodel has been widely used to evaluate the efficacy of agentsin the treatment ofOAdue to themodelrsquosmechanical instabil-ity which leads to cartilage degradation [24] As shown in thisstudy ACLT in the rabbit resulted in cartilage degradationand injection of Tet into the articular cavity (03mL 20mgL)for 6 weeks inhibited cartilage degradation as assessed byhistological evaluationThe results were consistentwith the invitro findings These observations provide evidence that Tetpossesses chondroprotective activity both in vitro and in vivo

Several signalling pathways are involved in the inductionofMMPs by IL-1120573 in chondrocytes Among them theWnt120573-catenin signalling pathway and proteins regulate organdevelopment tumourigenesis and bone homeostasis amongother functions and are believed to play important roles inthe induction of MMP expression [25 26] Given this agentsthat interfere with theWnt120573-catenin signalling pathwaymayaffect MMP expression In the present study we investigatedwhether Tet affected activation depending on the Wnt120573-catenin signalling pathway 120573-catenin is a multifunctionalprotein involved in the formation of adhesive ldquotaperdquo byinteracting with cadherin at cell junctions Furthermorefree 120573-catenin can penetrate into the nucleus to affect geneexpression [27]

Our in vitro and in vivo findings suggest that expressionof MMP and 120573-catenin genes showed a similar trend as didthe protein levels We demonstrated that Tet effectively tar-gets the Wnt120573-catenin signalling pathway in chondrocytessuggesting that inhibition of the Wnt120573-catenin signallingpathway may at least in part account for changes in thetrends in the expression of MMPs and TIMP-1 as well ascartilage protection This study is to our knowledge the firstto report the effects of Tet on Wnt120573-catenin signalling inchondrocytes and in an ACLT-based experimental model ofOA in rabbits These findings are consistent with previousstudies of the effects of Tet on the Wnt120573-catenin signallingpathway in other cell systems [14]The discrepancies betweenour findings and these reports may be related to differencesin cell type and stimulation conditions

In conclusionmodulation of cytokines such as IL-1120573 andTNF-120572 which control MMP gene overexpression appears tobe a fertile target for drug development for the treatment ofOA Several studies have illustrated the potential importance

Evidence-Based Complementary and Alternative Medicine 7

of modulating IL-1 activity as a means to reduce the progres-sion of structural changes in OA [24] In the present study wedemonstrated that Tet possessed chondroprotective effectsin IL-1120573-induced rabbit chondrocytes and an experimentalmodel of OA The inhibition of MMPs by Tet was associatedat least in part with inhibition of theWnt120573-catenin signallingpathway Our results indicate that Tet shows promise as atherapeutic agent for the treatment of OA However furtherstudies are needed to confirm and extend these preliminaryfindings

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

X Zhou and LWu designed the research X ZhouW Li andL Jiang performed the research X Zhou and J Bao L Taoand J Li analyzed the data and X Zhou wrote the paper

Acknowledgment

This study was supported by the National Natural ScienceFoundation of China (81071492)

References

[1] D T Felson ldquoEpidemiology of hip and knee osteoarthritisrdquoEpidemiologic Reviews vol 10 pp 1ndash28 1988

[2] M B Goldring ldquoThe role of the chondrocyte in osteoarthritisrdquoArthritis amp Rheumatism vol 43 no 9 pp 1916ndash1926 2000

[3] MGowenDDWood andRGG Russell ldquoStimulation of theproliferation of human bone cells in vitro by human monocyteproducts with interleukin-1 activityrdquo Journal of Clinical Investi-gation vol 75 no 4 pp 1223ndash1229 1985

[4] J A Mengshol M P Vincenti C I Coon A Barchowsky andC E Brinckerhoff ldquoInterleukin-1 induction of collagenase 3(matrix metalloproteinase 13) gene expression in chondrocytesrequires p38 c-Jun N-terminal kinase and nuclear factorkappaB differential regulation of collagenase 1 and collagenase3rdquo Arthritis amp Rheumatism vol 43 no 4 pp 801ndash811 2000

[5] N Ishiguro T Ito H Ito et al ldquoRelationship of matrix metal-loproteinases and their inhibitors to cartilage proteoglycan andcollagen turnover analyses of synovial fluid from patients withosteoarthritisrdquo Arthritis amp Rheumatism vol 42 no 1 pp 129ndash136 2001

[6] Y Okada M Shinmei O Tanaka et al ldquoLocalization of matrixmetalloproteinase 3 (stromelysin) in osteoarthritic cartilage andsynoviumrdquo Laboratory Investigation vol 66 no 6 pp 680ndash6901992

[7] R M Hembry M R Bagga J J Reynolds and D L HamblenldquoImmunolocalisation studies on six matrix metalloproteinasesand their inhibitors TIMP-1 and TIMP-2 in synovia frompatients with osteo- and rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 54 no 1 pp 25ndash32 1995

[8] I Uthman J P Raynauld and B Haraoui ldquoIntra-articulartherapy in osteoarthritisrdquo Postgraduate Medical Journal vol 79no 934 pp 449ndash453 2003

[9] N H Mashour G I Lin and W H Frishman ldquoHerbalmedicine for the treatment of cardiovascular disease clinicalconsiderationsrdquo Archives of Internal Medicine vol 158 no 20pp 2225ndash2234 1998

[10] X F Yu C Q Zou and M B Lin ldquoObservation of the effect oftetrandrine on experimental silicosis of ratsrdquo Ecotoxicology andEnvironmental Safety vol 7 no 3 pp 306ndash312 1983

[11] L J Ho and J H Lai ldquoChinese herbs as immunomodulators andpotential disease-modifying antirheumatic drugs in autoim-mune disordersrdquoCurrent DrugMetabolism vol 5 no 2 pp 181ndash192 2004

[12] H S Kim YH Zhang andY P Yun ldquoEffects of tetrandrine andfangchinoline on experimental thrombosis in mice and humanplatelet aggregationrdquo Planta Medica vol 65 no 2 pp 135ndash1381999

[13] F P Luyten P Tylzanowski and R J Lories ldquoWnt signaling andosteoarthritisrdquo Bone vol 44 no 4 pp 522ndash527 2009

[14] B C He J L Gao B Q Zhang et al ldquoTetrandrine inhibitsWntbeta-catenin signaling and suppresses tumor growth ofhuman colorectal cancerrdquoMolecular Pharmacology vol 79 no2 pp 211ndash219 2011

[15] L J Brunet J AMcMahonA PMcMahon andRMHarlandldquoNoggin cartilage morphogenesis and joint formation in themammalian skeletonrdquo Science vol 280 no 5368 pp 1455ndash14571998

[16] H J Mankin M E Johnson and L Lippiello ldquoBiochem-ical and metabolic abnormalities in articular cartilage fromosteoarthritic human hips III Distribution and metabolism ofamino sugar-containing macromoleculesrdquo Journal of Bone andJoint Surgery A vol 63 no 1 pp 131ndash139 1981

[17] J C Fernandes J Martel-Pelletier and J P Pelletier ldquoThe roleof cytokines in osteoarthritis pathophysiologyrdquoBiorheology vol39 no 1-2 pp 237ndash246 2002

[18] M Lotz ldquoCytokines in cartilage injury and repairrdquo ClinicalOrthopaedics and Related Research supplement 391 pp S108ndashS115 2001

[19] F Moldovan J P Pelletier F C Jolicoeur J M Cloutier and JMartel-Pelletier ldquoDiacerhein and rhein reduce the ICE-inducedIL-1120573 and IL-18 activation in human osteoarthritic cartilagerdquoOsteoarthritis and Cartilage vol 8 no 3 pp 186ndash196 2000

[20] P F Hu Y Chen P F Cai L F Jiang and L D WuldquoSphingosine-1-phosphate a potential therapeutic target forrheumatoid arthritisrdquoMolecular Biology Reports vol 38 no 6pp 4225ndash4230 2011

[21] M P Vincenti and C E Brinckerhoff ldquoTranscriptional reg-ulation of collagenase (MMP-1 MMP-13) genes in arthritisintegration of complex signaling pathways for the recruitmentof gene-specific transcription factorsrdquo Arthritis Research vol 4no 3 pp 157ndash164 2002

[22] L S Lohmander P J Neame and J D Sandy ldquoThe structureof aggrecan fragments in human synovial fluid evidence thataggrecanase mediates cartilage degradation in inflammatoryjoint disease joint injury and osteoarthritisrdquo Arthritis andRheumatism vol 36 no 9 pp 1214ndash1222 1993

[23] D D Dean J Martel-Pelletier J P Pelletier D S Howelland J F Woessner ldquoEvidence for metalloproteinase inhibitorimbalance in human osteoarthritic cartilagerdquo Journal of ClinicalInvestigation vol 84 no 2 pp 678ndash685 1989

[24] W P Chen P F Hu J P Bao and L D Wu ldquoMorin exertsantiosteoarthritic properties an in vitro and in vivo studyrdquoExperimental Biology andMedicine vol 237 no 4 pp 380ndash3862012

8 Evidence-Based Complementary and Alternative Medicine

[25] G Schett J Zwerina and J P David ldquoThe role of Wnt proteinsin arthritisrdquoNature Clinical Practice Rheumatology vol 4 no 9pp 473ndash480 2008

[26] T Yuasa T Otani T Koike M Iwamoto and M Enomoto-Iwamoto ldquoWnt120573-catenin signaling stimulates matrix catabolicgenes and activity in articular chondrocytes is possible role injoint degenerationrdquo Laboratory Investigation vol 88 no 3 pp264ndash274 2008

[27] M Corr ldquoWnt-120573-catenin signaling in the pathogenesis ofosteoarthritisrdquo Nature Clinical Practice Rheumatology vol 4no 10 pp 550ndash556 2008

4 Evidence-Based Complementary and Alternative Medicine

minus

minus minus

+ +5

+ +10 20

0

200

400

600

800

Gen

e exp

ress

ion

ofM

MP-

3

0

20

40

60

80

Gen

e exp

ress

ion

of

MM

P-13

01020304050

Gen

e exp

ress

ion

ofTI

MP-

1

0

5

10

15

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowast

lowast

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

minus

minus minus

+ +5

+ +10 20

IL-1120573 (10 ngmL)Tet (mgL)

0

20

40

60

80

Gen

e exp

ress

ion

ofM

MP-

1lowast

lowast

lowastlowast

Gen

e exp

ress

ion

of120573

-cat

enin

(a)

0

20

40

60

80

minus

minus

minus

+ ++

MM

P-3

0

20

40

60

80

TIM

P-1

0

20

40

60

IL-1120573 (10 ngmL)Tet (mgL) minus

minus

minus

+ ++

IL-1120573 (10 ngmL)Tet (mgL) minus

minus

minus

+ ++

IL-1120573 (10 ngmL)Tet (mgL)

lowast

lowast

lowast

lowastlowast

lowast

120573-c

aten

in

(b)

minus

minus

1

+ +

2 320minus

120573-catenin

TIMP-1

MMP-3

120573-actin

IL-1120573 (10 ngmL)Tet (mgL)

(c)

Figure 2 Effects of Tet on the expression of matrix metalloproteinase-1 (MMP-1) MMP-3 MMP-13 tissue inhibitors of metalloproteinase-1 (TIMP-1) and 120573-catenin in chondrocytes Cells were pretreated with Tet (0mgL 5mgL 10mgL 20mgL) for one hour followedby stimulation with interleukin-1120573 (IL-1120573) (10 ngmL) for 24 h qRT-PCR was performed to determine the expression levels of MMPs inchondrocytes in (a) The protein concentrations of MMP-3 TIMP-1 and 120573-catenin were detected by Western blot analysis with the optimalconcentration (b) The protein concentrations of MMP-3 MMP-13 and TIMP-1 were quantitated (c) Data are expressed as mean plusmn SDlowast119875 lt 005 compared with cells stimulated with IL-1120573 alone

33 Macroscopic Observations In the sham-operation (con-trol) group the cartilage on the femoral condyles was macro-scopically normal with a smooth glistening surface andno cartilage defect or osteophyte was observed In the OAgroup general characteristics of OA including erosion andosteophyte formation were seen on the side of the femoralcondyles after surgery The Tet group showed less bonewear than the OA group as determined by gross appearance(Figure 3(b))

34 Histopathological Changes in Articular Cartilage Histo-pathological changes in the rabbits after surgery centredmainly on the thinner cartilage layer abraded surface andreduced safranin O-fast green staining in the cartilage Tetinhibited the cartilage degradation which developed as OAprogressed However osteophyte proliferation could not bereversed (Figure 3(c)) Consistent with these findings theMankin score was reduced in the Tet group compared withthe OA group (Table 2 and Figure 4)

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

1

0

10

20

30

40

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

3

0

50

100

150

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

13

0

05

1

15

Normal OA Tet

Gen

e exp

ress

ion

ofTI

MP-

1

0

10

20

30

Normal OA Tet

lowast lowast

lowast

lowast

lowast

Gen

e exp

ress

ion

of120573

-cat

enin

(a)

(b)

Normal OA Tet

(c)

Figure 3 Effects of Tet on the expression of MMP-1 MMP-3 MMP-13 TIMP-1 and 120573-catenin in vivo experiment on cartilage in (a)The Tetand OA groups were treated with ACLT and only the Tet groupwas given treatment of Tet (20mgL) for 6 weeks after surgery Rabbits weresacrificed after last injection in articular cavity Rabbits receive the sham-operation regarding as the Normal group Representative picturesof macroscopic observations in (b) and Safranin O staining are shown in (c)

4 Discussion

OA may be of unknown origin (idiopathic primary) orrelated to a known medical condition or event the majorpathological changes occur in the structure of the hyalinecartilage with a variable degree of synovial inflammationThese changes are ascribed to a complex network of biochem-ical factors including proteolytic enzymes matrix metallo-proteinases and cytokines which interact and lead to thebreakdown of cartilage macromolecules Cytokines such as

TNF-120572 and IL-1 produced by mononuclear cells activatedsynoviocytes or even articular cartilage itself significantlyupregulate MMP gene expression [17] As a result cytokinesaffect compensatory chondrocyte synthesis pathways leadingto the degradation of the extracellular matrix (ECM) Manystudies have demonstrated that IL-1 and TNF-120572 inhibitchondrocyte compensatory biosynthesis pathways whichcan further compromise cartilage repair [18] IL-1120573 is knownto play a pivotal role in cartilage degradation through theinduction of MMPs secreted by chondrocytes Chondrocytes

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Histological score of articular cartilage

Femoral condyle Normal group Tet group OA groupStructural changes 02 plusmn 040 30 plusmn 063lowast 38 plusmn 117

Cellular changes 04 plusmn 049 16 plusmn 080lowast 18 plusmn 075

Safranin staining 04 plusmn 049 18 plusmn 075lowast 22 plusmn 075

Tide mark 02 plusmn 04 04 plusmn 049lowast 06 plusmn 049

Sum of score 12 plusmn 117 68 plusmn 133lowast 84 plusmn 185

Values are the means plusmn SD 119875 lt 005 when Normal group compared with OA group lowast119875 lt 005 when Normal group compared with Tet group

0

5

10

15

Normal Tet OA

Mod

ified

Man

kin

scor

e

lowast

Figure 4 The Mankin scores are also presented 119875 lt 005 whenNormal group compared with OA group lowast119875 lt 005 when Normalgroup compared with Tet group

stimulated with IL-1120573 in vitro have been used to mimic themicroenvironment that occurs in OA [19]

In this in vitro study we used IL-1120573 to induce MMP geneexpression based on the hypothesis above then evaluated theeffect of Tet on MMP induction in rabbit chondrocytes aswell as its effects on ACLT-induced OA in the rabbit model[20] We demonstrated that Tet inhibited the IL-1120573-mediatedinduction of MMP-1 MMP-3 and MMP-13 and induced theexpression of TIMP-1 a metalloproteinase inhibitor in rabbitarticular chondrocytes these effects were manifested at boththe mRNA and protein levels The MMPs are a family ofproteinases that are involved in ECM degradation MMP-1and MMP-13 in particular are interstitial collagenases thatdegrade type II collagen in cartilage a major component ofthe ECMwhich is a committed step in the progression ofOAAs a result targetingMMPs represents a promising approachto the treatment of OA MMPs can be induced by IL-1120573and increased expression of MMPs has been identified inosteoarthritic cartilage [21] Similarly serum concentrationsof MMP-3 are elevated in patients with OA compared withnormal subjects [22]

In the present study pretreatment with 20mgL Tetresulted in significantly decreased expression levels of MMP-1 MMP-3 and MMP-13 in IL-1120573-induced chondrocytes Tetalso induced the expression of TIMP-1 without causing cyto-toxicity Because TIMPs are endogenous inhibitors of MMPswe suggest that Tet may modulate the balance between

MMP and TIMP expression to exert its antiarthritic effectsin IL-1120573-treated rabbit articular chondrocytes [23] We alsoinvestigated in vivo the effects of Tet on cartilage degradationin an ACLT-based experimental model of OA in rabbitsThismodel has been widely used to evaluate the efficacy of agentsin the treatment ofOAdue to themodelrsquosmechanical instabil-ity which leads to cartilage degradation [24] As shown in thisstudy ACLT in the rabbit resulted in cartilage degradationand injection of Tet into the articular cavity (03mL 20mgL)for 6 weeks inhibited cartilage degradation as assessed byhistological evaluationThe results were consistentwith the invitro findings These observations provide evidence that Tetpossesses chondroprotective activity both in vitro and in vivo

Several signalling pathways are involved in the inductionofMMPs by IL-1120573 in chondrocytes Among them theWnt120573-catenin signalling pathway and proteins regulate organdevelopment tumourigenesis and bone homeostasis amongother functions and are believed to play important roles inthe induction of MMP expression [25 26] Given this agentsthat interfere with theWnt120573-catenin signalling pathwaymayaffect MMP expression In the present study we investigatedwhether Tet affected activation depending on the Wnt120573-catenin signalling pathway 120573-catenin is a multifunctionalprotein involved in the formation of adhesive ldquotaperdquo byinteracting with cadherin at cell junctions Furthermorefree 120573-catenin can penetrate into the nucleus to affect geneexpression [27]

Our in vitro and in vivo findings suggest that expressionof MMP and 120573-catenin genes showed a similar trend as didthe protein levels We demonstrated that Tet effectively tar-gets the Wnt120573-catenin signalling pathway in chondrocytessuggesting that inhibition of the Wnt120573-catenin signallingpathway may at least in part account for changes in thetrends in the expression of MMPs and TIMP-1 as well ascartilage protection This study is to our knowledge the firstto report the effects of Tet on Wnt120573-catenin signalling inchondrocytes and in an ACLT-based experimental model ofOA in rabbits These findings are consistent with previousstudies of the effects of Tet on the Wnt120573-catenin signallingpathway in other cell systems [14]The discrepancies betweenour findings and these reports may be related to differencesin cell type and stimulation conditions

In conclusionmodulation of cytokines such as IL-1120573 andTNF-120572 which control MMP gene overexpression appears tobe a fertile target for drug development for the treatment ofOA Several studies have illustrated the potential importance

Evidence-Based Complementary and Alternative Medicine 7

of modulating IL-1 activity as a means to reduce the progres-sion of structural changes in OA [24] In the present study wedemonstrated that Tet possessed chondroprotective effectsin IL-1120573-induced rabbit chondrocytes and an experimentalmodel of OA The inhibition of MMPs by Tet was associatedat least in part with inhibition of theWnt120573-catenin signallingpathway Our results indicate that Tet shows promise as atherapeutic agent for the treatment of OA However furtherstudies are needed to confirm and extend these preliminaryfindings

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

X Zhou and LWu designed the research X ZhouW Li andL Jiang performed the research X Zhou and J Bao L Taoand J Li analyzed the data and X Zhou wrote the paper

Acknowledgment

This study was supported by the National Natural ScienceFoundation of China (81071492)

References

[1] D T Felson ldquoEpidemiology of hip and knee osteoarthritisrdquoEpidemiologic Reviews vol 10 pp 1ndash28 1988

[2] M B Goldring ldquoThe role of the chondrocyte in osteoarthritisrdquoArthritis amp Rheumatism vol 43 no 9 pp 1916ndash1926 2000

[3] MGowenDDWood andRGG Russell ldquoStimulation of theproliferation of human bone cells in vitro by human monocyteproducts with interleukin-1 activityrdquo Journal of Clinical Investi-gation vol 75 no 4 pp 1223ndash1229 1985

[4] J A Mengshol M P Vincenti C I Coon A Barchowsky andC E Brinckerhoff ldquoInterleukin-1 induction of collagenase 3(matrix metalloproteinase 13) gene expression in chondrocytesrequires p38 c-Jun N-terminal kinase and nuclear factorkappaB differential regulation of collagenase 1 and collagenase3rdquo Arthritis amp Rheumatism vol 43 no 4 pp 801ndash811 2000

[5] N Ishiguro T Ito H Ito et al ldquoRelationship of matrix metal-loproteinases and their inhibitors to cartilage proteoglycan andcollagen turnover analyses of synovial fluid from patients withosteoarthritisrdquo Arthritis amp Rheumatism vol 42 no 1 pp 129ndash136 2001

[6] Y Okada M Shinmei O Tanaka et al ldquoLocalization of matrixmetalloproteinase 3 (stromelysin) in osteoarthritic cartilage andsynoviumrdquo Laboratory Investigation vol 66 no 6 pp 680ndash6901992

[7] R M Hembry M R Bagga J J Reynolds and D L HamblenldquoImmunolocalisation studies on six matrix metalloproteinasesand their inhibitors TIMP-1 and TIMP-2 in synovia frompatients with osteo- and rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 54 no 1 pp 25ndash32 1995

[8] I Uthman J P Raynauld and B Haraoui ldquoIntra-articulartherapy in osteoarthritisrdquo Postgraduate Medical Journal vol 79no 934 pp 449ndash453 2003

[9] N H Mashour G I Lin and W H Frishman ldquoHerbalmedicine for the treatment of cardiovascular disease clinicalconsiderationsrdquo Archives of Internal Medicine vol 158 no 20pp 2225ndash2234 1998

[10] X F Yu C Q Zou and M B Lin ldquoObservation of the effect oftetrandrine on experimental silicosis of ratsrdquo Ecotoxicology andEnvironmental Safety vol 7 no 3 pp 306ndash312 1983

[11] L J Ho and J H Lai ldquoChinese herbs as immunomodulators andpotential disease-modifying antirheumatic drugs in autoim-mune disordersrdquoCurrent DrugMetabolism vol 5 no 2 pp 181ndash192 2004

[12] H S Kim YH Zhang andY P Yun ldquoEffects of tetrandrine andfangchinoline on experimental thrombosis in mice and humanplatelet aggregationrdquo Planta Medica vol 65 no 2 pp 135ndash1381999

[13] F P Luyten P Tylzanowski and R J Lories ldquoWnt signaling andosteoarthritisrdquo Bone vol 44 no 4 pp 522ndash527 2009

[14] B C He J L Gao B Q Zhang et al ldquoTetrandrine inhibitsWntbeta-catenin signaling and suppresses tumor growth ofhuman colorectal cancerrdquoMolecular Pharmacology vol 79 no2 pp 211ndash219 2011

[15] L J Brunet J AMcMahonA PMcMahon andRMHarlandldquoNoggin cartilage morphogenesis and joint formation in themammalian skeletonrdquo Science vol 280 no 5368 pp 1455ndash14571998

[16] H J Mankin M E Johnson and L Lippiello ldquoBiochem-ical and metabolic abnormalities in articular cartilage fromosteoarthritic human hips III Distribution and metabolism ofamino sugar-containing macromoleculesrdquo Journal of Bone andJoint Surgery A vol 63 no 1 pp 131ndash139 1981

[17] J C Fernandes J Martel-Pelletier and J P Pelletier ldquoThe roleof cytokines in osteoarthritis pathophysiologyrdquoBiorheology vol39 no 1-2 pp 237ndash246 2002

[18] M Lotz ldquoCytokines in cartilage injury and repairrdquo ClinicalOrthopaedics and Related Research supplement 391 pp S108ndashS115 2001

[19] F Moldovan J P Pelletier F C Jolicoeur J M Cloutier and JMartel-Pelletier ldquoDiacerhein and rhein reduce the ICE-inducedIL-1120573 and IL-18 activation in human osteoarthritic cartilagerdquoOsteoarthritis and Cartilage vol 8 no 3 pp 186ndash196 2000

[20] P F Hu Y Chen P F Cai L F Jiang and L D WuldquoSphingosine-1-phosphate a potential therapeutic target forrheumatoid arthritisrdquoMolecular Biology Reports vol 38 no 6pp 4225ndash4230 2011

[21] M P Vincenti and C E Brinckerhoff ldquoTranscriptional reg-ulation of collagenase (MMP-1 MMP-13) genes in arthritisintegration of complex signaling pathways for the recruitmentof gene-specific transcription factorsrdquo Arthritis Research vol 4no 3 pp 157ndash164 2002

[22] L S Lohmander P J Neame and J D Sandy ldquoThe structureof aggrecan fragments in human synovial fluid evidence thataggrecanase mediates cartilage degradation in inflammatoryjoint disease joint injury and osteoarthritisrdquo Arthritis andRheumatism vol 36 no 9 pp 1214ndash1222 1993

[23] D D Dean J Martel-Pelletier J P Pelletier D S Howelland J F Woessner ldquoEvidence for metalloproteinase inhibitorimbalance in human osteoarthritic cartilagerdquo Journal of ClinicalInvestigation vol 84 no 2 pp 678ndash685 1989

[24] W P Chen P F Hu J P Bao and L D Wu ldquoMorin exertsantiosteoarthritic properties an in vitro and in vivo studyrdquoExperimental Biology andMedicine vol 237 no 4 pp 380ndash3862012

8 Evidence-Based Complementary and Alternative Medicine

[25] G Schett J Zwerina and J P David ldquoThe role of Wnt proteinsin arthritisrdquoNature Clinical Practice Rheumatology vol 4 no 9pp 473ndash480 2008

[26] T Yuasa T Otani T Koike M Iwamoto and M Enomoto-Iwamoto ldquoWnt120573-catenin signaling stimulates matrix catabolicgenes and activity in articular chondrocytes is possible role injoint degenerationrdquo Laboratory Investigation vol 88 no 3 pp264ndash274 2008

[27] M Corr ldquoWnt-120573-catenin signaling in the pathogenesis ofosteoarthritisrdquo Nature Clinical Practice Rheumatology vol 4no 10 pp 550ndash556 2008

Evidence-Based Complementary and Alternative Medicine 5

0

10

20

30

40

50

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

1

0

10

20

30

40

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

3

0

50

100

150

Normal OA Tet

Gen

e exp

ress

ion

ofM

MP-

13

0

05

1

15

Normal OA Tet

Gen

e exp

ress

ion

ofTI

MP-

1

0

10

20

30

Normal OA Tet

lowast lowast

lowast

lowast

lowast

Gen

e exp

ress

ion

of120573

-cat

enin

(a)

(b)

Normal OA Tet

(c)

Figure 3 Effects of Tet on the expression of MMP-1 MMP-3 MMP-13 TIMP-1 and 120573-catenin in vivo experiment on cartilage in (a)The Tetand OA groups were treated with ACLT and only the Tet groupwas given treatment of Tet (20mgL) for 6 weeks after surgery Rabbits weresacrificed after last injection in articular cavity Rabbits receive the sham-operation regarding as the Normal group Representative picturesof macroscopic observations in (b) and Safranin O staining are shown in (c)

4 Discussion

OA may be of unknown origin (idiopathic primary) orrelated to a known medical condition or event the majorpathological changes occur in the structure of the hyalinecartilage with a variable degree of synovial inflammationThese changes are ascribed to a complex network of biochem-ical factors including proteolytic enzymes matrix metallo-proteinases and cytokines which interact and lead to thebreakdown of cartilage macromolecules Cytokines such as

TNF-120572 and IL-1 produced by mononuclear cells activatedsynoviocytes or even articular cartilage itself significantlyupregulate MMP gene expression [17] As a result cytokinesaffect compensatory chondrocyte synthesis pathways leadingto the degradation of the extracellular matrix (ECM) Manystudies have demonstrated that IL-1 and TNF-120572 inhibitchondrocyte compensatory biosynthesis pathways whichcan further compromise cartilage repair [18] IL-1120573 is knownto play a pivotal role in cartilage degradation through theinduction of MMPs secreted by chondrocytes Chondrocytes

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Histological score of articular cartilage

Femoral condyle Normal group Tet group OA groupStructural changes 02 plusmn 040 30 plusmn 063lowast 38 plusmn 117

Cellular changes 04 plusmn 049 16 plusmn 080lowast 18 plusmn 075

Safranin staining 04 plusmn 049 18 plusmn 075lowast 22 plusmn 075

Tide mark 02 plusmn 04 04 plusmn 049lowast 06 plusmn 049

Sum of score 12 plusmn 117 68 plusmn 133lowast 84 plusmn 185

Values are the means plusmn SD 119875 lt 005 when Normal group compared with OA group lowast119875 lt 005 when Normal group compared with Tet group

0

5

10

15

Normal Tet OA

Mod

ified

Man

kin

scor

e

lowast

Figure 4 The Mankin scores are also presented 119875 lt 005 whenNormal group compared with OA group lowast119875 lt 005 when Normalgroup compared with Tet group

stimulated with IL-1120573 in vitro have been used to mimic themicroenvironment that occurs in OA [19]

In this in vitro study we used IL-1120573 to induce MMP geneexpression based on the hypothesis above then evaluated theeffect of Tet on MMP induction in rabbit chondrocytes aswell as its effects on ACLT-induced OA in the rabbit model[20] We demonstrated that Tet inhibited the IL-1120573-mediatedinduction of MMP-1 MMP-3 and MMP-13 and induced theexpression of TIMP-1 a metalloproteinase inhibitor in rabbitarticular chondrocytes these effects were manifested at boththe mRNA and protein levels The MMPs are a family ofproteinases that are involved in ECM degradation MMP-1and MMP-13 in particular are interstitial collagenases thatdegrade type II collagen in cartilage a major component ofthe ECMwhich is a committed step in the progression ofOAAs a result targetingMMPs represents a promising approachto the treatment of OA MMPs can be induced by IL-1120573and increased expression of MMPs has been identified inosteoarthritic cartilage [21] Similarly serum concentrationsof MMP-3 are elevated in patients with OA compared withnormal subjects [22]

In the present study pretreatment with 20mgL Tetresulted in significantly decreased expression levels of MMP-1 MMP-3 and MMP-13 in IL-1120573-induced chondrocytes Tetalso induced the expression of TIMP-1 without causing cyto-toxicity Because TIMPs are endogenous inhibitors of MMPswe suggest that Tet may modulate the balance between

MMP and TIMP expression to exert its antiarthritic effectsin IL-1120573-treated rabbit articular chondrocytes [23] We alsoinvestigated in vivo the effects of Tet on cartilage degradationin an ACLT-based experimental model of OA in rabbitsThismodel has been widely used to evaluate the efficacy of agentsin the treatment ofOAdue to themodelrsquosmechanical instabil-ity which leads to cartilage degradation [24] As shown in thisstudy ACLT in the rabbit resulted in cartilage degradationand injection of Tet into the articular cavity (03mL 20mgL)for 6 weeks inhibited cartilage degradation as assessed byhistological evaluationThe results were consistentwith the invitro findings These observations provide evidence that Tetpossesses chondroprotective activity both in vitro and in vivo

Several signalling pathways are involved in the inductionofMMPs by IL-1120573 in chondrocytes Among them theWnt120573-catenin signalling pathway and proteins regulate organdevelopment tumourigenesis and bone homeostasis amongother functions and are believed to play important roles inthe induction of MMP expression [25 26] Given this agentsthat interfere with theWnt120573-catenin signalling pathwaymayaffect MMP expression In the present study we investigatedwhether Tet affected activation depending on the Wnt120573-catenin signalling pathway 120573-catenin is a multifunctionalprotein involved in the formation of adhesive ldquotaperdquo byinteracting with cadherin at cell junctions Furthermorefree 120573-catenin can penetrate into the nucleus to affect geneexpression [27]

Our in vitro and in vivo findings suggest that expressionof MMP and 120573-catenin genes showed a similar trend as didthe protein levels We demonstrated that Tet effectively tar-gets the Wnt120573-catenin signalling pathway in chondrocytessuggesting that inhibition of the Wnt120573-catenin signallingpathway may at least in part account for changes in thetrends in the expression of MMPs and TIMP-1 as well ascartilage protection This study is to our knowledge the firstto report the effects of Tet on Wnt120573-catenin signalling inchondrocytes and in an ACLT-based experimental model ofOA in rabbits These findings are consistent with previousstudies of the effects of Tet on the Wnt120573-catenin signallingpathway in other cell systems [14]The discrepancies betweenour findings and these reports may be related to differencesin cell type and stimulation conditions

In conclusionmodulation of cytokines such as IL-1120573 andTNF-120572 which control MMP gene overexpression appears tobe a fertile target for drug development for the treatment ofOA Several studies have illustrated the potential importance

Evidence-Based Complementary and Alternative Medicine 7

of modulating IL-1 activity as a means to reduce the progres-sion of structural changes in OA [24] In the present study wedemonstrated that Tet possessed chondroprotective effectsin IL-1120573-induced rabbit chondrocytes and an experimentalmodel of OA The inhibition of MMPs by Tet was associatedat least in part with inhibition of theWnt120573-catenin signallingpathway Our results indicate that Tet shows promise as atherapeutic agent for the treatment of OA However furtherstudies are needed to confirm and extend these preliminaryfindings

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

X Zhou and LWu designed the research X ZhouW Li andL Jiang performed the research X Zhou and J Bao L Taoand J Li analyzed the data and X Zhou wrote the paper

Acknowledgment

This study was supported by the National Natural ScienceFoundation of China (81071492)

References

[1] D T Felson ldquoEpidemiology of hip and knee osteoarthritisrdquoEpidemiologic Reviews vol 10 pp 1ndash28 1988

[2] M B Goldring ldquoThe role of the chondrocyte in osteoarthritisrdquoArthritis amp Rheumatism vol 43 no 9 pp 1916ndash1926 2000

[3] MGowenDDWood andRGG Russell ldquoStimulation of theproliferation of human bone cells in vitro by human monocyteproducts with interleukin-1 activityrdquo Journal of Clinical Investi-gation vol 75 no 4 pp 1223ndash1229 1985

[4] J A Mengshol M P Vincenti C I Coon A Barchowsky andC E Brinckerhoff ldquoInterleukin-1 induction of collagenase 3(matrix metalloproteinase 13) gene expression in chondrocytesrequires p38 c-Jun N-terminal kinase and nuclear factorkappaB differential regulation of collagenase 1 and collagenase3rdquo Arthritis amp Rheumatism vol 43 no 4 pp 801ndash811 2000

[5] N Ishiguro T Ito H Ito et al ldquoRelationship of matrix metal-loproteinases and their inhibitors to cartilage proteoglycan andcollagen turnover analyses of synovial fluid from patients withosteoarthritisrdquo Arthritis amp Rheumatism vol 42 no 1 pp 129ndash136 2001

[6] Y Okada M Shinmei O Tanaka et al ldquoLocalization of matrixmetalloproteinase 3 (stromelysin) in osteoarthritic cartilage andsynoviumrdquo Laboratory Investigation vol 66 no 6 pp 680ndash6901992

[7] R M Hembry M R Bagga J J Reynolds and D L HamblenldquoImmunolocalisation studies on six matrix metalloproteinasesand their inhibitors TIMP-1 and TIMP-2 in synovia frompatients with osteo- and rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 54 no 1 pp 25ndash32 1995

[8] I Uthman J P Raynauld and B Haraoui ldquoIntra-articulartherapy in osteoarthritisrdquo Postgraduate Medical Journal vol 79no 934 pp 449ndash453 2003

[9] N H Mashour G I Lin and W H Frishman ldquoHerbalmedicine for the treatment of cardiovascular disease clinicalconsiderationsrdquo Archives of Internal Medicine vol 158 no 20pp 2225ndash2234 1998

[10] X F Yu C Q Zou and M B Lin ldquoObservation of the effect oftetrandrine on experimental silicosis of ratsrdquo Ecotoxicology andEnvironmental Safety vol 7 no 3 pp 306ndash312 1983

[11] L J Ho and J H Lai ldquoChinese herbs as immunomodulators andpotential disease-modifying antirheumatic drugs in autoim-mune disordersrdquoCurrent DrugMetabolism vol 5 no 2 pp 181ndash192 2004

[12] H S Kim YH Zhang andY P Yun ldquoEffects of tetrandrine andfangchinoline on experimental thrombosis in mice and humanplatelet aggregationrdquo Planta Medica vol 65 no 2 pp 135ndash1381999

[13] F P Luyten P Tylzanowski and R J Lories ldquoWnt signaling andosteoarthritisrdquo Bone vol 44 no 4 pp 522ndash527 2009

[14] B C He J L Gao B Q Zhang et al ldquoTetrandrine inhibitsWntbeta-catenin signaling and suppresses tumor growth ofhuman colorectal cancerrdquoMolecular Pharmacology vol 79 no2 pp 211ndash219 2011

[15] L J Brunet J AMcMahonA PMcMahon andRMHarlandldquoNoggin cartilage morphogenesis and joint formation in themammalian skeletonrdquo Science vol 280 no 5368 pp 1455ndash14571998

[16] H J Mankin M E Johnson and L Lippiello ldquoBiochem-ical and metabolic abnormalities in articular cartilage fromosteoarthritic human hips III Distribution and metabolism ofamino sugar-containing macromoleculesrdquo Journal of Bone andJoint Surgery A vol 63 no 1 pp 131ndash139 1981

[17] J C Fernandes J Martel-Pelletier and J P Pelletier ldquoThe roleof cytokines in osteoarthritis pathophysiologyrdquoBiorheology vol39 no 1-2 pp 237ndash246 2002

[18] M Lotz ldquoCytokines in cartilage injury and repairrdquo ClinicalOrthopaedics and Related Research supplement 391 pp S108ndashS115 2001

[19] F Moldovan J P Pelletier F C Jolicoeur J M Cloutier and JMartel-Pelletier ldquoDiacerhein and rhein reduce the ICE-inducedIL-1120573 and IL-18 activation in human osteoarthritic cartilagerdquoOsteoarthritis and Cartilage vol 8 no 3 pp 186ndash196 2000

[20] P F Hu Y Chen P F Cai L F Jiang and L D WuldquoSphingosine-1-phosphate a potential therapeutic target forrheumatoid arthritisrdquoMolecular Biology Reports vol 38 no 6pp 4225ndash4230 2011

[21] M P Vincenti and C E Brinckerhoff ldquoTranscriptional reg-ulation of collagenase (MMP-1 MMP-13) genes in arthritisintegration of complex signaling pathways for the recruitmentof gene-specific transcription factorsrdquo Arthritis Research vol 4no 3 pp 157ndash164 2002

[22] L S Lohmander P J Neame and J D Sandy ldquoThe structureof aggrecan fragments in human synovial fluid evidence thataggrecanase mediates cartilage degradation in inflammatoryjoint disease joint injury and osteoarthritisrdquo Arthritis andRheumatism vol 36 no 9 pp 1214ndash1222 1993

[23] D D Dean J Martel-Pelletier J P Pelletier D S Howelland J F Woessner ldquoEvidence for metalloproteinase inhibitorimbalance in human osteoarthritic cartilagerdquo Journal of ClinicalInvestigation vol 84 no 2 pp 678ndash685 1989

[24] W P Chen P F Hu J P Bao and L D Wu ldquoMorin exertsantiosteoarthritic properties an in vitro and in vivo studyrdquoExperimental Biology andMedicine vol 237 no 4 pp 380ndash3862012

8 Evidence-Based Complementary and Alternative Medicine

[25] G Schett J Zwerina and J P David ldquoThe role of Wnt proteinsin arthritisrdquoNature Clinical Practice Rheumatology vol 4 no 9pp 473ndash480 2008

[26] T Yuasa T Otani T Koike M Iwamoto and M Enomoto-Iwamoto ldquoWnt120573-catenin signaling stimulates matrix catabolicgenes and activity in articular chondrocytes is possible role injoint degenerationrdquo Laboratory Investigation vol 88 no 3 pp264ndash274 2008

[27] M Corr ldquoWnt-120573-catenin signaling in the pathogenesis ofosteoarthritisrdquo Nature Clinical Practice Rheumatology vol 4no 10 pp 550ndash556 2008

6 Evidence-Based Complementary and Alternative Medicine

Table 2 Histological score of articular cartilage

Femoral condyle Normal group Tet group OA groupStructural changes 02 plusmn 040 30 plusmn 063lowast 38 plusmn 117

Cellular changes 04 plusmn 049 16 plusmn 080lowast 18 plusmn 075

Safranin staining 04 plusmn 049 18 plusmn 075lowast 22 plusmn 075

Tide mark 02 plusmn 04 04 plusmn 049lowast 06 plusmn 049

Sum of score 12 plusmn 117 68 plusmn 133lowast 84 plusmn 185

Values are the means plusmn SD 119875 lt 005 when Normal group compared with OA group lowast119875 lt 005 when Normal group compared with Tet group

0

5

10

15

Normal Tet OA

Mod

ified

Man

kin

scor

e

lowast

Figure 4 The Mankin scores are also presented 119875 lt 005 whenNormal group compared with OA group lowast119875 lt 005 when Normalgroup compared with Tet group

stimulated with IL-1120573 in vitro have been used to mimic themicroenvironment that occurs in OA [19]

In this in vitro study we used IL-1120573 to induce MMP geneexpression based on the hypothesis above then evaluated theeffect of Tet on MMP induction in rabbit chondrocytes aswell as its effects on ACLT-induced OA in the rabbit model[20] We demonstrated that Tet inhibited the IL-1120573-mediatedinduction of MMP-1 MMP-3 and MMP-13 and induced theexpression of TIMP-1 a metalloproteinase inhibitor in rabbitarticular chondrocytes these effects were manifested at boththe mRNA and protein levels The MMPs are a family ofproteinases that are involved in ECM degradation MMP-1and MMP-13 in particular are interstitial collagenases thatdegrade type II collagen in cartilage a major component ofthe ECMwhich is a committed step in the progression ofOAAs a result targetingMMPs represents a promising approachto the treatment of OA MMPs can be induced by IL-1120573and increased expression of MMPs has been identified inosteoarthritic cartilage [21] Similarly serum concentrationsof MMP-3 are elevated in patients with OA compared withnormal subjects [22]

In the present study pretreatment with 20mgL Tetresulted in significantly decreased expression levels of MMP-1 MMP-3 and MMP-13 in IL-1120573-induced chondrocytes Tetalso induced the expression of TIMP-1 without causing cyto-toxicity Because TIMPs are endogenous inhibitors of MMPswe suggest that Tet may modulate the balance between

MMP and TIMP expression to exert its antiarthritic effectsin IL-1120573-treated rabbit articular chondrocytes [23] We alsoinvestigated in vivo the effects of Tet on cartilage degradationin an ACLT-based experimental model of OA in rabbitsThismodel has been widely used to evaluate the efficacy of agentsin the treatment ofOAdue to themodelrsquosmechanical instabil-ity which leads to cartilage degradation [24] As shown in thisstudy ACLT in the rabbit resulted in cartilage degradationand injection of Tet into the articular cavity (03mL 20mgL)for 6 weeks inhibited cartilage degradation as assessed byhistological evaluationThe results were consistentwith the invitro findings These observations provide evidence that Tetpossesses chondroprotective activity both in vitro and in vivo

Several signalling pathways are involved in the inductionofMMPs by IL-1120573 in chondrocytes Among them theWnt120573-catenin signalling pathway and proteins regulate organdevelopment tumourigenesis and bone homeostasis amongother functions and are believed to play important roles inthe induction of MMP expression [25 26] Given this agentsthat interfere with theWnt120573-catenin signalling pathwaymayaffect MMP expression In the present study we investigatedwhether Tet affected activation depending on the Wnt120573-catenin signalling pathway 120573-catenin is a multifunctionalprotein involved in the formation of adhesive ldquotaperdquo byinteracting with cadherin at cell junctions Furthermorefree 120573-catenin can penetrate into the nucleus to affect geneexpression [27]

Our in vitro and in vivo findings suggest that expressionof MMP and 120573-catenin genes showed a similar trend as didthe protein levels We demonstrated that Tet effectively tar-gets the Wnt120573-catenin signalling pathway in chondrocytessuggesting that inhibition of the Wnt120573-catenin signallingpathway may at least in part account for changes in thetrends in the expression of MMPs and TIMP-1 as well ascartilage protection This study is to our knowledge the firstto report the effects of Tet on Wnt120573-catenin signalling inchondrocytes and in an ACLT-based experimental model ofOA in rabbits These findings are consistent with previousstudies of the effects of Tet on the Wnt120573-catenin signallingpathway in other cell systems [14]The discrepancies betweenour findings and these reports may be related to differencesin cell type and stimulation conditions

In conclusionmodulation of cytokines such as IL-1120573 andTNF-120572 which control MMP gene overexpression appears tobe a fertile target for drug development for the treatment ofOA Several studies have illustrated the potential importance

Evidence-Based Complementary and Alternative Medicine 7

of modulating IL-1 activity as a means to reduce the progres-sion of structural changes in OA [24] In the present study wedemonstrated that Tet possessed chondroprotective effectsin IL-1120573-induced rabbit chondrocytes and an experimentalmodel of OA The inhibition of MMPs by Tet was associatedat least in part with inhibition of theWnt120573-catenin signallingpathway Our results indicate that Tet shows promise as atherapeutic agent for the treatment of OA However furtherstudies are needed to confirm and extend these preliminaryfindings

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

X Zhou and LWu designed the research X ZhouW Li andL Jiang performed the research X Zhou and J Bao L Taoand J Li analyzed the data and X Zhou wrote the paper

Acknowledgment

This study was supported by the National Natural ScienceFoundation of China (81071492)

References

[1] D T Felson ldquoEpidemiology of hip and knee osteoarthritisrdquoEpidemiologic Reviews vol 10 pp 1ndash28 1988

[2] M B Goldring ldquoThe role of the chondrocyte in osteoarthritisrdquoArthritis amp Rheumatism vol 43 no 9 pp 1916ndash1926 2000

[3] MGowenDDWood andRGG Russell ldquoStimulation of theproliferation of human bone cells in vitro by human monocyteproducts with interleukin-1 activityrdquo Journal of Clinical Investi-gation vol 75 no 4 pp 1223ndash1229 1985

[4] J A Mengshol M P Vincenti C I Coon A Barchowsky andC E Brinckerhoff ldquoInterleukin-1 induction of collagenase 3(matrix metalloproteinase 13) gene expression in chondrocytesrequires p38 c-Jun N-terminal kinase and nuclear factorkappaB differential regulation of collagenase 1 and collagenase3rdquo Arthritis amp Rheumatism vol 43 no 4 pp 801ndash811 2000

[5] N Ishiguro T Ito H Ito et al ldquoRelationship of matrix metal-loproteinases and their inhibitors to cartilage proteoglycan andcollagen turnover analyses of synovial fluid from patients withosteoarthritisrdquo Arthritis amp Rheumatism vol 42 no 1 pp 129ndash136 2001

[6] Y Okada M Shinmei O Tanaka et al ldquoLocalization of matrixmetalloproteinase 3 (stromelysin) in osteoarthritic cartilage andsynoviumrdquo Laboratory Investigation vol 66 no 6 pp 680ndash6901992

[7] R M Hembry M R Bagga J J Reynolds and D L HamblenldquoImmunolocalisation studies on six matrix metalloproteinasesand their inhibitors TIMP-1 and TIMP-2 in synovia frompatients with osteo- and rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 54 no 1 pp 25ndash32 1995

[8] I Uthman J P Raynauld and B Haraoui ldquoIntra-articulartherapy in osteoarthritisrdquo Postgraduate Medical Journal vol 79no 934 pp 449ndash453 2003

[9] N H Mashour G I Lin and W H Frishman ldquoHerbalmedicine for the treatment of cardiovascular disease clinicalconsiderationsrdquo Archives of Internal Medicine vol 158 no 20pp 2225ndash2234 1998

[10] X F Yu C Q Zou and M B Lin ldquoObservation of the effect oftetrandrine on experimental silicosis of ratsrdquo Ecotoxicology andEnvironmental Safety vol 7 no 3 pp 306ndash312 1983

[11] L J Ho and J H Lai ldquoChinese herbs as immunomodulators andpotential disease-modifying antirheumatic drugs in autoim-mune disordersrdquoCurrent DrugMetabolism vol 5 no 2 pp 181ndash192 2004

[12] H S Kim YH Zhang andY P Yun ldquoEffects of tetrandrine andfangchinoline on experimental thrombosis in mice and humanplatelet aggregationrdquo Planta Medica vol 65 no 2 pp 135ndash1381999

[13] F P Luyten P Tylzanowski and R J Lories ldquoWnt signaling andosteoarthritisrdquo Bone vol 44 no 4 pp 522ndash527 2009

[14] B C He J L Gao B Q Zhang et al ldquoTetrandrine inhibitsWntbeta-catenin signaling and suppresses tumor growth ofhuman colorectal cancerrdquoMolecular Pharmacology vol 79 no2 pp 211ndash219 2011

[15] L J Brunet J AMcMahonA PMcMahon andRMHarlandldquoNoggin cartilage morphogenesis and joint formation in themammalian skeletonrdquo Science vol 280 no 5368 pp 1455ndash14571998

[16] H J Mankin M E Johnson and L Lippiello ldquoBiochem-ical and metabolic abnormalities in articular cartilage fromosteoarthritic human hips III Distribution and metabolism ofamino sugar-containing macromoleculesrdquo Journal of Bone andJoint Surgery A vol 63 no 1 pp 131ndash139 1981

[17] J C Fernandes J Martel-Pelletier and J P Pelletier ldquoThe roleof cytokines in osteoarthritis pathophysiologyrdquoBiorheology vol39 no 1-2 pp 237ndash246 2002

[18] M Lotz ldquoCytokines in cartilage injury and repairrdquo ClinicalOrthopaedics and Related Research supplement 391 pp S108ndashS115 2001

[19] F Moldovan J P Pelletier F C Jolicoeur J M Cloutier and JMartel-Pelletier ldquoDiacerhein and rhein reduce the ICE-inducedIL-1120573 and IL-18 activation in human osteoarthritic cartilagerdquoOsteoarthritis and Cartilage vol 8 no 3 pp 186ndash196 2000

[20] P F Hu Y Chen P F Cai L F Jiang and L D WuldquoSphingosine-1-phosphate a potential therapeutic target forrheumatoid arthritisrdquoMolecular Biology Reports vol 38 no 6pp 4225ndash4230 2011

[21] M P Vincenti and C E Brinckerhoff ldquoTranscriptional reg-ulation of collagenase (MMP-1 MMP-13) genes in arthritisintegration of complex signaling pathways for the recruitmentof gene-specific transcription factorsrdquo Arthritis Research vol 4no 3 pp 157ndash164 2002

[22] L S Lohmander P J Neame and J D Sandy ldquoThe structureof aggrecan fragments in human synovial fluid evidence thataggrecanase mediates cartilage degradation in inflammatoryjoint disease joint injury and osteoarthritisrdquo Arthritis andRheumatism vol 36 no 9 pp 1214ndash1222 1993

[23] D D Dean J Martel-Pelletier J P Pelletier D S Howelland J F Woessner ldquoEvidence for metalloproteinase inhibitorimbalance in human osteoarthritic cartilagerdquo Journal of ClinicalInvestigation vol 84 no 2 pp 678ndash685 1989

[24] W P Chen P F Hu J P Bao and L D Wu ldquoMorin exertsantiosteoarthritic properties an in vitro and in vivo studyrdquoExperimental Biology andMedicine vol 237 no 4 pp 380ndash3862012

8 Evidence-Based Complementary and Alternative Medicine

[25] G Schett J Zwerina and J P David ldquoThe role of Wnt proteinsin arthritisrdquoNature Clinical Practice Rheumatology vol 4 no 9pp 473ndash480 2008

[26] T Yuasa T Otani T Koike M Iwamoto and M Enomoto-Iwamoto ldquoWnt120573-catenin signaling stimulates matrix catabolicgenes and activity in articular chondrocytes is possible role injoint degenerationrdquo Laboratory Investigation vol 88 no 3 pp264ndash274 2008

[27] M Corr ldquoWnt-120573-catenin signaling in the pathogenesis ofosteoarthritisrdquo Nature Clinical Practice Rheumatology vol 4no 10 pp 550ndash556 2008

Evidence-Based Complementary and Alternative Medicine 7

of modulating IL-1 activity as a means to reduce the progres-sion of structural changes in OA [24] In the present study wedemonstrated that Tet possessed chondroprotective effectsin IL-1120573-induced rabbit chondrocytes and an experimentalmodel of OA The inhibition of MMPs by Tet was associatedat least in part with inhibition of theWnt120573-catenin signallingpathway Our results indicate that Tet shows promise as atherapeutic agent for the treatment of OA However furtherstudies are needed to confirm and extend these preliminaryfindings

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

X Zhou and LWu designed the research X ZhouW Li andL Jiang performed the research X Zhou and J Bao L Taoand J Li analyzed the data and X Zhou wrote the paper

Acknowledgment

This study was supported by the National Natural ScienceFoundation of China (81071492)

References

[1] D T Felson ldquoEpidemiology of hip and knee osteoarthritisrdquoEpidemiologic Reviews vol 10 pp 1ndash28 1988

[2] M B Goldring ldquoThe role of the chondrocyte in osteoarthritisrdquoArthritis amp Rheumatism vol 43 no 9 pp 1916ndash1926 2000

[3] MGowenDDWood andRGG Russell ldquoStimulation of theproliferation of human bone cells in vitro by human monocyteproducts with interleukin-1 activityrdquo Journal of Clinical Investi-gation vol 75 no 4 pp 1223ndash1229 1985

[4] J A Mengshol M P Vincenti C I Coon A Barchowsky andC E Brinckerhoff ldquoInterleukin-1 induction of collagenase 3(matrix metalloproteinase 13) gene expression in chondrocytesrequires p38 c-Jun N-terminal kinase and nuclear factorkappaB differential regulation of collagenase 1 and collagenase3rdquo Arthritis amp Rheumatism vol 43 no 4 pp 801ndash811 2000

[5] N Ishiguro T Ito H Ito et al ldquoRelationship of matrix metal-loproteinases and their inhibitors to cartilage proteoglycan andcollagen turnover analyses of synovial fluid from patients withosteoarthritisrdquo Arthritis amp Rheumatism vol 42 no 1 pp 129ndash136 2001

[6] Y Okada M Shinmei O Tanaka et al ldquoLocalization of matrixmetalloproteinase 3 (stromelysin) in osteoarthritic cartilage andsynoviumrdquo Laboratory Investigation vol 66 no 6 pp 680ndash6901992

[7] R M Hembry M R Bagga J J Reynolds and D L HamblenldquoImmunolocalisation studies on six matrix metalloproteinasesand their inhibitors TIMP-1 and TIMP-2 in synovia frompatients with osteo- and rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 54 no 1 pp 25ndash32 1995

[8] I Uthman J P Raynauld and B Haraoui ldquoIntra-articulartherapy in osteoarthritisrdquo Postgraduate Medical Journal vol 79no 934 pp 449ndash453 2003

[9] N H Mashour G I Lin and W H Frishman ldquoHerbalmedicine for the treatment of cardiovascular disease clinicalconsiderationsrdquo Archives of Internal Medicine vol 158 no 20pp 2225ndash2234 1998

[10] X F Yu C Q Zou and M B Lin ldquoObservation of the effect oftetrandrine on experimental silicosis of ratsrdquo Ecotoxicology andEnvironmental Safety vol 7 no 3 pp 306ndash312 1983

[11] L J Ho and J H Lai ldquoChinese herbs as immunomodulators andpotential disease-modifying antirheumatic drugs in autoim-mune disordersrdquoCurrent DrugMetabolism vol 5 no 2 pp 181ndash192 2004

[12] H S Kim YH Zhang andY P Yun ldquoEffects of tetrandrine andfangchinoline on experimental thrombosis in mice and humanplatelet aggregationrdquo Planta Medica vol 65 no 2 pp 135ndash1381999

[13] F P Luyten P Tylzanowski and R J Lories ldquoWnt signaling andosteoarthritisrdquo Bone vol 44 no 4 pp 522ndash527 2009

[14] B C He J L Gao B Q Zhang et al ldquoTetrandrine inhibitsWntbeta-catenin signaling and suppresses tumor growth ofhuman colorectal cancerrdquoMolecular Pharmacology vol 79 no2 pp 211ndash219 2011

[15] L J Brunet J AMcMahonA PMcMahon andRMHarlandldquoNoggin cartilage morphogenesis and joint formation in themammalian skeletonrdquo Science vol 280 no 5368 pp 1455ndash14571998

[16] H J Mankin M E Johnson and L Lippiello ldquoBiochem-ical and metabolic abnormalities in articular cartilage fromosteoarthritic human hips III Distribution and metabolism ofamino sugar-containing macromoleculesrdquo Journal of Bone andJoint Surgery A vol 63 no 1 pp 131ndash139 1981

[17] J C Fernandes J Martel-Pelletier and J P Pelletier ldquoThe roleof cytokines in osteoarthritis pathophysiologyrdquoBiorheology vol39 no 1-2 pp 237ndash246 2002

[18] M Lotz ldquoCytokines in cartilage injury and repairrdquo ClinicalOrthopaedics and Related Research supplement 391 pp S108ndashS115 2001

[19] F Moldovan J P Pelletier F C Jolicoeur J M Cloutier and JMartel-Pelletier ldquoDiacerhein and rhein reduce the ICE-inducedIL-1120573 and IL-18 activation in human osteoarthritic cartilagerdquoOsteoarthritis and Cartilage vol 8 no 3 pp 186ndash196 2000

[20] P F Hu Y Chen P F Cai L F Jiang and L D WuldquoSphingosine-1-phosphate a potential therapeutic target forrheumatoid arthritisrdquoMolecular Biology Reports vol 38 no 6pp 4225ndash4230 2011

[21] M P Vincenti and C E Brinckerhoff ldquoTranscriptional reg-ulation of collagenase (MMP-1 MMP-13) genes in arthritisintegration of complex signaling pathways for the recruitmentof gene-specific transcription factorsrdquo Arthritis Research vol 4no 3 pp 157ndash164 2002

[22] L S Lohmander P J Neame and J D Sandy ldquoThe structureof aggrecan fragments in human synovial fluid evidence thataggrecanase mediates cartilage degradation in inflammatoryjoint disease joint injury and osteoarthritisrdquo Arthritis andRheumatism vol 36 no 9 pp 1214ndash1222 1993

[23] D D Dean J Martel-Pelletier J P Pelletier D S Howelland J F Woessner ldquoEvidence for metalloproteinase inhibitorimbalance in human osteoarthritic cartilagerdquo Journal of ClinicalInvestigation vol 84 no 2 pp 678ndash685 1989

[24] W P Chen P F Hu J P Bao and L D Wu ldquoMorin exertsantiosteoarthritic properties an in vitro and in vivo studyrdquoExperimental Biology andMedicine vol 237 no 4 pp 380ndash3862012

8 Evidence-Based Complementary and Alternative Medicine

[25] G Schett J Zwerina and J P David ldquoThe role of Wnt proteinsin arthritisrdquoNature Clinical Practice Rheumatology vol 4 no 9pp 473ndash480 2008

[26] T Yuasa T Otani T Koike M Iwamoto and M Enomoto-Iwamoto ldquoWnt120573-catenin signaling stimulates matrix catabolicgenes and activity in articular chondrocytes is possible role injoint degenerationrdquo Laboratory Investigation vol 88 no 3 pp264ndash274 2008

[27] M Corr ldquoWnt-120573-catenin signaling in the pathogenesis ofosteoarthritisrdquo Nature Clinical Practice Rheumatology vol 4no 10 pp 550ndash556 2008

8 Evidence-Based Complementary and Alternative Medicine

[25] G Schett J Zwerina and J P David ldquoThe role of Wnt proteinsin arthritisrdquoNature Clinical Practice Rheumatology vol 4 no 9pp 473ndash480 2008

[26] T Yuasa T Otani T Koike M Iwamoto and M Enomoto-Iwamoto ldquoWnt120573-catenin signaling stimulates matrix catabolicgenes and activity in articular chondrocytes is possible role injoint degenerationrdquo Laboratory Investigation vol 88 no 3 pp264ndash274 2008

[27] M Corr ldquoWnt-120573-catenin signaling in the pathogenesis ofosteoarthritisrdquo Nature Clinical Practice Rheumatology vol 4no 10 pp 550ndash556 2008