ligustrazine inhibits cartilage endplate hypertrophy via

Research ArticleLigustrazine Inhibits Cartilage Endplate Hypertrophy viaSuppression of TGF-1205731

Shufen Liu12 Bizeng Zhao3 Huipeng Shi3 Qianqian Liang12 Yishan Fu3 Zhu Yang12

Leqin Xu12 Yongjun Wang12 and Qin Bian12

1Department of Orthopaedics amp Traumatology Longhua Hospital Shanghai University of Traditional Chinese MedicineNo 725 South Wanping Road Shanghai 200032 China2Institute of Spine Shanghai University of Traditional Chinese Medicine No 725 South Wanping Road Shanghai 200032 China3Department of Orthopaedics Sixth Peoplersquos Hospital Jiaotong University No 600 Yi Shan Road Shanghai 200233 China

Correspondence should be addressed to Yongjun Wang yjwang88126com and Qin Bian qbian1jhmiedu

Received 18 April 2016 Accepted 27 June 2016

Academic Editor Hyunsu Bae

Copyright copy 2016 Shufen Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

CEP hypertrophy is one of the characteristics of intervertebral disc degeneration (IDD) LIG exerts a protective effect on IDDin animal model The effect of LIG on CEP hypertrophy is further investigated in the present study Cells were isolated fromhypertrophic samples obtained from patients during vertebral fusion surgery Cellular proliferation and the expression of type Icollagen (Col I) and TGF-1205731 were tested In the bipedal rats the edges of the CEP and the sizes of noncartilaginous outgrowth aswell as the expression of osteogenic markers Col1a ALP Runx2 and TGF-1205731 were detected Within two passages the condensedhypertrophic CEP cells exhibited osteogenic capacity by bony-like nodules and ALP positive staining along with increasedexpression of Col I and TGF-1205731 LIG inhibited proliferation of CEP cells and downregulated the expression of Col I and TGF-1205731 in vitro Furthermore LIG attenuated CEP hypertrophy on the lumbar spine of bipedal rats by reducing Col1a ALP Runx2 andTGF-1205731 mRNA expression and TGF-1205731 distribution in vivo We concluded LIG exerted a preventive effect on CEP hypertrophyvia suppression of TGF-1205731 levels This information could be used to develop alternative therapeutic methods to treat spinal CEPhypertrophy

1 Introduction

Cartilaginous endplate (CEP) hypertrophy a distinct featureof intervertebral disc degeneration (IDD) is considered asthe early stage of osteophyte formation which is adaptivebone remodeling in response to progressive changes in themechanical environment [1 2] More evidence has shown thedegree of IDD to be positively correlated with the size of bonyoutgrowths or calcified hypertrophy [3 4]

Although therapies suitable to the relief of pain and im-provement of function in IDD patients such as nonsteroidalanti-inflammatory drugs (NSAIDs) nonopioid analgesicsopioid analgesics glucosamine and chondroitin have beenstudied only a few have been verified to have an effect onCEP hypertrophy or following osteophyte development Forexample long-term estrogen replacement therapy (ERT)

was found to reduce the prevalence of abaxial osteophytesin the lateral tibial plateau but not the medial plateauin an ovariectomized (OVX) cynomolgus macaques modelof naturally occurring OA [5] Bisphosphonates may haveboth chondroprotective and osteophyte-preventive effects asdetermined by analysis of data from a randomized controlledtrial [6]The level of beta-carotene was low in elderly patientswith lumbar osteophytes suggesting that this antioxidant hadan inhibitory on lumbar spine degeneration [7] Howevernewer and safer drugs that are also effective in the preventionof CEP hypertrophy and osteophytes development are stillpursued

Ligustrazine (LIG) extracted from Ligusticum chuangx-iong hort has been reported to have a therapeutic effect onOA and one study observed few adverse effects during 5weeks for treatment and 3 months of follow-up [8] In our

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 1042489 9 pageshttpdxdoiorg10115520161042489

2 Evidence-Based Complementary and Alternative Medicine

previous studies we found LIG exerts a preventive effect onIDD in animalmodel [9] Moreover the qishejingkang recipe(QSJKR) of which LIG is the major constituent was foundto decrease the activity of alkaline phosphatase (ALP) indegenerated vertebral discs and inhibit CEPhypertrophy [10]Therefore we entered the present project expecting LIG tohave an inhibitory effect on CEP hypertrophy

To determine the inhibitory effect of LIG in vitro wecollected hypertrophic CEP samples from patient during ver-tebral fusion surgery Cells were isolated and cultured Cellu-lar proliferation and the mRNA and protein levels of Col1aand TGF-1205731 were assayed using MTT qPCR immunofluo-rescence and western blot analysis To investigated whetherLIG could attenuate CEP hypertrophy in vivo we employeda CEP hypertrophy rat model as previously reported [411] Safranin O fast green staining and morphometry wereperformed to determine the edges of the CEP and the sizesof noncartilaginous outgrowths The mRNA expressions oftype I collagen (Col1a) ALP runt-related transcription factor2 (Runx2) and TGF-1205731 were detected by qPCR TGF-1205731protein distributions were observed using immunohisto-chemistry Our results revealed that LIG has a preventiveeffect on CEP hypertrophy via the suppression of TGF-1205731levels This information could be used to develop alternativetherapeutic for spinal degeneration disease accompanied byCEP hypertrophy

2 Materials and Methods

21 Cell Culture and Drug Preparation Samples were ob-tained from ten patients during vertebral fusion surgery(with previous oral informed consent approved by EthicsCommittee of the Sixth Peoplersquos hospital Shanghai CN)The samples were washed with Dulbecco modified Eaglersquosmedium with high glucose content (DMEM-HG BiowestNuaille France) Then the tissues were dissected into 1-2-mm3 pieces and cultured in a medium containing DMEM-HG 20 fetal bovine serum (FBS Gibco Langley OKUSA)and 1 penicillin-streptomycin (Gibco Langley OK USA)

Ligustrazine phosphate (purity gt 99 MW 25221)was purchased from the Chinese Medicines and BiologicalProducts Institute (Beijing CN)The solutions of ligustrazinephosphate were prepared in dimethylsulfoxide (DMSOSigma USA) for the in vitro experiments The final concen-tration of DMSO was 01

22 Animal Models and Drug Administration Male Sprague-Dawley (SD) rats aged 1 month (119899 = 30) were providedby the Shanghai Laboratory Animal Center (SYXK2003-0002 Science and Technology Commission of ShanghaiMunicipality gave approval for this experimental study onanimals) were randomly divided into Sham Vehicle (Veh)and LIG groups In the Veh and LIG groups rats were forcedto stand up by forelimbs surgery as previously described [12]In the Sham group the rats did not receive any treatmentand were maintained in standard cages Eight months afterthe surgery ligustrazine hydrochloride (Nanning Maple LeafPharmaceutical Co Ltd CN (lot number 051125)) was

intraperitoneally injected into rats of LIG group (16mLkgsdotd10mL sterile saline 40mg ligustrazine hydrochloride) whilesterile saline of equal volume was injected into rats of Vehgroup once a day for one month Rats (119899 = 10) in each groupwere euthanized at 9 months after the surgery Their lumbarspines were dissected for analysis

23 ALP Assay The cells were fixed with 10 formalin andstainedwith 1-StepNBT-BCIP (Pierce USA) for 30minutesLyons blue represents the positive staining The images werescanned (CanoScan 8800F Japan)

24 MTT Detection Passage III hypertrophic CEP cells werecultured in 96-well plates at 1 times 105mL with 200120583L medium(the medium contained LIG and 20 FBS) for 8 wells pergroup After being cultured for 1 5 9 and 13 days 20120583L of5mgmLMTT reagent was added to each well and they wereincubated for 4 hours at 37∘CMedia were removed followedby adding 150120583L MTT solvent

The OD values were tested at 590 nm after shaking for15min A cellular growth curve was produced to reflect theOD values

25 qPCR RNA was extracted from L1-2 L2-3 and L3-4 mar-ginal discs (noncartilaginous outgrowth) or hypertrophicCEP cells treated by LIG for 48 hours using 1mL TRIzolreagent (Sigma St Louis MO USA) according to the man-ufacturerrsquos instruction Cellstissues were directly processedfollowing RNA preparation employing the PURE Prep Kitprotocol One microgram of total RNA was reverse-tran-scribed using the Advantage RT-for-PCR Kit (Qiagen Valen-cia CA USA) according to the manufacturerrsquos instructionsFreshly transcribed cDNA (1120583L) was employed for qPCRusing SYBR Green (Bio-Bad Hercules CA USA) to monitorDNA synthesis with specific primers (Tables 1 and 2) designedby TaKaRa Biotechnology Co Ltd Gene expression wasnormalized to the housekeeping gene 120573-actin PCR productswere subjected to melting-curve analysis and data wereanalyzed and quantified with the RotorGene 60 analysissoftware

26 Western Blotting Cells were cultured in basal mediumor 10minus7M LIG for 10 days At the end of the study the cellswere washed with PBS scraped and resuspended in lysate(Beyotime Hangzhou CN) for 1 hour on ice The cell lysateswere centrifuged at 12000 rpm for 10 minutes at 4∘C Thesupernatant was transferred to a new microcentrifuge tubeand protein concentration wasmeasured by Bradford proteinassay (Beyotime Hangzhou CN) Proteins were added with4x sample buffer (001 bromophenol blue 0125M TrispH 68 10 glycerol and 2 SDS) and denaturized at 95∘Cfor 10 minutes Equal amounts of protein (50 120583glane) weresolubilized in Laemmli sample buffer and loaded onto amini-SDS-PAGE system Following electrophoresis the proteinswere transferred to a polyvinylidene difluoride (PVDF)membrane (Millipore Temecula CA USA) using a Bio-Radwet transfer system Protein transfer efficiency was verifiedusing prestained protein markers The membranes were then

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Sequences of primers for rats used in the qPCR

Genes Forward primer Reverse primer Product length (bp)120573-actin GGAGATTACTGCCCTGGCTCCTA GACTCATCGTACTCCTGCTTGCTG 150Col 11205722 TCCTGGCAATCGTGGTTCAA ACCAGCTGGGCCAACATTTC 133Runx2 CCATAACGGTCTTCACAAATCCT TCTGTCTGTGCCTTCTTGGTTC 99TGF-1205731 TGCGCCTGCAGAGATTCAAG AGGTAACGCCAGGAATTGTTGCTA 82ALP TTGAATCGGAACAACCTGACTGAC GATGGCCTCATCCATCTCCAC 183

Table 2 Sequences of primers for humans used in the qRT-PCR

Genes Forward primer Reverse primer Product length (bp)120573-actin CCTGTACGCCAACACAGTGC ATACTCCTGCTTGCTGATCC 211Col 11205721 AGAGGGCAGCCGCAAGAAC CTGGCCGCCATACTCGAACT 280TGF-1205731 CCGACTACTACGCCAAGGA CTGAGGTATCGCCAGGAAT 247

blocked with 5 nonfat milk for 1 hour at room temperatureand subsequently incubated overnight at 4∘C with TGF-1205731 antibodies directed against the protein (1 500 LifespanBioSciences Seattle WA USA) Chemiluminescence imageswere obtained by application of LAS-4000 (Fujinfilm TokyoJapan) and Image Reader LAS-3000 software in precisionmode

27 Safranin O Fast Green Staining The lumbar spines of therats were fixed in 4 paraformaldehyde for 24 hours andwashed for 2 hoursThen they were decalcified in 20 EDTAfor 4 weeks and the fluid was changed once a week Lumbarspines were dehydrated and embedded in paraffin wax Atleast four consecutive sections of 7 120583m in thickness wereobtained from the sagittal planes and stained with safranin Oand fast green A morphometric study was performed usingan image auto-analysis system (Olympus BX50 Japan) L

4

were examined

28 Immunohistochemical Analysis and Cytoimmunofluores-cence Sections were pretreated and incubated with anti-body against TGF-1205731 (1 100 Cell Signaling Inc MA USA)overnight at 4∘C and incubated with biotinylated goat anti-rabbit-IgG for 15 minutes at 37∘C This was followed bystreptavidin-HRP for 10 minutes at 37∘C Bound complexeswere visualized using 331015840-diaminobenzidine (DAB) reagentcounterstained with hematoxylin dehydrated and mountedwith gummy for immunohistochemical assays The datawere quantified using a medical image management system(Cmias CN)

In cytoimmunofluorescence assays samples were incu-bated with antibody against Col I (1 500 Abcam Ltd Cam-bridge UK) overnight at 4∘CThey were then incubated withthe secondary fluorescent antibodies for 1 hour at 37∘C andcounterstained with 2-(4-amidinophenyl)-6-indolecarbami-dine dihydrochloride (DAPI) Light (Olympus DP71 Japan)and fluorescence microscopes (Leica DM3000B Germany)were used

29 Statistical Analyses The data are expressed as means plusmnSE and statistical significance was calculated using one-wayANOVA followed by a post-hoc LSD test (homogeneity ofvariance) and Tukeyrsquos test (heterogeneity of variance) usingSPSS software (SPSS Inc Chicago USA) The significancelevel was defined as 119901 lt 005

3 Results

31 Isolation and Characteristics of Cells from HypertrophicCEP Samples We collected hypertrophic CEP samples frompatients who underwent vertebral fusion surgery and cul-tured these tissues of small pieces Hypertrophic CEP-derivedcells could be seen to slough off from the side of the tissue onday 16

To obtain adequate cells for experiments we identifiedthat passage had little effect on cellular morphology Wefound that these cells showed polygonal and spear-likemorphology following initial passage After the first andsecond passages several bony nodules could be seen eventhough the tissue pieces had been removed off Cells retainedthese shapes and began to grow from or up to the nodules(Figure 1(a))

As these bony nodules were seen when the cells reachedto a high concentration we hypothesized that hypertrophicCEP cells have osteogenic capacity when they undergo con-densation as hypertrophic chondrocytes do during primaryossification [13] To test this hypothesis ALP assay was usedThe results showed negativeALP-staining for cells of low con-centration on day 3 of culture However the condensed cellsspontaneously showed strong positive staining on day 26of culture indicating these cells had an osteogenic potential(Figure 1(b))

To further identify the characteristics of hypertrophicCEP cells in these two states low concentration and highconcentration (condensation) we detected function proteinof osteoblasts type I collagen The mRNA level of Col1a1 of


times240 times120

1st passage 2nd passage

times120

16th day ex vivo

(a)

3d 26d

(b)

0

1

2

3

4

Fold

indu

ctio

n

Col I mRNA

lowastlowast

3d 26d(c)

0

1

2

3

Fold

indu

ctio

n

TGF-1205731 mRNA

lowast

3d 26d(d)

GAPDH

3d 26d

TGF-1205731

(e)

Figure 1 Characteristics of hypertrophic CEP cells from patient samples (a) Hypertrophic CEP-derived cells could be seen sloughing offthe tissue on day 16 After the first and second passages several bony-like nodules could be seen although the CEP pieces had been removedoff (b) Cells with low concentration were negative for ALP-staining on day 3 On day 26 the condensed cells spontaneously showed strongpositive ALP staining (c d) mRNA levels of (c) Col1a1 and (d) TGF-120573 were detected by qPCR (e) TGF-120573 protein level was tested by westernblot Each column represents the mean plusmn SE of three independent experiments lowast119901 lt 005 lowastlowast119901 lt 001

the condensed cells (26 d) was higher than that in the cells atlow concentration (3 d) (119901 lt 001) (Figure 1(c))

Then we tested TGF-1205731 expression of hypertrophic CEPcells since TGF-1205731 has been reported to contribute to CEPhypertrophy [14] We found condensed cells expressed moreTGF-1205731 and protein than cells of low concentration did(Figures 1(d) and 1(e))

Taken together the results suggested that condensed hy-pertrophic CEP cells have osteogenic capacities

32 Effects of LIG on Proliferation and Col1a and TGF-1205731Expression in Hypertrophic CEP Cells To investigate if LIGhas effect on proliferation of hypertrophic CEP cells we didMTT assays The results showed a decrease in cell prolifera-tion by LIG treatment for 13 days at two dosesThe inhibitoryeffect of LIG was more pronounced at a dose of 10minus5M than10minus7M (Figure 2(a))

Then we tested the effect of LIG on Col1a and TGF-1205731expression of condensed hypertrophic CEP cells As a result


MTT

0

05

1

15

2

OD

val

ue

0M10minus5 M10minus7 M

1d 5d 9d 13d

(a)

0

05

1

15 Col I mRNA

Fold

indu

ctio

n

lowastlowast

0 10minus5 10minus7MM M

(b)

Col I

0M 10minus7 M

(c)

120573-actin

0M 10minus7 M

TGF-1205731

(d)

Figure 2 Effects of LIG on cell proliferation and Col1a and TGF-120573 expression in vitro (a) MTT assays showed that passage-III hypertrophicCEP cell proliferation decreased on day 13 after LIG treatment at two doses compared to the control (0M) The inhibitory effect ofLIG was more pronounced at a dose of 10minus5M than at 10minus7M (bndashd) Condensed cells were treated with LIG (b) Col1a1 expression wassignificantly decreased by LIG of 10minus7M LIG also showed inhibitory tendency at a dose of 10minus5M with no statistical significance (c)Cytoimmunofluorescence results showed attenuated positive staining for Col I in the extracellular matrix with LIG of 10minus7M comparedto the nontreatment (d) Western blot analysis of TGF-120573 expression by 10 days of LIG treatment at a dose of 10minus7M Each column representsthe mean plusmn SE of three independent experiments lowastlowast119901 lt 001 versus 0M

LIG treatment decreased Col1a1 expression of the condensedcells at the dose of 10minus7M (119901 lt 001) A dose of 10minus5MLIG also showed a decreased tendency but no significantdifferences were found (Figure 2(b)) The results have beenidentified by cytoimmunofluorescence examination whichdemonstrated attenuated positive staining for Col I in theextracellular matrix of hypertrophic CEP cells by treatmentwith 10minus7M LIG (Figure 2(c)) In addition the protein levelof TGF-1205731 was significantly suppressedwith LIG at same doseby western blot analysis (Figure 2(d))

33 The Effects of LIG on CEP Hypertrophy on Lumbar Spineof Rats To investigate the effect of LIG on CEP hypertrophyin vivo we employed a rat model as previously reported toinduce CEP hypertrophy [14] After 1-month treatment ofLIG the lumbar spines of rats were collected for examina-tion The distance between marginal articular cartilage (redstaining) and marginal noncartilaginous outgrowth (green

staining) was measured which is defined as the thickness oflimbic hypertrophy (LHT) as described previously Resultsshowed a significant increase in LHT in rats kept in an uprightposture for 9months LIGwas found to significantly decreaseLHT (Figures 3(a) and 3(b))

To further examine the inhibitory effects of LIG onCEP hypertrophy through inhibition of osteogenic func-tion three osteogenesis-related markers were detected OurqPCR studies demonstrated that the expression levels of thethree osteogenesis-related markers Col1 ALP and Runx2increased in Veh samples relative to Sham However LIGtreatment caused significant decreases in expression of thesethree genes as compared to Veh treatment (Figures 1(c)ndash1(e))

34The Effects of LIG on the TGF-1205731 Expression in CEP Tissueof Rats As increased TGF-1205731 expression was found in vitrowe further confirm it in vivo Strong positive matrix stainingfor TGF-1205731 was detected in the marginal area in the Veh


Sham Veh LIG

LHT

times200(a)

0

1

2

3 LHT

Fold

indu

ctio

n

Sham Veh LIG

lowastlowast

(b)

Fold

indu

ctio

n0

10

20

30 Col1a2 mRNA

Sham Veh LIG

lowastlowast

(c)

Sham Veh LIG0

2

4

6

8

10

12

Fold

indu

ctio

n

Runx2 mRNAlowastlowast

(d)Sham Veh LIG

0

5

10

15

20 ALP mRNA

Fold

indu

ctio

n

lowastlowast

(e)

Figure 3 Effects of LIG onCEP hypertrophy of the lumbar spine in bipedal rats (a b) Representative images of SafraninO fast green stainingof L4section showed a significant increase in OFT at the marginal disc of bipedal rats LIG partially reduced the noncartilaginous outgrowth

(cndashe)The expression of the three osteogenesis-relatedmarkers (c) Col1a2 (d) ALP (e) Runx2 in the Sham Veh and LIG groups Each columnrepresents the mean plusmn SE lowastlowast119901 lt 001 versus Sham and 119901 lt 001 versus Veh

group This was much weaker in the Sham and LIG groups(Figures 4(a) and 4(b)) TGF-1205731 mRNA levels showed thesame tendency as TGF-1205731 protein levels in all three groups(119901 lt 001) (Figure 4(c))

4 Discussion

In this study we revealed the inhibitory effect of LIG onCEP hypertrophy via suppression of TGF-1205731 expression bothin vivo and in vitro Our study is the first to report a pre-ventive effect of LIG From these results it seems suitableas an alternative therapeutic method for spinal degenerativedisease accompanied by CEP hypertrophy

Pathologic characteristic manifestations of spinal degen-eration and CEP hypertrophy and following osteophyteformation are commonly seen An experimental model ofcervical spondylosis in rabbits showed a process of osteophyteformation involving endochondral calcification and ossifi-cation established by resection of the cervical supraspinousand interspinous ligaments and detachment of the poste-rior paravertebral muscles from cervical vertebrae [10 15]Puncture of a lumbar disc based on the presence of nucleuspulposus contributed to the formation of disc nodules andosteophytes [16] It was reported by Gloobe that osteophyteformation occurred in experimental bipedal rats [11] Inour previous study calcified hypertrophy considered to beosteophyte formations low rate of progression was induced


times200

Sham Veh LIG

TGF-1205731

(a)

0

002

004

006

008

LIG

A

rpos

itive

Sham Veh

TGF-1205731 distribution

lowastlowast

(b)LIGSham Veh

0

1

2

3

4

5

6

Fold

indu

ctio

n

TGF-1205731 mRNAlowastlowast

(c)

Figure 4 LIG suppresses TGF-1205731 distribution and expression in CEP tissue of bipedal rats (a) Immunohistochemical staining demonstratedstronger positivematrix staining for TGF-1205731 (brown) in themarginal area in theVeh group compared to that in the Shamgroup LIG treatmentweakens the positive staining for TGF-1205731 (b) Quantitative data of (a) (c) LIG reduced the increased mRNA level of TGF-1205731 in bipedal ratsby qPCR Each column represents the mean plusmn SE lowastlowast119901 lt 001 versus Sham and 119901 lt 001 versus Veh

in the regions of lumbar vertebral column by prolongedupright posture [4 17] This model was chosen for the in vivoobservation in this project Because the gradually increasedthickness of the marginal CEP could be observed over time(at 5 7 and 9 months after the surgery) decreases were takento indicate preventive and therapeutic effects of LIG in theparameters of LHT and changes in the expressions of threeosteogenesis-related markers at mRNA levels Col1 ALP andRunx2 at 9 months after the surgery with one month oftreatment

Transforming growth factor-beta 1 (TGF-1205731) has beenfound to play a crucial role in endplate hypertrophy andbony outgrowths [18] The expression of TGF-1205731 mRNA ishigher in the early-mid stages of osteophyte development[19] Overexpression of TGF-1205731 can induce local outgrowthssimilar to those observed under experimental conditions[20ndash23] Inhibition of endogenous TGF-120573 can nearly com-pletely prevent hypertrophy development by scavenging sol-uble TGF-120573-RII [24 25] However synovial lining cellssuch as macrophages might contribute to TGF-1205731-mediatedbony outgrowths [23 26 27] Consistent with several reportsstating that TGF-1205731 contributes to the process of osteophyteformation our findings have shown TGF-1205731 to be distributedin hypertrophyic CEP tissues and cells To show this we usedthe same immunochemicalmethods thatUchino et al used to

locate TGF-1205731 in the superficial cells of hypertrophic cartilage[28 29]

This study has some limitations First several kinds ofcells have been observed in newly formed hypertrophic CEPtissue These include fibroblastic mesenchymal cells fibro-chondrocytes chondrocytes and osteoblasts [30] Osteo-phyte-derived mesenchymal stem cells (oMSCs) have beencharacterized with regard to their distinct properties ofproliferation differentiation and immunomodulation [31]Our results indicated hypertrophic CEP cells have osteogenicpotential which acted as oMSCs However we did not isolatedifferent cell types from the hypertrophic CEP tissues Theeffects of LIG on specific cell types could not be verifiedSecond the data showing that LIG inhibits CEP hypertrophywere obtained from animal models in vivo and humanhypertrophic CEP samples in vitro Future studies will haveto gather clinical evidence

5 Conclusions

In conclusion LIG exerted a preventive effect on CEP hyper-trophy via suppression of TGF-1205731 levels LIG can be suggestedas an alternative therapeutic method to treat spinal CEP hy-pertrophy


Disclosure

Shufen Liu and Bizeng Zhao are co-first authors and con-tributed equally to this study

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the Project of National NaturalScience Foundation of China (81573992 81528022 8157400181473701 and 81403416) National Basic Research Programin China (973 Plan 2010CB530400) and the Program forChangjiang Scholars and Innovative Research Team in Uni-versity (PCSIRT IRT1270)

References

[1] J AWheeldon N Yoganandan and F A Pintar ldquoStrain energydensity used as the biomechanical signal for osteophyte growthin the cervical spinemdashbiomed 2009rdquoBiomedical Sciences Instru-mentation vol 45 pp 143ndash148 2009

[2] GHe and Z Xinghua ldquoThe numerical simulation of osteophyteformation on the edge of the vertebral body using quantitativebone remodeling theoryrdquo Joint Bone Spine vol 73 no 1 pp 95ndash101 2006

[3] S Kumaresan N Yoganandan F A Pintar D J Maiman andV K Goel ldquoContribution of disc degeneration to osteophyteformation in the cervical spine a biomechanical inverstigationrdquoJournal of Orthopaedic Research vol 19 no 5 pp 977ndash984 2001

[4] J-H Huang W-W Li Q Bian and Z-Y Shen ldquoA logicalframework derived from philosophy of language for analysis ofthe terms of traditional Chinese medicine and an example foranalysis of lsquokidney essencersquordquo Zhong Xi Yi Jie He Xue Bao vol 9no 9 pp 929ndash932 2011

[5] E J Olson B R Lindgren and C S Carlson ldquoEffects of long-term estrogen replacement therapy on the prevalence and areaof periarticular tibial osteophytes in surgically postmenopausalcynomolgus monkeysrdquo Bone vol 41 no 2 pp 282ndash289 2007

[6] T Neogi M C Nevitt K E Ensrud D Bauer and D T FelsonldquoThe effect of alendronate on progression of spinal osteophytesand disc-space narrowingrdquo Annals of the Rheumatic Diseasesvol 67 no 10 pp 1427ndash1430 2008

[7] S Imagama Y Hasegawa T Seki et al ldquoThe effect of120573-caroteneon lumbar osteophyte formationrdquo Spine vol 36 no 26 pp2293ndash2298 2011

[8] J-Z Hu C-Y Luo M Kang H-B Lu G-H Lei and Z DaildquoTherapeutic effects of intraarticular injection of ligustrazine onknee osteoarthritisrdquo Zhong Nan Da Xue Xue Bao Yi Xue Banvol 31 no 4 pp 591ndash594 2006

[9] Q-Q Liang D-F Ding Z-J Xi et al ldquoProtective effectof ligustrazine on lumbar intervertebral disc degeneration ofrats induced by prolonged upright posturerdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID508461 9 pages 2014

[10] Y Wang Q Shi and P Shen ldquoExperimental study on effectof qishejingkang recipe on activity of alkaline phosphatases

in osteophyte formation of degenerated cervical vertebrae ofrabbitsrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 20 no 3 pp199ndash201 2000

[11] H Gloobe and H Nathan ldquoOsteophyte formation in experi-mental bipedal ratsrdquo Journal of Comparative Pathology vol 83no 1 pp 133ndash141 1973

[12] Q-Q Liang Q Zhou M Zhang et al ldquoProlonged uprightposture induces degenerative changes in intervertebral discs inrat lumbar spinerdquo Spine vol 33 no 19 pp 2052ndash2058 2008

[13] E Kozhemyakina A B Lassar and E Zelzer ldquoA pathway tobone signaling molecules and transcription factors involved inchondrocyte development and maturationrdquo Development vol142 no 5 pp 817ndash831 2015

[14] Q Bian Q-Q Liang C Wan et al ldquoProlonged upright postureinduces calcified hypertrophy in the cartilage end plate in ratlumbar spinerdquo Spine vol 36 no 24 pp 2011ndash2020 2011

[15] B Peng S Hou Q Shi and L Jia ldquoExperimental study onmechanism of vertebral osteophyte formationrdquo Chinese Journalof Traumatology vol 3 no 4 pp 202ndash205 2000

[16] K Olmarker ldquoPuncture of a disc and application of nucleuspulposus induces disc herniation-like changes and osteophytesAn experimental study in ratsrdquoTheOpen Orthopaedics Journalvol 5 pp 154ndash159 2011

[17] C-J Menkes and N E Lane ldquoAre osteophytes good or badrdquoOsteoarthritis and Cartilage vol 12 pp S53ndashS54 2004

[18] Q Bian A Jain X Xu et al ldquoExcessive activation of TGF120573 byspinal instability causes vertebral endplate sclerosisrdquo ScientificReports vol 6 Article ID 27093 2016

[19] J W Li X S Weng G X Qiu et al ldquoGene expression oftransforming growth factor-1205731 in osteophyte developmentrdquoZhongguo Yi Xue Ke Xue Yuan Xue Bao vol 29 no 4 pp 522ndash527 2007

[20] W B van den Berg G J V M van Osch P M van derKraan and H M van Beuningen ldquoCartilage destruction andosteophytes in instability-inducedmurine osteoarthritis role ofTGF120573 in osteophyte formationrdquoAgents andActions vol 40 no3-4 pp 215ndash219 1993

[21] H M van Beuningen P M van der Kraan O J Arntz andW B van den Berg ldquoTransforming growth factor-1205731 stimu-lates articular chondrocyte proteoglycan synthesis and inducesosteophyte formation in the murine knee jointrdquo LaboratoryInvestigation vol 71 no 2 pp 279ndash290 1994

[22] A Scharstuhl E L Vitters P M van der Kraan and W Bvan den Berg ldquoReduction of osteophyte formation and syn-ovial thickening by adenoviral overexpression of transforminggrowth factor 120573bone morphogenetic protein inhibitors duringexperimental osteoarthritisrdquoArthritis and Rheumatism vol 48no 12 pp 3442ndash3451 2003

[23] A C Bakker F A J van de Loo H M van Beuningen et alldquoOverexpression of active TGF-beta-1 in the murine knee jointevidence for synovial-layer-dependent chondro-osteophyte for-mationrdquo Osteoarthritis and Cartilage vol 9 no 2 pp 128ndash1362001

[24] A Scharstuhl H L Glansbeek H M van Beuningen E LVitters P M van der Kraan and W B van den Berg ldquoInhibi-tion of endogenous TGF-120573 during experimental osteoarthritisprevents osteophyte formation and impairs cartilage repairrdquoTheJournal of Immunology vol 169 no 1 pp 507ndash514 2002

[25] E N Blaney Davidson E L Vitters H M van Beuningen F AJ van de Loo W B van den Berg and P M van der KraanldquoResemblance of osteophytes in experimental osteoarthritis


to transforming growth factor 120573-induced osteophytes limitedrole of bone morphogenetic protein in early osteoarthriticosteophyte formationrdquo Arthritis and Rheumatism vol 56 no12 pp 4065ndash4073 2007

[26] A B Blom P L EM van Lent A EMHolthuysen et al ldquoSyn-ovial lining macrophages mediate osteophyte formation duringexperimental osteoarthritisrdquo Osteoarthritis and Cartilage vol12 no 8 pp 627ndash635 2004

[27] P L E M van Lent A B Blom P van der Kraan et al ldquoCrucialrole of synovial lining macrophages in the promotion oftransforming growth factor 120573-mediated osteophyte formationrdquoArthritis amp Rheumatism vol 50 no 1 pp 103ndash111 2004

[28] M Uchino T Izumi T Tominaga et al ldquoGrowth factorexpression in the osteophytes of the human femoral head inosteoarthritisrdquo Clinical Orthopaedics and Related Research no377 pp 119ndash125 2000

[29] HM van BeuningenH L Glansbeek PM van der Kraan andW B van den Berg ldquoDifferential effects of local application ofBMP-2 or TGF-1205731 on both articular cartilage composition andosteophyte formationrdquo Osteoarthritis and Cartilage vol 6 no5 pp 306ndash317 1998

[30] T Nakase T Miyaji T Tomita et al ldquoLocalization of bonemor-phogenetic protein-2 in human osteoarthritic cartilage andosteophyterdquo Osteoarthritis and Cartilage vol 11 no 4 pp 278ndash284 2003

[31] S Singh B J Jones R Crawford and Y Xiao ldquoCharacterizationof a mesenchymal-like stem cell population from osteophytetissuerdquo Stem Cells and Development vol 17 no 2 pp 245ndash2542008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


previous studies we found LIG exerts a preventive effect onIDD in animalmodel [9] Moreover the qishejingkang recipe(QSJKR) of which LIG is the major constituent was foundto decrease the activity of alkaline phosphatase (ALP) indegenerated vertebral discs and inhibit CEPhypertrophy [10]Therefore we entered the present project expecting LIG tohave an inhibitory effect on CEP hypertrophy

To determine the inhibitory effect of LIG in vitro wecollected hypertrophic CEP samples from patient during ver-tebral fusion surgery Cells were isolated and cultured Cellu-lar proliferation and the mRNA and protein levels of Col1aand TGF-1205731 were assayed using MTT qPCR immunofluo-rescence and western blot analysis To investigated whetherLIG could attenuate CEP hypertrophy in vivo we employeda CEP hypertrophy rat model as previously reported [411] Safranin O fast green staining and morphometry wereperformed to determine the edges of the CEP and the sizesof noncartilaginous outgrowths The mRNA expressions oftype I collagen (Col1a) ALP runt-related transcription factor2 (Runx2) and TGF-1205731 were detected by qPCR TGF-1205731protein distributions were observed using immunohisto-chemistry Our results revealed that LIG has a preventiveeffect on CEP hypertrophy via the suppression of TGF-1205731levels This information could be used to develop alternativetherapeutic for spinal degeneration disease accompanied byCEP hypertrophy

2 Materials and Methods

21 Cell Culture and Drug Preparation Samples were ob-tained from ten patients during vertebral fusion surgery(with previous oral informed consent approved by EthicsCommittee of the Sixth Peoplersquos hospital Shanghai CN)The samples were washed with Dulbecco modified Eaglersquosmedium with high glucose content (DMEM-HG BiowestNuaille France) Then the tissues were dissected into 1-2-mm3 pieces and cultured in a medium containing DMEM-HG 20 fetal bovine serum (FBS Gibco Langley OKUSA)and 1 penicillin-streptomycin (Gibco Langley OK USA)

Ligustrazine phosphate (purity gt 99 MW 25221)was purchased from the Chinese Medicines and BiologicalProducts Institute (Beijing CN)The solutions of ligustrazinephosphate were prepared in dimethylsulfoxide (DMSOSigma USA) for the in vitro experiments The final concen-tration of DMSO was 01

22 Animal Models and Drug Administration Male Sprague-Dawley (SD) rats aged 1 month (119899 = 30) were providedby the Shanghai Laboratory Animal Center (SYXK2003-0002 Science and Technology Commission of ShanghaiMunicipality gave approval for this experimental study onanimals) were randomly divided into Sham Vehicle (Veh)and LIG groups In the Veh and LIG groups rats were forcedto stand up by forelimbs surgery as previously described [12]In the Sham group the rats did not receive any treatmentand were maintained in standard cages Eight months afterthe surgery ligustrazine hydrochloride (Nanning Maple LeafPharmaceutical Co Ltd CN (lot number 051125)) was

intraperitoneally injected into rats of LIG group (16mLkgsdotd10mL sterile saline 40mg ligustrazine hydrochloride) whilesterile saline of equal volume was injected into rats of Vehgroup once a day for one month Rats (119899 = 10) in each groupwere euthanized at 9 months after the surgery Their lumbarspines were dissected for analysis

23 ALP Assay The cells were fixed with 10 formalin andstainedwith 1-StepNBT-BCIP (Pierce USA) for 30minutesLyons blue represents the positive staining The images werescanned (CanoScan 8800F Japan)

24 MTT Detection Passage III hypertrophic CEP cells werecultured in 96-well plates at 1 times 105mL with 200120583L medium(the medium contained LIG and 20 FBS) for 8 wells pergroup After being cultured for 1 5 9 and 13 days 20120583L of5mgmLMTT reagent was added to each well and they wereincubated for 4 hours at 37∘CMedia were removed followedby adding 150120583L MTT solvent

The OD values were tested at 590 nm after shaking for15min A cellular growth curve was produced to reflect theOD values

25 qPCR RNA was extracted from L1-2 L2-3 and L3-4 mar-ginal discs (noncartilaginous outgrowth) or hypertrophicCEP cells treated by LIG for 48 hours using 1mL TRIzolreagent (Sigma St Louis MO USA) according to the man-ufacturerrsquos instruction Cellstissues were directly processedfollowing RNA preparation employing the PURE Prep Kitprotocol One microgram of total RNA was reverse-tran-scribed using the Advantage RT-for-PCR Kit (Qiagen Valen-cia CA USA) according to the manufacturerrsquos instructionsFreshly transcribed cDNA (1120583L) was employed for qPCRusing SYBR Green (Bio-Bad Hercules CA USA) to monitorDNA synthesis with specific primers (Tables 1 and 2) designedby TaKaRa Biotechnology Co Ltd Gene expression wasnormalized to the housekeeping gene 120573-actin PCR productswere subjected to melting-curve analysis and data wereanalyzed and quantified with the RotorGene 60 analysissoftware

26 Western Blotting Cells were cultured in basal mediumor 10minus7M LIG for 10 days At the end of the study the cellswere washed with PBS scraped and resuspended in lysate(Beyotime Hangzhou CN) for 1 hour on ice The cell lysateswere centrifuged at 12000 rpm for 10 minutes at 4∘C Thesupernatant was transferred to a new microcentrifuge tubeand protein concentration wasmeasured by Bradford proteinassay (Beyotime Hangzhou CN) Proteins were added with4x sample buffer (001 bromophenol blue 0125M TrispH 68 10 glycerol and 2 SDS) and denaturized at 95∘Cfor 10 minutes Equal amounts of protein (50 120583glane) weresolubilized in Laemmli sample buffer and loaded onto amini-SDS-PAGE system Following electrophoresis the proteinswere transferred to a polyvinylidene difluoride (PVDF)membrane (Millipore Temecula CA USA) using a Bio-Radwet transfer system Protein transfer efficiency was verifiedusing prestained protein markers The membranes were then








4

were examined




3 Results






times240 times120


times120

16th day ex vivo

(a)

3d 26d

(b)

0

1

2

3

4

Fold

indu

ctio

n

Col I mRNA

lowastlowast

3d 26d(c)

0

1

2

3

Fold

indu

ctio

n

TGF-1205731 mRNA

lowast

3d 26d(d)

GAPDH

3d 26d

TGF-1205731

(e)








MTT

0

05

1

15

2

OD

val

ue


1d 5d 9d 13d

(a)

0

05

1

15 Col I mRNA

Fold

indu

ctio

n

lowastlowast


(b)

Col I

0M 10minus7 M

(c)

120573-actin

0M 10minus7 M

TGF-1205731

(d)








Sham Veh LIG

LHT

times200(a)

0

1

2

3 LHT

Fold

indu

ctio

n

Sham Veh LIG

lowastlowast

(b)

Fold

indu

ctio

n0

10

20

30 Col1a2 mRNA

Sham Veh LIG

lowastlowast

(c)

Sham Veh LIG0

2

4

6

8

10

12

Fold

indu

ctio

n


(d)Sham Veh LIG

0

5

10

15

20 ALP mRNA

Fold

indu

ctio

n

lowastlowast

(e)




4 Discussion




times200

Sham Veh LIG

TGF-1205731

(a)

0

002

004

006

008

LIG

A

rpos

itive

Sham Veh


lowastlowast

(b)LIGSham Veh

0

1

2

3

4

5

6

Fold

indu

ctio

n


(c)






5 Conclusions



Disclosure


Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4

were examined




3 Results






times240 times120


times120

16th day ex vivo

(a)

3d 26d

(b)

0

1

2

3

4

Fold

indu

ctio

n

Col I mRNA

lowastlowast

3d 26d(c)

0

1

2

3

Fold

indu

ctio

n

TGF-1205731 mRNA

lowast

3d 26d(d)

GAPDH

3d 26d

TGF-1205731

(e)








MTT

0

05

1

15

2

OD

val

ue


1d 5d 9d 13d

(a)

0

05

1

15 Col I mRNA

Fold

indu

ctio

n

lowastlowast


(b)

Col I

0M 10minus7 M

(c)

120573-actin

0M 10minus7 M

TGF-1205731

(d)








Sham Veh LIG

LHT

times200(a)

0

1

2

3 LHT

Fold

indu

ctio

n

Sham Veh LIG

lowastlowast

(b)

Fold

indu

ctio

n0

10

20

30 Col1a2 mRNA

Sham Veh LIG

lowastlowast

(c)

Sham Veh LIG0

2

4

6

8

10

12

Fold

indu

ctio

n


(d)Sham Veh LIG

0

5

10

15

20 ALP mRNA

Fold

indu

ctio

n

lowastlowast

(e)




4 Discussion




times200

Sham Veh LIG

TGF-1205731

(a)

0

002

004

006

008

LIG

A

rpos

itive

Sham Veh


lowastlowast

(b)LIGSham Veh

0

1

2

3

4

5

6

Fold

indu

ctio

n


(c)






5 Conclusions



Disclosure


Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















times240 times120


times120

16th day ex vivo

(a)

3d 26d

(b)

0

1

2

3

4

Fold

indu

ctio

n

Col I mRNA

lowastlowast

3d 26d(c)

0

1

2

3

Fold

indu

ctio

n

TGF-1205731 mRNA

lowast

3d 26d(d)

GAPDH

3d 26d

TGF-1205731

(e)








MTT

0

05

1

15

2

OD

val

ue


1d 5d 9d 13d

(a)

0

05

1

15 Col I mRNA

Fold

indu

ctio

n

lowastlowast


(b)

Col I

0M 10minus7 M

(c)

120573-actin

0M 10minus7 M

TGF-1205731

(d)








Sham Veh LIG

LHT

times200(a)

0

1

2

3 LHT

Fold

indu

ctio

n

Sham Veh LIG

lowastlowast

(b)

Fold

indu

ctio

n0

10

20

30 Col1a2 mRNA

Sham Veh LIG

lowastlowast

(c)

Sham Veh LIG0

2

4

6

8

10

12

Fold

indu

ctio

n


(d)Sham Veh LIG

0

5

10

15

20 ALP mRNA

Fold

indu

ctio

n

lowastlowast

(e)




4 Discussion




times200

Sham Veh LIG

TGF-1205731

(a)

0

002

004

006

008

LIG

A

rpos

itive

Sham Veh


lowastlowast

(b)LIGSham Veh

0

1

2

3

4

5

6

Fold

indu

ctio

n


(c)






5 Conclusions



Disclosure


Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MTT

0

05

1

15

2

OD

val

ue


1d 5d 9d 13d

(a)

0

05

1

15 Col I mRNA

Fold

indu

ctio

n

lowastlowast


(b)

Col I

0M 10minus7 M

(c)

120573-actin

0M 10minus7 M

TGF-1205731

(d)








Sham Veh LIG

LHT

times200(a)

0

1

2

3 LHT

Fold

indu

ctio

n

Sham Veh LIG

lowastlowast

(b)

Fold

indu

ctio

n0

10

20

30 Col1a2 mRNA

Sham Veh LIG

lowastlowast

(c)

Sham Veh LIG0

2

4

6

8

10

12

Fold

indu

ctio

n


(d)Sham Veh LIG

0

5

10

15

20 ALP mRNA

Fold

indu

ctio

n

lowastlowast

(e)




4 Discussion




times200

Sham Veh LIG

TGF-1205731

(a)

0

002

004

006

008

LIG

A

rpos

itive

Sham Veh


lowastlowast

(b)LIGSham Veh

0

1

2

3

4

5

6

Fold

indu

ctio

n


(c)






5 Conclusions



Disclosure


Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham Veh LIG

LHT

times200(a)

0

1

2

3 LHT

Fold

indu

ctio

n

Sham Veh LIG

lowastlowast

(b)

Fold

indu

ctio

n0

10

20

30 Col1a2 mRNA

Sham Veh LIG

lowastlowast

(c)

Sham Veh LIG0

2

4

6

8

10

12

Fold

indu

ctio

n


(d)Sham Veh LIG

0

5

10

15

20 ALP mRNA

Fold

indu

ctio

n

lowastlowast

(e)




4 Discussion




times200

Sham Veh LIG

TGF-1205731

(a)

0

002

004

006

008

LIG

A

rpos

itive

Sham Veh


lowastlowast

(b)LIGSham Veh

0

1

2

3

4

5

6

Fold

indu

ctio

n


(c)






5 Conclusions



Disclosure


Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















times200

Sham Veh LIG

TGF-1205731

(a)

0

002

004

006

008

LIG

A

rpos

itive

Sham Veh


lowastlowast

(b)LIGSham Veh

0

1

2

3

4

5

6

Fold

indu

ctio

n


(c)






5 Conclusions



Disclosure


Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Disclosure


Competing Interests


Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ligustrazine inhibits cartilage endplate hypertrophy via

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