research article effect of pulsed electromagnetic field on...

Research ArticleEffect of Pulsed Electromagnetic Field on Bone Formation andLipid Metabolism of Glucocorticoid-Induced Osteoporosis Ratsthrough Canonical Wnt Signaling Pathway

Yuan Jiang Hui Gou Sanrong Wang Jiang Zhu Si Tian and Lehua Yu

Department of Rehabilitation Medicine and Physical Therapy Second Affiliated Hospital Chongqing Medical UniversityChongqing 400010 China

Correspondence should be addressed to Lehua Yu yulehuacqmueducn

Received 2 December 2015 Accepted 31 December 2015

Academic Editor Settimio Grimaldi

Copyright copy 2016 Yuan Jiang 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

Pulsed electromagnetic field (PEMF) has been suggested as a promising method alternative to drug-based therapies for treatingosteoporosis (OP) but the role of PEMF in GIOP animal models still remains unknown This study was performed to investigatethe effect of PEMF on bone formation and lipid metabolism and further explored the several important components and targetsof canonical Wnt signaling pathway in GIOP rats After 12 weeks of intervention bone mineral density (BMD) level of the wholebody increased significantly serum lipid levels decreased significantly and trabeculae were thicker in GIOP rats of PEMF groupPEMF stimulation upregulated themRNAandprotein expression ofWnt10b LRP5120573-cateninOPG andRunx2 and downregulatedAxin2 PPAR-120574 CEBP120572 FABP4 and Dkk-1 The results of this study suggested that PEMF stimulation can prevent bone loss andimprove lipid metabolism disorders in GIOP rats CanonicalWnt signaling pathway plays an important role in bone formation andlipid metabolism during PEMF stimulation

1 Introduction

Glucocorticoids (GCs) are frequently used to treat variousdiseases such as bronchial asthma rheumatoid arthritischronic renal diseases collagen diseases and pulmonary andskin diseases [1 2] Long-term use and (or) higher dosesof GCs are inevitably in clinics due to the improvementsin the outcome of these diseases Nevertheless approxi-mately 30ndash50 of patients receiving long-term GCs therapydevelop glucocorticoid-induced osteoporosis (GIOP) andosteonecrosis [3] GIOP is themost frequently occurring typeof secondary OP [4]The risk of fractures in GIOP patients ischaracterized by closely correlating with the daily and cumu-lative doses of GCs but it does not seem to correlate with thespecific underlying disease [5] Lipid metabolism disordersspecifically hyperlipidemia are frequently accompanied byGIOP as well as postmenopausal OP Lipid metabolismdisorders are deemed to be a complication of GIOP [6] andit can exacerbate the degree of GIOP because GCs induceadipocyte formation in bone marrow and further negatively

affect the bone status Unfortunately the patients often haveto tolerate the GIOP and hyperlipidemia during long-termGCs treatment because treatment options are limited [7]Therefore to choose an effective therapy of complications ofGCs therapy is very important

Currently the clinical pharmacological treatments ofGIOP rely on medications similar to the drugs which areused for the treatment of postmenopausalOP including thosewith bisphosphonates raloxifene hormone replacementparathyroid hormone (PTH) calcium vitamin D calcitoninfluoride testosterone and anabolic steroids [7 8] Theseantiosteoporosis drugs also can modulate lipid metabolismin OP patients but the risks of potential complications inlong-term treatment cannot be ignored For example useof bisphosphonates is accompanied by some potential sideeffects such as gastrointestinal complaints osteonecrosis ofthe jaw and atypical subtrochanteric or diaphyseal femoralfractures [9] The prolonged use of hormone replacement isrestricted because of potential complications such as breastcancer uterine bleeding and cardiovascular events [1]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 4927035 13 pageshttpdxdoiorg10115520164927035

2 Evidence-Based Complementary and Alternative Medicine

Therefore safe and noninvasive biophysical countermeasuresfor complications ofGCs therapymight bemore promising inclinical application

Pulsed electromagnetic fields (PEMFs) have been sug-gested as promising method alternatives to drug-based ther-apies for treating a wide range of bone disorders suchas fresh fracture delayed and nonunion fractures diabeticosteopenia osteoporosis osteonecrosis and osteoarthritis[10 11] PEMF is useful in enhancing BMD in OP patientsand preventing bone loss in animal models of disuse OPtail-suspension OP ovariectomy-induced OP and diabetes-mellitus-induced OP [12ndash14] Several previous studies wereusing PEMF to prevent steroid-associated osteonecrosis andfound that PEMF can improve serum lipid levels [15 16]

Wingless-type MMTV integration site (Wnt) signalingpathway plays a critical role inmaintenance of bonemass andis able to suppress adipogenesis and promote osteoblastoge-nesis of bone marrow mesenchymal stromal cells (BMSCs)[17] PEMF stimulation can activate Wnt signaling pathwayduring treatment of bone loss in ovariectomized rats [18 19]However the skeletal dynamics and pathogenesis of GIOPare distinctly different from other OP that associated withageing estrogen deprivation and immobilization [5 20] Fewstudies have investigated the effects of PEMF on bone statuslipid metabolism and related signaling pathway mechanismsin GIOP animal models Particularly GCs can directlyinduce differentiation of BMSCs into adipocytes and inhibitosteogenic differentiation through downregulating the Wntsignaling pathway [16 21 22] In this present study weestablished GIOP SD rats model by intramuscular injectionwith dexamethasone to investigate the effect of PEMF onbone formation and lipid metabolism and further exploredthe several important components and targets of canonicalWnt signaling pathway in GIOP rats

2 Materials and Methods

21 Establishment of GIOP Rats Model All animal experi-mental protocol and care were approved by the InstitutionalAnimal Care and Use Committee of Chongqing MedicalUniversity Totally 40 female Sprague Dawley rats wereemployed aged 3 months weighing 210 plusmn 20 g and housedindividually and maintained under controlled environmen-tal conditions (12-hour light-dark cycle temperature 22∘Cwith humidity of 50 plusmn 5) All rats had unrestricted accessto water and food After one week of acclimatization 10 ratswere randomly divided into control groups The rest of therats were injected with dexamethasone sodium phosphateinjection (DXMT 25mgkg) into their right haunchmusclesto establish GIOP rat models twice a week 12 weeks in a rowAfter 12 weeks those rats received no DXMT interventionand were randomly divided into GIOP group (119899 = 10)calcium supplement group (calcium group 119899 = 10) andPEMF group (119899 = 10) The rats in the control groupwere injected with equivalent dose of normal saline intotheir right haunch muscles twice a week 12 weeks in arow Subsequently the fasting rats in each group receivedintraperitoneal anesthesia of 10 chloral hydrate (35mLkg)The rats were positioned on the operation platform in supine

position After the fur on the neck of rat was removed andskin was sterilized a midline 1ndash15 cm longitudinal incisionwas performed The left cephalic artery of rat was found totake blood (15ndash2mL) and then the incision was stitchedBlood specimens were centrifuged to get serum Bone min-eral density (BMD) and bone mineral content (BMC) weremeasured by using dual energy X-ray absorptiometry (LunariDXA GE Healthcare) equipped with dedicated software forsmall animalmeasurements and serumbiochemical analyseswere operated to investigate whether the GIOP rat model wassuccessfully induced by injecting DXMT and associated withlipid metabolism disorders

22 GIOP Rats Treatments The rats in calcium groupreceived oral calcium (5625mgkg calcium gluconic tabletsHainan Pharmaceutical Factory Co Ltd China) once a day12 weeks in a row At the same time the rats in control groupGIOP group and PEMF group were perfused with the samevolume of saline as the criteria of body mass The rats inPEMF group were exposed to PEMF which was generatedby the ZH-21 osteoporosis treatment system (ChongqingZhonghuan Electronic Technology Co Ltd China) with afrequency of 50Hz and an intensity of 40mT once a day 40-minute treatment every day 12 weeks in a rowThe waveformis square wave with pulse width 200 120583s At the same time therats in control group calcium group and GIOP group werealso placed in the ZH-21 osteoporosis treatment system butthe treatment systemwas not running to provide shamPEMFstimulation

23 BoneMineral Density andBoneMineral ContentMeasure-ment After a period of 12 weeksrsquo treatment all rats receivedintraperitoneal anesthesia of 10 chloral hydrate (35mLkg)Then the rats were positioned on the platform of dual energyX-ray absorptiometry (Lunar iDXA GEHealthcare) in proneposition The BMD and BMC of head upper limb femurtrunk rib pelvis spine and the whole body were detectedand recorded

24 Serum Biochemical Analysis The fasting rats in eachgroup received intraperitoneal anesthesia with 10 chlo-ral hydrate (35mLkg) Then the rats were positioned onthe operation platform before they were executed The furon the abdomen was removed and skin was sterilized amidline 4-5 cm longitudinal incision was performed Theblood specimens were withdrawn from aorta abdominalisand then centrifuged to get serum The serum calcium (Ca)phosphorus (P) alkaline phosphatase (ALP) triglyceride(TG) total cholesterol (TCHO) high density lipoproteincholesterol (HDL) and low density lipoprotein cholesterol(LDL) were determined by automatic biochemical analyzer(TBA-120FR Toshiba) The serum tartrate resistant acidphosphatase (TRAP) was determined by tartrate resistantacid phosphatase assay kit (Beyotime China) according tothe protocol of the manufacturer

25 Histomorphometrical and Histopathological AnalysisAfter all rats were executed the fourth lumbar (L4) vertebralbodies were collected Each fourth lumbar was carefully

Evidence-Based Complementary and Alternative Medicine 3

cleaned and then decalcified by EDTA decalcifying solution(BOSTER China) for 6 weeks The vertebral body sampleswere put into the optimum cutting temperature compound(OCT compound Sakura) and quick freezing then flat-placed in the cryostat mould and cut into serial sectionswith cryostat (15120583m per section) The serial sections werestained with Safranin-OFast green for histomorphometricalanalysis and stained with hematoxylin-eosin (HE) stainingsolution for histopathological analysis Finally the trabec-ular bone structure of L4 vertebral bodies in each groupwas observed by inverted microscope Histomorphometricalparameters were quantified by using the Image-Pro Plus60 software (Media Cybernetics) The static parameters arecalculated using the following formula [19] percentage oftrabecular area (TbAr) = trabecular area (TbAr)bone area(TAr) times 100 trabecular width (TbWi) = (20001199) times(TbArtrabecular perimeter [TbPm]) trabecular number(TbN) = (11992) times (TbPmTAr) trabecular separation(TbSp) = (20001199) times (TAr minus TbAr)TbPm

26 Real-Time PCR Analysis After all rats were execut-ed the right thighbones were collected Total RNA was ex-tracted from caput femoris using the TRIzol reagent(Beyotime China) according to the manufacturerrsquosinstruction and quantified by spectrophotometry at awavelength of 260 nm Reverse transcription actions andPCR were performed using reverse transcriptase oligo(DT) primers and Taq DNA polymerase The specificsequences of the primers for Wnt10b low density lipoproteinreceptor-related protein 5 (LRP5) 120573-catenin axis inhibitionprotein 2 (Axin2) osteoprotegerin (OPG) receptor activatorof nuclear factor 120581B ligand (RANKL) dickkopf1 (Dkk-1)Sclerostin (SOST) Runt-related transcription factor 2(Runx2) peroxisome proliferator-activated receptor-120574(PPAR-120574) CCAATenhancer binding protein-120572 (CEBP120572)and fatty acid binding protein 4 (FABP4) are listed inTable 1 GAPDH was used as an internal control RelativemRNA expressions were defined as the ratio of target genesexpression to GAPDH expression

27 Western Blot Analysis The caput femoris samples weremilled and lysed with RIPA lysis buffer (Beyotime China)for extracting total proteins Protein samples were sepa-rated by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis and electrotransferred onto a polyvinylidenedifluoride (PVDF) membrane The PVDF membrane wasblocked for 2 h at room temperature in TBS-Tween 20(TBST) buffer containing 5 BSA washed with TBST threetimes and incubated overnight at 4∘C with 1500 dilution ofWnt10b antibodies LRP5 antibodies 120573-catenin antibodiesAxin2 antibodies OPG antibodies RANKL antibodies Dkk-1 antibodies SOST antibodies Runx2 antibodies PPAR-120574 antibodies CEBP120572 antibodies FABP4 antibodies (allpurchased from Santa Cruz Biotech) and GAPDH antibody(1 1000 BOSTER China) respectively After being washedwith TBST the membranes were incubated with the sec-ondary biotin-conjugated antibody and then with anti-biotinhorseradish peroxidase- (HRP-) linked antibody (1 1000)Protein signals were detected using SuperSignal West Pico

Table 1 Primer sequences for real-time PCR analysis

Gene ID Gene 51015840-31015840 Sequence

315294 Wnt10b Forward CAGGCTTTGTGTGGAGTCATTReverse GAGGTTCTGGGCTGTAGTGG

293649 LRP5 Forward GACCTGATGGGACTCAAAGCReverse GGGTGAAGAAGCACAGATGG

84353 120573-catenin Forward CTTACGGCAATCAGGAAAGCReverse GACAGACAGCACCTTCAGCA

25341 OPG Forward TCAAGAATGCCACAGAAReverse GTCACGAAGCGGGTGTAGT

117516 RANKL Forward GGGAGCACTAAGAACTGGTCAReverse TTGGACACCTGGACGCTAAT

29134 Axin2 Forward AGTCAGCAGAGGGACAGGAAReverse CTTGGAGTGCGTGGACACTA

293897 Dkk-1 Forward TGACCACAGCCATTTACCTCReverse ACAGAGCCTTCTTGCCCTTT

80722 SOST Forward GAATGGTAGGTGCCAGGAGCAReverse TTAGGTAGGTGCCAGGAGCA

367218 Runx2 Forward CCTCTGACTTCTGCCTCTGGReverse GATGAAATGCCTGGGAACTG

25664 PPAR-120574 Forward CGGTTGATTTCTCCAGCATTReverse TCGCACTTTGGTATTCTTGG

24252 CEBP120572 Forward AGTTGACCAGTGACAATGACCGReverse TCAGGCAGCTGGCGGAAGAT

79451 FABP4 Forward CGACCACCATAAAGAGGAGACReverse AAACCACCAAATCCCATCAA

24383 GAPDH Forward CAGGAGGCATTGCTGATGATReverse GAAGGCTGGGGCTCATTT

Chemiluminescent Substrate Trial kit (Thermo Scientific)and quantified by densitometry using Quantity One software(Bio-Rad)

28 Statistical Analysis The statistical analysis was con-ducted using SPSS 200 for Windows software Data werepresented as mean plusmn standard deviation (SD) Differences ingroup were analyzed by using repeated measure analysis ofvariance (ANOVA) Intergroup comparisonswere performedusing the least significant difference (LSD) test for multiplecomparisons 119875 lt 005 was considered to be statisticalsignificance

3 Results

31 GIOP Rats Model After a period of 12 weeks with DXMTor normal saline intervention the rats in each group weredetected by dual energy X-ray absorptiometry The results ofBMD are shown in Table 2 Compared with the normal ratsin control group the BMD values of head femur trunk ribpelvis spine and whole body of GIOP rats models in GIOPgroup calcium group and PEMF group were significantlydeclined respectively (all 119875 lt 005) As shown in Table 3the BMC values of trunk rib pelvis spine and whole bodyin GIOP rats models were significantly lower than those in


Table2Bo

nemineraldensity

(BMD)o

feachgrou

pbefore

andaft

ertre

atment(gcm2

)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

0237plusmn0010

0153plusmn0032

0142plusmn0011

0125plusmn000

90113plusmn000

60132plusmn0017

0132plusmn0008

0149plusmn0005

Posttreatment

0243plusmn0016

0144plusmn0026

0156plusmn0012

O0125plusmn0008

0110plusmn0007

0134plusmn0017

0137plusmn000

90153plusmn0007

GIO

Pgrou

pPriortotre

atment

0225plusmn0015lowast

0131plusmn

0031






0132plusmn000

4lowastPo

sttreatment

0235plusmn0021

0123plusmn0032

0132plusmn0011

0114plusmn0007

0102plusmn0007

0117plusmn0016

0111plusmn0015

0135plusmn000

9

Calcium

grou

pPriortotre

atment

0221plusmn

0010lowast

0147plusmn0035


0106plusmn000

9lowast0102plusmn0012lowast



0131plusmn

0005lowast

Posttreatment

0237plusmn0024

0119plusmn0033

0129plusmn0019

0113plusmn0014

0100plusmn0008

0123plusmn0018O

0125plusmn0018

O0141plusmn

0013

PEMFgrou

pPriortotre

atment

0219plusmn000

6lowast0138plusmn000

90120plusmn000



0101plusmn

0015lowast


0133plusmn000

6lowastPo

sttreatment

0234plusmn0024

0112plusmn0035

0133plusmn0018O

0117plusmn0008O

0104plusmn0008

0117plusmn0018O

0130plusmn0013

O0148plusmn0008

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Table3Bo

nemineralcontent(BM

C)of

each

grou

pbefore

andaft

ertre

atment(g)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

202plusmn023

049plusmn025

233plusmn10

0382plusmn10

512

5plusmn049

140plusmn023

173plusmn049

819plusmn064

Posttreatment

204plusmn028

049plusmn023

279plusmn069

343plusmn097

12

3plusmn060

13

5plusmn053

185plusmn075

831plusmn095

GIO

Pgrou

pPriortotre

atment

180plusmn013lowast

039plusmn027

231plusmn073

260plusmn064lowast

067plusmn032lowast

115plusmn026lowast

080plusmn025lowast

721plusmn

063lowast

Posttreatment

185plusmn018

034plusmn010

232plusmn069

232plusmn073

058plusmn031

110plusmn037

080plusmn028

706plusmn057

Calcium

grou

pPriortotre

atment

182plusmn010lowast

042plusmn023

262plusmn10

1212plusmn033lowast

053plusmn011lowast

100plusmn034lowast

081plusmn025lowast

714plusmn077lowast

Posttreatment

190plusmn032

035plusmn019

258plusmn065

244plusmn041

061plusmn020

124plusmn032

O091plusmn033

752plusmn071

O

PEMFgrou

pPriortotre

atment

188plusmn016

053plusmn027

225plusmn097

227plusmn040lowast

053plusmn012lowast

095plusmn026lowast

086plusmn032lowast

709plusmn080lowast

Posttreatment

204plusmn021

O046plusmn023

273plusmn068

233plusmn039

063plusmn018

O10

2plusmn027

099plusmn034

768plusmn064

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Beforetreatment

Aftertreatment

OPG

RA

NKL

mRN

A

1000

2000

3000

ALP

(UL

)

10

20

30

TRA

P (U

L)

10

20

30

40

expr

essio

n

OPG

RA

NKL

pro

tein

expr

essio

n

05

10

15

20

25

10

20

30

40

(mm

olL

)

10

20

30

40(m

mol

L)

P TG TCHO HDL LDLCaBefore treatment

P TG TCHO HDL LDLCaAfter treatment

Beforetreatment

Aftertreatment

Control groupGIOP group

Calcium groupPEMF group



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowastlowastlowastlowast

(G) (H)

(F)

(D)

(E)

(C)

(A) (B)

(A) (B)

(A) (C)

(D)(B)(a)

(b)

(c)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

∘

∘

∘

∘

∘

∘

lowast∘ lowast∘

00 00

0000

00 00

Figure 1 The serum concentrations of alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) in each group beforeand after treatment ((a) A B) The results of serum biochemical analysis in each group before and after treatment include serum calcium(Ca) phosphorus (P) triglyceride (TG) total cholesterol (TCHO) high density lipoprotein cholesterol (HDL) and low density lipoproteincholesterol (LDL) ((a) C D) The L4 vertebral bodies were stained with Safranin-OFast green ((b) A) control group ((b) B) GIOP group((b) C) calcium group and ((b) D) PEMF group bone tissue was stained in grayish-green or blue cartilage tissue was stained in red TheL4 vertebral bodies also were stained with HE staining solution ((b) E) control group ((b) F) GIOP group ((b) G) calcium group and ((b)H) PEMF group The rate of OPG to RANKL mRNA and protein expressions in each group after treatment ((c) A B) Compared with thecontrol group lowast119875 lt 005 compared with the GIOP group 119875 lt 005 compared with calcium group O119875 lt 005

the normal rats (119875 lt 005) The serum ALP level of theGIOP rats models was lower and the serum TRAP levelwas higher compared to the normal rats (Figure 1(a)(A B))In contrast with the control group the serum Ca and phos-phorus level of GIOP group calcium group and PEMF grouphad increased but there was no significant difference amongthe four groups On the contrary the serum TG TCHO andLDL of GIOP rats models in GIOP group calcium groupand PEMF group were significantly higher than normalrats in control group (Figure 1(a)(C)) All the abovemen-tioned results demonstrated that the GIOP rats model wassuccessfully established and disorder of lipid metabolism(hyperlipemia) was accompanied by bone mass loss

32 Body Weight Rats were weighed every week BeforePEMF stimulation or oral calcium treatment the bodyweights of rats in control group GIOP group calciumgroup and PEMF group were 2662 plusmn 192 g 2601 plusmn 261 g2596 plusmn 142 g and 2618 plusmn 104 g There was no significantdifference in body weight among the four groups beforePEMF stimulation or oral calcium treatment (119875 gt 005)After 12 weeks of different treatment the body weights ofrats in control group GIOP group calcium group and PEMFgroup were 2941 plusmn 179 g 2816 plusmn 235 g 2837 plusmn 106 gand 2859 plusmn 87 g There was no significant difference inbody weight among the four groups after applying differenttreatment methods (119875 gt 005)


Table 4 Histomorphometrical analysis of the fourth lumbar (L4) vertebral bodies

Group TbAr () TbWi (120583m) TbN (nmm) TbSp (120583m)Control group 5423 plusmn 248 5541 plusmn 569 985 plusmn 101 4687 plusmn 629GIOP group 2695 plusmn 555lowast 5270 plusmn 467 525 plusmn 063lowast 13980 plusmn 2568lowast

Calcium group 4269 plusmn 630lowast 6223 plusmn 505 688 plusmn 106lowast 8532 plusmn 1991lowast

PEMF group 4816 plusmn 428 6142 plusmn 263 784 plusmn 052lowast 6655 plusmn 964

Data were expressed as mean plusmn SDTbAr trabecular area TbWi trabecular width TbN trabecular number TbSp trabecular separationCompared with the control group lowast119875 lt 005 compared with the GIOP group 119875 lt 005

33 BoneMineral Density andBoneMineral ContentMeasure-ment As shown inTable 2 after 12weeks of PEMF treatmentthe BMD values of femur trunk pelvis spine and the wholebody in PEMF group were significantly higher than thosebefore treatment (119875 lt 005) and the BMD values of spineand the whole body were significantly higher than those inGIOP group (119875 lt 005) The BMC values of head rib andthe whole body were significantly higher than those beforetreatment (119875 lt 005) After 12 weeks of calcium supplementtreatment the BMD values of pelvis and spine in calciumgroup were significantly higher than those before treatment(119875 lt 005) and only the BMD value of spine was significantlyhigher than that inGIOP group (119875 lt 005)TheBMCvalue ofpelvis and the whole body was significantly higher than thatbefore treatment (119875 lt 005)

34 Serum Biochemical Analysis As shown in Figure 1(a)the serum ALP level of PEMF group after treatment wassignificantly higher than before treatment and GIOP grouprespectively (119875 lt 005) The serum ALP level of GIOP groupwas still lower than control group after 12 weeks of shamtreatment period free of DXMT (119875 lt 005)The serumTRAPlevel of PEMF group after treatment was significantly lowerthan before treatment and GIOP group respectively (119875 lt005) There was no significant difference in the serum TRAPlevel between the GIOP group and control group after shamtreatment (119875 gt 005) In contrast to GIOP group theserum Ca and phosphorus level of calcium group and PEMFgroup have declined after 12 weeks of intervention treatmentwhile only phosphorus level of PEMF group had a significantreduction in contrast to before treatment (119875 lt 005) Thelevels of serum TG and LDL of GIOP group were stilllower than control group after 12 weeks of sham treatmentperiod free of DXMT (119875 lt 005) The level of serumTG TCHO and LDL of PEMF group after treatment hada significant reduction in contrast to before treatment andGIOP group respectively (119875 lt 005) However the above-mentioned blood lipids index in calcium group after treat-ment had decreased tendency but there was no statisticaldifference after compared with before treatment and GIOPgroup respectively (119875 gt 005)

35 Histomorphometrical and Histopathological AnalysisAfter 12 weeks of different intervention treatment the L4vertebral bodies of four groups were stained with Safranin-OFast green for histomorphometrical analysis and shown inFigure 1(b)(AndashD) Bone tissue was stained in grayish-greenor blue and cartilage tissue was stained in red The results of

histomorphometrical analysis were shown in Table 4 TbArTbN significantly declined and TbSp significantly increasedin GIOP group in contrast with normal rats in control group(119875 lt 005) By contrast PEMF stimulation and calciumsupplement increased TbAr TbN and decreased TbSp TheL4 vertebral bodieswere stainedwithHE staining solution forhistopathological analysis and shown in Figure 1(b)(EndashH) Incontrast with control group the trabeculae were thinner andsparse and cracks and breaks were observed in GIOP groupThe trabeculae number in PEMF group and calcium groupwas slightly increased and the trabeculae were thicker thanGIOP group

36 Real-Time PCRAnalysis The relativemRNA expressionsof target genes were estimated using real-time PCR analysisAs shown in Figure 2 in contrast with GIOP group themRNA expressions of Wnt10b LRP5 120573-catenin and OPGwere significantly increased (all 119875 lt 005) and the mRNAexpressions of Axin2 RANKL PPAR-120574 CEBP120572 FABP4and Dkk-1 were significantly decreased (all 119875 lt 005) inPEMF group after 12 weeks of PEMF stimulation Onlythe mRNA expression of FABP4 was significantly decreased(119875 lt 005) in calcium group after 12 weeks of calciumsupplement treatment Moreover the rate of OPGRANKLmRNA expression level was significantly increased in PEMFgroup compared to the other three groups (119875 lt 005Figure 1(c)(A))

37 Western Blot Analysis The protein expressions of targetgenes were estimated using Western blot analysis As shownin Figure 3 the protein expressions of PPAR-120574 and FABP4were significantly increased (all 119875 lt 005) in GIOP groupcompared with control group after 12 weeks of sham treat-ment period free of DXMT In contrast with GIOP groupthe protein expressions of Wnt10b LRP5 and Runx2 weresignificantly increased (119875 lt 005) and the protein expressionsof Axin2 RANKL PPAR-120574 CEBP120572 FABP4 andDkk-1 weresignificantly decreased in PEMF group (119875 lt 005) Only theprotein expression of FABP4was significantly decreased (119875 lt005) in calcium group Moreover the rate of OPGRANKLprotein expression level was significantly increased in PEMFgroup compared to the other three groups (119875 lt 005Figure 1(c)(B))

4 Discussion

It is necessary to search for a suitable therapeutic methodfor GIOP with minor side effects and lower cost due to the


GIOP Calcium PEMFControlgroup group groupgroup












05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

Wnt10b LRP5 120573-catenin

OPG RANKL Runx2

PPAR-120574 CEBP120572 FABP4

Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000

Figure 2The relative mRNA expressions of target genes were estimated using real-time PCR analysis after 12-week interventions Comparedwith the control group lowast119875 lt 005 compared with the GIOP group 119875 lt 005 compared with calcium group O119875 lt 005


Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

Figure 3The protein expressions of target genes were estimated usingWestern blot analysis after 12-week interventions Compared with thecontrol group lowast119875 lt 005 compared with the GIOP group 119875 lt 005 compared with calcium group O119875 lt 005

serious side effects andor high cost of currently availabletherapies PEMF is a safe and effective method of treatingpostmenopausal OP and steroid-associated osteonecrosis Atpresent few studies have investigated the effects of PEMF onGIOP animal models or GIOP patients The role of PEMFin GIOP needs further investigation The laboratory rat isthe preferred animal for most research due to the similaritiesin pathophysiologic responses between the human and ratskeleton [23] Moreover rat can be used for building the reli-able animal model of GCs-induced osteopeniaosteoporosis

and massive formation of adipocytes and attempting tomimic the bone changes seen in humans [6 24] Certainlythe phenotypes of GIOP rats depend on the age and dosageand the period of GC administration [25] In this study wechose female SD rats aged 3 months as the animal modelDexamethasone was given to rats by intramuscular injectionbecause dexamethasone causes more skeletal complicationsthan prednisone [23] The detection results of BMC BMDserum ALP serum TRAP and serum lipid levels suggestedthat intramuscular injection with DXMT (25mgkg twice a


week) for 12 weeks can induce the GIOP and be accompaniedby hyperlipidemia in experimental rats BMD measurementis considered a testing standard for diagnosis of OP andoften used to evaluate BMD and BMC in animal models[12 26] Dual energy X-ray absorptiometry (Lunar iDXAGE Healthcare) with small animal software can be used tomeasure both total and regional BMD and BMC in ratMany studies have shown GCs administration in humansincreases the risk of skeletal fractures particularly in the ribsand spine which are mainly composed of trabecular bone[27 28] In this study the BMD values of ribs and spine inGIOP rats were significantly lower than normal rats Thoseresults further demonstrated that GIOP rats can mimic thebone changes seen in GIOP patients In addition we foundthat the osteoporosis degree of GIOP rats has not improveddramatically after 12 weeks of sham treatment period free ofDXMT We suspect that GIOP rats find it hard to recoverbone mass loss on their own steam if without any effectivetreatment method By contrast PEMF stimulation for 12weeks increased the values of BMD and BMC of GIOP ratseffectively and the curative effect of calcium supplementtreatment was less marked than PEMF stimulation Thetrabecular bone microarchitecture is generally consideredas a good predictor of bone mass loss and bone structuredeterioration [29] and bone loss in GIOP is most obviousin trabecular bone structural changes [28] In this studyhistopathological analysis showed GCs caused thinning oftrabeculae and deteriorated architecture of trabecular bonesuggesting that the trabecular bone structural changes ofGIOP rat have not significantly improved after DXMT injec-tion was stopped However PEMF stimulation improved thechanges of trabecular bone as well as calcium supplementtreatment after 12-week interventions Histomorphometricalanalysis shows that PEMF stimulation increased trabecularwidth and trabecular number The results of the abovemen-tioned analysis indicated that PEMF stimulation markedlyimproved the bone loss in GIOP rats

ALP is amarker of early stage of osteoblast differentiationand it is known to be importantly involved in the regulation ofosteoblastic cell differentiation proliferation and migrationduring bone formation [8 30] TRAP is amarker of osteoclastactivity and it is used to measure the changes in bone resorp-tion In this study PEMF stimulation significantly improvedserum ALP level and reduced serum TRAP level after 12-week interventions suggesting that PEMF can activate theosteoblast differentiation and bone formation meanwhileit can inhibit osteoclast function and bone resorption TGTCHO LDL and HDL are the commonly observed param-eter to measure lipid metabolism in clinical practice [3]Compared with normal rats GIOP rats are characterized byincreased levels of serum TG TCHO and LDL in this studysuggesting that long-termDXMT administration causes lipidmetabolism disorders Moreover the levels of serum TGTCHO and LDLhave not significantly improved afterDXMTinjection was stopped However PEMF stimulation signifi-cantly reduced levels of serum TG TCHO and LDL after12-week interventions suggesting that PEMF improved thelipid metabolism disorders and the improvement effect wassuperior to calcium supplement treatment in GIOP rats

In order to clarify the mechanism of PEMF stimulationfurther experiments were in progress to evaluate the role ofcanonicalWnt signaling pathway in bone formation and lipidmetabolism in GIOP rats The maintenance of bone mass isdetermined by bone remodeling activity which is character-ized by a dynamic balance between osteoblastic bone forma-tion and osteoclastic bone resorption [31] Therefore regula-tion of the functions of osteoblasts and osteoclasts is essentialfor the maintenance of bone mass Osteoblasts and osteo-clasts are differentiated from bone marrow mesenchymalstem cells (BMSCs) Activation of canonical Wnt signalingpathway promotes the differentiation of BMSCs into matureosteoblasts suppresses the apoptosis of osteoblasts andenhances the proliferation and mineralization of osteoblasts[11] Wnt10b LRP5 and 120573-catenin are a key link of canonicalWnt signaling pathway Wnt10b is a positive modulator ofbone formation and it is expressed in bone marrow Thelevels of Wnt10b are directly correlated with bone densityand indirectly related to marrow adiposity [32] LRP5 is acritical coreceptor for Wnt signaling pathway and upstreamof 120573-catenin and it plays an important role in skeletaldevelopment and bone maintenance [30] 120573-catenin is anessentialmediator of signals emanating fromLRP5 and it canpromote the survival and differentiation of osteoblasts [30]In this study the mRNA and protein expressions of Wnt10bLRP5 and120573-cateninwere significantly increased in theGIOPrats after PEMF stimulation for 12 weeks suggesting thatcanonicalWnt signaling pathway was activated during PEMFstimulation which is in agreement with previous reports[16 18] RANKRANKLOPG signaling pathway plays a keyrole in differentiation and functional activation of osteoclasts[10 12] OPG and RANKL are mainly secreted by osteoblastsOPG is a decoy receptor for the RANKL and it preventsRANKL specifically from binding with RANK to promoteosteoclast differentiation and activation [11] Osteoclast activ-ity is likely to depend on the relative balance of OPG andRANKL and the OPGRANKL ratio is an essential factorin bone resorption [30] The OPGRANKL ratio in PEMFgroup was significantly higher than GIOP group suggestingthat PEMF stimulation can promote the OPGRANKL ratiofor regulating osteoclast differentiation and preventing boneresorption Canonical Wnt signaling pathway increases OPGsecretionwhich is likely to depend on activation of120573-catenin120573-catenin can upregulate OPG expression and increasesthe OPGRANKL ratio in osteoblasts [32] Spencer et al[33] suggested that RANKL is a direct target of canonicalWnt signaling pathway Taken together different from theresearch results obtained before [12 18 19] we speculated thatactivation of canonical Wnt signaling pathway can promoteRANKRANKLOPG signaling pathway during PEMF stim-ulation and further regulate the dynamic balance betweenosteoblastic bone formation and osteoclastic bone resorptionin GIOP rats

BMSCs can differentiate into osteoblasts adipocytesmyocytes and chondrocytes [34] Excessive use of GCscan disturb lipid metabolism homeostasis directly by GCsinducing BMSCs differentiation into adipocytes GCs alsocan upregulate the expression of PPAR-120574 downregulate theRunx2 to break the dynamic balance between adipogenesis


and osteogenesis of BMSCs and lead to fat tissue accumula-tion in bone marrow eventually the degree of osteoporosiswas increased [7 15] PPAR-120574 is an adipogenic gene and itplays a pivotal role in the regulation of adipogenesis and lipidmetabolism homeostasis in synergy with another key adi-pogenic transcription factor CEBP120572 PPAR-120574 and CEBP120572positively activate the transcription of each other [17] FABP4is a marker of mature adipocyte On the contrary Runx2 isan osteogenic gene and it is a master regulator of BMSCsdifferentiation into osteoblast and RANKL expression inosteoblasts [35] Furthermore Runx2 is a transcriptionaltarget of canonical Wnt signaling pathway and it involvesnegative regulation of PPAR-120574 expression [10]The inhibitionof Runx2 by GCs is a critical mechanism of GIOP [36]In this study we found that PEMF stimulation significantlydeclined the mRNA and protein expressions of PPAR-120574CEBP120572 and FABP4 and significantly increased the mRNAand protein expressions of Runx2 Canonical Wnt signalingpathway can suppress PPAR-120574 and CEBP120572 expression andupregulate the expression of Runx2 [6] Axin2 a masterscaffolding protein is an intracellular inhibitor of canonicalWnt signaling pathway and it is likely to involve positive reg-ulation of PPAR-120574 expression Naito et al [37] suggested thatDEX promoted adipocyte differentiation by upregulation ofAxin2 expression In this study PEMF stimulation decreasedthe expressions of Axin2 in GIOP rats We speculated thatPEMF stimulation activated canonical Wnt signaling path-way for preventing adipogenesis in BMSCs by suppressingthe expression of adipogenic gene and eventually improvedthe lipid metabolism disorders of GIOP rats Improvement oflipid metabolism disorders can prevent the development ofosteoporosis to a certain extent

GCs depress canonical Wnt signaling pathway throughincreasing the expression of Wnt-antagonists Dkk-1 andSOST in rodents and cell cultures [12] Dkk-1 and SOST bindto LRP56 receptors to inactivateWnt signaling In this studythe expressions of Dkk-1 and SOST have not significantlyincreased in GIOP rats after DXMT injection was stoppedit may be due to GCs absence PEMF stimulation effectivelysuppressed the expressions of Dkk-1 and slightly suppressedthe expressions of SOST SOST is almost exclusively expressedin osteocytes and late osteoblasts [21] The relative higherexpression of SOST inPEMFgroupmaybe due to the numberof osteocytes and late osteoblasts were increased duringPEMF stimulation We speculated that PEMF stimulationactivates canonical Wnt signaling pathway which may bepartially via suppression of Dkk-1

Adequate calcium and vitamin D supplementation is im-portant for GIOP patients because GCs induce an over-all negative calcium balance [12] In this study calciumsupplement treatment can prevent bone loss to a certainextent in GIOP rats perhaps because the calcium gluconateabsorption rate of rats is better than humans We foundthat the moderate prevention effect seemed to have nothingto do with canonical Wnt signaling pathway In additionhyperlipidemia was improved in GIOP rats after calciumsupplement treatment for 12 weeks and our results showedthat calcium gluconate had a comparatively faint inhibitionto the expressions of PPAR-120574 and FABP4 Jensen et al

[38] demonstrated that high extracellular calcium plays anegative role in adipocyte differentiation Parra et al [39]found the antiobesity effect of dietary calcium in male miceCalcium supplementation may have some potential benefitsof overcoming lipid metabolism disorders in GIOP rats

In summary our study clearly demonstrated that GIOPrats still need effective antiosteoporosis therapy after GCsadministration was stopped PEMF stimulation can preventbone loss and improve lipid metabolism disorders with noapparent side effects in GIOP rats Wnt10bLRP5120573-cateninsignaling pathway played an important role during PEMFstimulation Certainly other signaling pathwaysmay possiblybe activated byPEMF such as parathyroid hormone pathwaysand insulin-like growth factor The influence of inactivationof canonical Wnt signaling pathway on bone formation andlipid metabolism during PEMFs stimulation needs furtherinvestigation However our study suggests that PEMF treat-ment may be a suitable therapeutic method for GIOP and itwould offer some potential benefits for GIOP patients

Conflict of Interests

Yuan Jiang Lehua Yu Hui Gou Sanrong Wang Jiang Zhuand Si Tian declare that they have no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the grants fromNational NaturalScience Foundation of China (no 81171859) and ChongqingMunicipal Healthcare Department Medical Research Grant(no 2010-1-202) The authors also express their sincerethanks to Dr Lei Yuan Dr Mingyuan Tian Dr Yi Yuan andMs Zunzhen Zhou for their helpful assistance

References

[1] R Feng L Feng Z Yuan et al ldquoIcariin protects againstglucocorticoid-induced osteoporosis in vitro and preventsglucocorticoid-induced osteocyte apoptosis in vivordquo Cell Bio-chemistry and Biophysics vol 67 no 1 pp 189ndash197 2013

[2] Y Fujita K Watanabe S Uchikanbori and K Maki ldquoEffectsof risedronate on cortical and trabecular bone of the mandiblein glucocorticoid-treated growing ratsrdquo American Journal ofOrthodontics and Dentofacial Orthopedics vol 139 no 3 ppe267ndashe277 2011

[3] H-Y Yoon Y-S Cho Q Jin H-G Kim E-R Woo and Y-S Chung ldquoEffects of ethyl acetate extract of Poncirus trifoliatafruit for glucocorticoid-induced osteoporosisrdquo Biomoleculesand Therapeutics vol 20 no 1 pp 89ndash95 2012

[4] J Folwarczna M Pytlik L Sliwinski U Cegieła B Nowinskaand M Rajda ldquoEffects of propranolol on the development ofglucocorticoid-induced osteoporosis in male ratsrdquo Pharmaco-logical Reports vol 63 no 4 pp 1040ndash1049 2011

[5] A Pranic-Kragic M Radic D Martinovic-Kaliterna and JRadic ldquoGlucocorticoid induced osteoporosisrdquo Acta ClinicaCroatica vol 50 no 4 pp 563ndash566 2011

[6] L Tian and X Yu ldquoLipid metabolism disorders and bonedysfunctionmdashinterrelated and mutually regulated (review)rdquoMolecular Medicine Reports vol 12 no 1 pp 783ndash794 2015


[7] L Cui T Li Y Liu et al ldquoSalvianolic acid B prevents bone lossin prednisone-treated rats through stimulation of osteogenesisand bonemarrow angiogenesisrdquo PLoS ONE vol 7 no 4 ArticleID e34647 2012

[8] Z Zhang L Xiang D Bai et al ldquoTreatment with RhizomaDioscoreae extract has protective effect on osteopenia in ovari-ectomized ratsrdquo The Scientific World Journal vol 2014 ArticleID 645975 12 pages 2014

[9] R S Weinstein ldquoGlucocorticoid-induced osteoporosis andosteonecrosisrdquo Endocrinology and Metabolism Clinics of NorthAmerica vol 41 no 3 pp 595ndash611 2012

[10] C Zhong T-F Zhao Z-J Xu and R-X He ldquoEffects of elec-tromagnetic fields on bone regeneration in experimental andclinical studies a review of the literaturerdquo Chinese MedicalJournal vol 125 no 2 pp 367ndash372 2012

[11] J Zhou X Li Y Liao W Feng C Fu and X Guo ldquoPulsedelectromagnetic fields inhibit bone loss in streptozotocin-induced diabetic ratsrdquo Endocrine vol 49 no 1 pp 258ndash2662015

[12] D Jing J Cai Y Wu et al ldquoPulsed electromagnetic fields par-tially preserve bone mass microarchitecture and strengthby promoting bone formation in hindlimb-suspended ratsrdquoJournal of Bone and Mineral Research vol 29 no 10 pp 2250ndash2261 2014

[13] J Zhou S Chen H Guo et al ldquoPulsed electromagnetic fieldstimulates osteoprotegerin and reduces RANKL expression inovariectomized ratsrdquo Rheumatology International vol 33 no 5pp 1135ndash1141 2013

[14] D Jing J Cai G Shen et al ldquoThe preventive effects of pulsedelectromagnetic fields on diabetic bone loss in streptozotocin-treated ratsrdquo Osteoporosis International vol 22 no 6 pp 1885ndash1895 2011

[15] J-P Li S Chen H Peng J-L Zhou and H-S Fang ldquoPulsedelectromagnetic fields protect the balance between adipogenesisand osteogenesis on steroid-induced osteonecrosis of femoralhead at the pre-collapse stage in ratsrdquo Bioelectromagnetics vol35 no 3 pp 170ndash180 2014

[16] S Ding H Peng H-S Fang J-L Zhou and Z Wang ldquoPulsedelectromagnetic fields stimulation prevents steroid-inducedosteonecrosis in ratsrdquo BMC Musculoskeletal Disorders vol 12article 215 2011

[17] M Kawai S Mushiake K Bessho et al ldquoWntLrp120573-cateninsignaling suppresses adipogenesis by inhibiting mutual acti-vation of PPAR120574 and CEBP120572rdquo Biochemical and BiophysicalResearch Communications vol 363 no 2 pp 276ndash282 2007

[18] D Jing F Li M Jiang et al ldquoPulsed electromagnetic fieldsimprove bone microstructure and strength in ovariectomizedrats through a WntLrp5120573-catenin signaling-associated mech-anismrdquo PLoS ONE vol 8 no 11 Article ID e79377 2013

[19] J Zhou H He L Yang et al ldquoEffects of pulsed electromagneticfields on bone mass and Wnt120573-catenin signaling pathway inovariectomized ratsrdquo Archives of Medical Research vol 43 no4 pp 274ndash282 2012

[20] H H Ahmed N Y S Morcos E F Eskander D M S SeoudiandA B Shalby ldquoPotential role of leptin against glucocorticoid-induced secondary osteoporosis in adult female ratsrdquo EuropeanReview for Medical and Pharmacological Sciences vol 16 no 10pp 1446ndash1452 2012

[21] N Guanabens L Gifre and P Peris ldquoThe role of Wnt signalingand sclerostin in the pathogenesis of glucocorticoid-inducedosteoporosisrdquo Current Osteoporosis Reports vol 12 no 1 pp90ndash97 2014

[22] S Rahman P J Czernik Y Lu and B Lecka-Czernik ldquo120573-catenin directly sequesters adipocytic and insulin sensitizingactivities but not osteoblastic activity of PPAR1205742 in marrowmesenchymal stem cellsrdquo PLoS ONE vol 7 no 12 Article IDe51746 2012

[23] R S Weinstein ldquoGlucocorticoid-induced osteonecrosisrdquoEndocrine vol 41 no 2 pp 183ndash190 2012

[24] W S Jee and W Yao ldquoOverview animal models of osteopeniaand osteoporosisrdquo Journal of Musculoskeletal and NeuronalInteractions vol 1 no 3 pp 193ndash207 2001

[25] T Komori ldquoAnimal models for osteoporosisrdquo European Journalof Pharmacology vol 759 pp 287ndash294 2015

[26] P P Lelovas T T Xanthos S E Thorma G P Lyritis and I ADontas ldquoThe laboratory rat as an animalmodel for osteoporosisresearchrdquo Comparative Medicine vol 58 no 5 pp 424ndash4302008

[27] A Bitto B P Burnett F Polito et al ldquoGenistein aglyconereverses glucocorticoid-induced osteoporosis and increasesbone breaking strength in rats a comparative study withalendronaterdquo British Journal of Pharmacology vol 156 no 8 pp1287ndash1295 2009

[28] L C Hofbauer U Zeitz M Schoppet et al ldquoPrevention ofglucocorticoid-induced bone loss in mice by inhibition ofRANKLrdquo Arthritis and Rheumatism vol 60 no 5 pp 1427ndash1437 2009

[29] D Jing G Shen J Huang et al ldquoCircadian rhythm affectsthe preventive role of pulsed electromagnetic fields onovariectomy-induced osteoporosis in ratsrdquo Bone vol 46 no 2pp 487ndash495 2010

[30] C Zhang J Peng S Wu et al ldquoDioscin promotes osteoblasticproliferation and differentiation via Lrp5 and ER pathwayin mouse and human osteoblast-like cell linesrdquo Journal ofBiomedical Science vol 21 article 30 2014

[31] J Zhou J-Q Wang B-F Ge et al ldquoDifferent electromagneticfield waveforms have different effects on proliferation differ-entiation and mineralization of osteoblasts in vitrordquo Bioelectro-magnetics vol 35 no 1 pp 30ndash38 2014

[32] D G Monroe M E McGee-Lawrence M J Oursler and J JWestendorf ldquoUpdate onWnt signaling in bone cell biology andbone diseaserdquo Gene vol 492 no 1 pp 1ndash18 2012

[33] G J Spencer J C Utting S L Etheridge T R Arnett and PG Genever ldquoWnt signalling in osteoblasts regulates expressionof the receptor activator of NF-kappaB ligand and inhibitsosteoclastogenesis in vitrordquo Journal of Cell Science vol 119 part7 pp 1283ndash1296 2006

[34] A W James ldquoReview of signaling pathways governing MSCosteogenic and adipogenic differentiationrdquo Scientifica vol 2013Article ID 684736 17 pages 2013

[35] J Albers J Keller A Baranowsky et al ldquoCanonical Wnt signal-ing inhibits osteoclastogenesis independent of osteoprotegerinrdquoJournal of Cell Biology vol 200 no 4 pp 537ndash549 2013

[36] TKoromila S K Baniwal Y S SongAMartin J Xiong andBFrenkel ldquoGlucocorticoids antagonize RUNX2 during osteoblastdifferentiation in cultures of ST2 pluripotent mesenchymalcellsrdquo Journal of Cellular Biochemistry vol 115 no 1 pp 27ndash332014

[37] M Naito Y Mikami M Takagi and T Takahashi ldquoUp-regulation of Axin2 by dexamethasone promotes adipocytedifferentiation in ROB-C26mesenchymal progenitor cellsrdquoCelland Tissue Research vol 354 no 3 pp 761ndash770 2013


[38] B Jensen M C Farach-Carson E Kenaley and K A AkanbildquoHigh extracellular calcium attenuates adipogenesis in 3T3-L1preadipocytesrdquo Experimental Cell Research vol 301 no 2 pp280ndash292 2004

[39] P Parra G Bruni A Palou and F Serra ldquoDietary calciumattenuation of body fat gain during high-fat feeding in micerdquoJournal of Nutritional Biochemistry vol 19 no 2 pp 109ndash1172008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


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



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Therefore safe and noninvasive biophysical countermeasuresfor complications ofGCs therapymight bemore promising inclinical application

Pulsed electromagnetic fields (PEMFs) have been sug-gested as promising method alternatives to drug-based ther-apies for treating a wide range of bone disorders suchas fresh fracture delayed and nonunion fractures diabeticosteopenia osteoporosis osteonecrosis and osteoarthritis[10 11] PEMF is useful in enhancing BMD in OP patientsand preventing bone loss in animal models of disuse OPtail-suspension OP ovariectomy-induced OP and diabetes-mellitus-induced OP [12ndash14] Several previous studies wereusing PEMF to prevent steroid-associated osteonecrosis andfound that PEMF can improve serum lipid levels [15 16]

Wingless-type MMTV integration site (Wnt) signalingpathway plays a critical role inmaintenance of bonemass andis able to suppress adipogenesis and promote osteoblastoge-nesis of bone marrow mesenchymal stromal cells (BMSCs)[17] PEMF stimulation can activate Wnt signaling pathwayduring treatment of bone loss in ovariectomized rats [18 19]However the skeletal dynamics and pathogenesis of GIOPare distinctly different from other OP that associated withageing estrogen deprivation and immobilization [5 20] Fewstudies have investigated the effects of PEMF on bone statuslipid metabolism and related signaling pathway mechanismsin GIOP animal models Particularly GCs can directlyinduce differentiation of BMSCs into adipocytes and inhibitosteogenic differentiation through downregulating the Wntsignaling pathway [16 21 22] In this present study weestablished GIOP SD rats model by intramuscular injectionwith dexamethasone to investigate the effect of PEMF onbone formation and lipid metabolism and further exploredthe several important components and targets of canonicalWnt signaling pathway in GIOP rats

2 Materials and Methods

21 Establishment of GIOP Rats Model All animal experi-mental protocol and care were approved by the InstitutionalAnimal Care and Use Committee of Chongqing MedicalUniversity Totally 40 female Sprague Dawley rats wereemployed aged 3 months weighing 210 plusmn 20 g and housedindividually and maintained under controlled environmen-tal conditions (12-hour light-dark cycle temperature 22∘Cwith humidity of 50 plusmn 5) All rats had unrestricted accessto water and food After one week of acclimatization 10 ratswere randomly divided into control groups The rest of therats were injected with dexamethasone sodium phosphateinjection (DXMT 25mgkg) into their right haunchmusclesto establish GIOP rat models twice a week 12 weeks in a rowAfter 12 weeks those rats received no DXMT interventionand were randomly divided into GIOP group (119899 = 10)calcium supplement group (calcium group 119899 = 10) andPEMF group (119899 = 10) The rats in the control groupwere injected with equivalent dose of normal saline intotheir right haunch muscles twice a week 12 weeks in arow Subsequently the fasting rats in each group receivedintraperitoneal anesthesia of 10 chloral hydrate (35mLkg)The rats were positioned on the operation platform in supine

position After the fur on the neck of rat was removed andskin was sterilized a midline 1ndash15 cm longitudinal incisionwas performed The left cephalic artery of rat was found totake blood (15ndash2mL) and then the incision was stitchedBlood specimens were centrifuged to get serum Bone min-eral density (BMD) and bone mineral content (BMC) weremeasured by using dual energy X-ray absorptiometry (LunariDXA GE Healthcare) equipped with dedicated software forsmall animalmeasurements and serumbiochemical analyseswere operated to investigate whether the GIOP rat model wassuccessfully induced by injecting DXMT and associated withlipid metabolism disorders

22 GIOP Rats Treatments The rats in calcium groupreceived oral calcium (5625mgkg calcium gluconic tabletsHainan Pharmaceutical Factory Co Ltd China) once a day12 weeks in a row At the same time the rats in control groupGIOP group and PEMF group were perfused with the samevolume of saline as the criteria of body mass The rats inPEMF group were exposed to PEMF which was generatedby the ZH-21 osteoporosis treatment system (ChongqingZhonghuan Electronic Technology Co Ltd China) with afrequency of 50Hz and an intensity of 40mT once a day 40-minute treatment every day 12 weeks in a rowThe waveformis square wave with pulse width 200 120583s At the same time therats in control group calcium group and GIOP group werealso placed in the ZH-21 osteoporosis treatment system butthe treatment systemwas not running to provide shamPEMFstimulation

23 BoneMineral Density andBoneMineral ContentMeasure-ment After a period of 12 weeksrsquo treatment all rats receivedintraperitoneal anesthesia of 10 chloral hydrate (35mLkg)Then the rats were positioned on the platform of dual energyX-ray absorptiometry (Lunar iDXA GEHealthcare) in proneposition The BMD and BMC of head upper limb femurtrunk rib pelvis spine and the whole body were detectedand recorded

24 Serum Biochemical Analysis The fasting rats in eachgroup received intraperitoneal anesthesia with 10 chlo-ral hydrate (35mLkg) Then the rats were positioned onthe operation platform before they were executed The furon the abdomen was removed and skin was sterilized amidline 4-5 cm longitudinal incision was performed Theblood specimens were withdrawn from aorta abdominalisand then centrifuged to get serum The serum calcium (Ca)phosphorus (P) alkaline phosphatase (ALP) triglyceride(TG) total cholesterol (TCHO) high density lipoproteincholesterol (HDL) and low density lipoprotein cholesterol(LDL) were determined by automatic biochemical analyzer(TBA-120FR Toshiba) The serum tartrate resistant acidphosphatase (TRAP) was determined by tartrate resistantacid phosphatase assay kit (Beyotime China) according tothe protocol of the manufacturer

25 Histomorphometrical and Histopathological AnalysisAfter all rats were executed the fourth lumbar (L4) vertebralbodies were collected Each fourth lumbar was carefully






















3 Results



Table2Bo

nemineraldensity

(BMD)o

feachgrou

pbefore

andaft

ertre

atment(gcm2

)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

0237plusmn0010

0153plusmn0032

0142plusmn0011

0125plusmn000

90113plusmn000

60132plusmn0017

0132plusmn0008

0149plusmn0005

Posttreatment

0243plusmn0016

0144plusmn0026

0156plusmn0012

O0125plusmn0008

0110plusmn0007

0134plusmn0017

0137plusmn000

90153plusmn0007

GIO

Pgrou

pPriortotre

atment


0131plusmn

0031






0132plusmn000

4lowastPo

sttreatment

0235plusmn0021

0123plusmn0032

0132plusmn0011

0114plusmn0007

0102plusmn0007

0117plusmn0016

0111plusmn0015

0135plusmn000

9

Calcium

grou

pPriortotre

atment

0221plusmn

0010lowast

0147plusmn0035


0106plusmn000




0131plusmn

0005lowast

Posttreatment

0237plusmn0024

0119plusmn0033

0129plusmn0019

0113plusmn0014

0100plusmn0008

0123plusmn0018O

0125plusmn0018

O0141plusmn

0013

PEMFgrou

pPriortotre

atment

0219plusmn000


90120plusmn000



0101plusmn

0015lowast


0133plusmn000

6lowastPo

sttreatment

0234plusmn0024

0112plusmn0035

0133plusmn0018O

0117plusmn0008O

0104plusmn0008

0117plusmn0018O

0130plusmn0013

O0148plusmn0008

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Table3Bo

nemineralcontent(BM

C)of

each

grou

pbefore

andaft

ertre

atment(g)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

202plusmn023

049plusmn025

233plusmn10

0382plusmn10

512

5plusmn049

140plusmn023

173plusmn049

819plusmn064

Posttreatment

204plusmn028

049plusmn023

279plusmn069

343plusmn097

12

3plusmn060

13

5plusmn053

185plusmn075

831plusmn095

GIO

Pgrou

pPriortotre

atment

180plusmn013lowast

039plusmn027

231plusmn073

260plusmn064lowast

067plusmn032lowast

115plusmn026lowast

080plusmn025lowast

721plusmn

063lowast

Posttreatment

185plusmn018

034plusmn010

232plusmn069

232plusmn073

058plusmn031

110plusmn037

080plusmn028

706plusmn057

Calcium

grou

pPriortotre

atment

182plusmn010lowast

042plusmn023

262plusmn10

1212plusmn033lowast

053plusmn011lowast

100plusmn034lowast

081plusmn025lowast

714plusmn077lowast

Posttreatment

190plusmn032

035plusmn019

258plusmn065

244plusmn041

061plusmn020

124plusmn032

O091plusmn033

752plusmn071

O

PEMFgrou

pPriortotre

atment

188plusmn016

053plusmn027

225plusmn097

227plusmn040lowast

053plusmn012lowast

095plusmn026lowast

086plusmn032lowast

709plusmn080lowast

Posttreatment

204plusmn021

O046plusmn023

273plusmn068

233plusmn039

063plusmn018

O10

2plusmn027

099plusmn034

768plusmn064

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Beforetreatment

Aftertreatment

OPG

RA

NKL

mRN

A

1000

2000

3000

ALP

(UL

)

10

20

30

TRA

P (U

L)

10

20

30

40

expr

essio

n

OPG

RA

NKL

pro

tein

expr

essio

n

05

10

15

20

25

10

20

30

40

(mm

olL

)

10

20

30

40(m

mol

L)



Beforetreatment

Aftertreatment





lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(G) (H)

(F)

(D)

(E)

(C)

(A) (B)

(A) (B)

(A) (C)

(D)(B)(a)

(b)

(c)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

∘

∘

∘

∘

∘

∘

lowast∘ lowast∘

00 00

0000

00 00
















4 Discussion















05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n


OPG RANKL Runx2


Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000



Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































3 Results



Table2Bo

nemineraldensity

(BMD)o

feachgrou

pbefore

andaft

ertre

atment(gcm2

)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

0237plusmn0010

0153plusmn0032

0142plusmn0011

0125plusmn000

90113plusmn000

60132plusmn0017

0132plusmn0008

0149plusmn0005

Posttreatment

0243plusmn0016

0144plusmn0026

0156plusmn0012

O0125plusmn0008

0110plusmn0007

0134plusmn0017

0137plusmn000

90153plusmn0007

GIO

Pgrou

pPriortotre

atment


0131plusmn

0031






0132plusmn000

4lowastPo

sttreatment

0235plusmn0021

0123plusmn0032

0132plusmn0011

0114plusmn0007

0102plusmn0007

0117plusmn0016

0111plusmn0015

0135plusmn000

9

Calcium

grou

pPriortotre

atment

0221plusmn

0010lowast

0147plusmn0035


0106plusmn000




0131plusmn

0005lowast

Posttreatment

0237plusmn0024

0119plusmn0033

0129plusmn0019

0113plusmn0014

0100plusmn0008

0123plusmn0018O

0125plusmn0018

O0141plusmn

0013

PEMFgrou

pPriortotre

atment

0219plusmn000


90120plusmn000



0101plusmn

0015lowast


0133plusmn000

6lowastPo

sttreatment

0234plusmn0024

0112plusmn0035

0133plusmn0018O

0117plusmn0008O

0104plusmn0008

0117plusmn0018O

0130plusmn0013

O0148plusmn0008

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Table3Bo

nemineralcontent(BM

C)of

each

grou

pbefore

andaft

ertre

atment(g)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

202plusmn023

049plusmn025

233plusmn10

0382plusmn10

512

5plusmn049

140plusmn023

173plusmn049

819plusmn064

Posttreatment

204plusmn028

049plusmn023

279plusmn069

343plusmn097

12

3plusmn060

13

5plusmn053

185plusmn075

831plusmn095

GIO

Pgrou

pPriortotre

atment

180plusmn013lowast

039plusmn027

231plusmn073

260plusmn064lowast

067plusmn032lowast

115plusmn026lowast

080plusmn025lowast

721plusmn

063lowast

Posttreatment

185plusmn018

034plusmn010

232plusmn069

232plusmn073

058plusmn031

110plusmn037

080plusmn028

706plusmn057

Calcium

grou

pPriortotre

atment

182plusmn010lowast

042plusmn023

262plusmn10

1212plusmn033lowast

053plusmn011lowast

100plusmn034lowast

081plusmn025lowast

714plusmn077lowast

Posttreatment

190plusmn032

035plusmn019

258plusmn065

244plusmn041

061plusmn020

124plusmn032

O091plusmn033

752plusmn071

O

PEMFgrou

pPriortotre

atment

188plusmn016

053plusmn027

225plusmn097

227plusmn040lowast

053plusmn012lowast

095plusmn026lowast

086plusmn032lowast

709plusmn080lowast

Posttreatment

204plusmn021

O046plusmn023

273plusmn068

233plusmn039

063plusmn018

O10

2plusmn027

099plusmn034

768plusmn064

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Beforetreatment

Aftertreatment

OPG

RA

NKL

mRN

A

1000

2000

3000

ALP

(UL

)

10

20

30

TRA

P (U

L)

10

20

30

40

expr

essio

n

OPG

RA

NKL

pro

tein

expr

essio

n

05

10

15

20

25

10

20

30

40

(mm

olL

)

10

20

30

40(m

mol

L)



Beforetreatment

Aftertreatment





lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(G) (H)

(F)

(D)

(E)

(C)

(A) (B)

(A) (B)

(A) (C)

(D)(B)(a)

(b)

(c)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

∘

∘

∘

∘

∘

∘

lowast∘ lowast∘

00 00

0000

00 00
















4 Discussion















05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n


OPG RANKL Runx2


Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000



Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Bo

nemineraldensity

(BMD)o

feachgrou

pbefore

andaft

ertre

atment(gcm2

)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

0237plusmn0010

0153plusmn0032

0142plusmn0011

0125plusmn000

90113plusmn000

60132plusmn0017

0132plusmn0008

0149plusmn0005

Posttreatment

0243plusmn0016

0144plusmn0026

0156plusmn0012

O0125plusmn0008

0110plusmn0007

0134plusmn0017

0137plusmn000

90153plusmn0007

GIO

Pgrou

pPriortotre

atment


0131plusmn

0031






0132plusmn000

4lowastPo

sttreatment

0235plusmn0021

0123plusmn0032

0132plusmn0011

0114plusmn0007

0102plusmn0007

0117plusmn0016

0111plusmn0015

0135plusmn000

9

Calcium

grou

pPriortotre

atment

0221plusmn

0010lowast

0147plusmn0035


0106plusmn000




0131plusmn

0005lowast

Posttreatment

0237plusmn0024

0119plusmn0033

0129plusmn0019

0113plusmn0014

0100plusmn0008

0123plusmn0018O

0125plusmn0018

O0141plusmn

0013

PEMFgrou

pPriortotre

atment

0219plusmn000


90120plusmn000



0101plusmn

0015lowast


0133plusmn000

6lowastPo

sttreatment

0234plusmn0024

0112plusmn0035

0133plusmn0018O

0117plusmn0008O

0104plusmn0008

0117plusmn0018O

0130plusmn0013

O0148plusmn0008

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Table3Bo

nemineralcontent(BM

C)of

each

grou

pbefore

andaft

ertre

atment(g)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

202plusmn023

049plusmn025

233plusmn10

0382plusmn10

512

5plusmn049

140plusmn023

173plusmn049

819plusmn064

Posttreatment

204plusmn028

049plusmn023

279plusmn069

343plusmn097

12

3plusmn060

13

5plusmn053

185plusmn075

831plusmn095

GIO

Pgrou

pPriortotre

atment

180plusmn013lowast

039plusmn027

231plusmn073

260plusmn064lowast

067plusmn032lowast

115plusmn026lowast

080plusmn025lowast

721plusmn

063lowast

Posttreatment

185plusmn018

034plusmn010

232plusmn069

232plusmn073

058plusmn031

110plusmn037

080plusmn028

706plusmn057

Calcium

grou

pPriortotre

atment

182plusmn010lowast

042plusmn023

262plusmn10

1212plusmn033lowast

053plusmn011lowast

100plusmn034lowast

081plusmn025lowast

714plusmn077lowast

Posttreatment

190plusmn032

035plusmn019

258plusmn065

244plusmn041

061plusmn020

124plusmn032

O091plusmn033

752plusmn071

O

PEMFgrou

pPriortotre

atment

188plusmn016

053plusmn027

225plusmn097

227plusmn040lowast

053plusmn012lowast

095plusmn026lowast

086plusmn032lowast

709plusmn080lowast

Posttreatment

204plusmn021

O046plusmn023

273plusmn068

233plusmn039

063plusmn018

O10

2plusmn027

099plusmn034

768plusmn064

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Beforetreatment

Aftertreatment

OPG

RA

NKL

mRN

A

1000

2000

3000

ALP

(UL

)

10

20

30

TRA

P (U

L)

10

20

30

40

expr

essio

n

OPG

RA

NKL

pro

tein

expr

essio

n

05

10

15

20

25

10

20

30

40

(mm

olL

)

10

20

30

40(m

mol

L)



Beforetreatment

Aftertreatment





lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(G) (H)

(F)

(D)

(E)

(C)

(A) (B)

(A) (B)

(A) (C)

(D)(B)(a)

(b)

(c)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

∘

∘

∘

∘

∘

∘

lowast∘ lowast∘

00 00

0000

00 00
















4 Discussion















05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n


OPG RANKL Runx2


Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000



Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table3Bo

nemineralcontent(BM

C)of

each

grou

pbefore

andaft

ertre

atment(g)

Group

sPart

Head

Upp

erlim

bFemur

Trun

kRib

Pelvis

Spine

Who

lebo

dy

Con

trolgroup

Priortotre

atment

202plusmn023

049plusmn025

233plusmn10

0382plusmn10

512

5plusmn049

140plusmn023

173plusmn049

819plusmn064

Posttreatment

204plusmn028

049plusmn023

279plusmn069

343plusmn097

12

3plusmn060

13

5plusmn053

185plusmn075

831plusmn095

GIO

Pgrou

pPriortotre

atment

180plusmn013lowast

039plusmn027

231plusmn073

260plusmn064lowast

067plusmn032lowast

115plusmn026lowast

080plusmn025lowast

721plusmn

063lowast

Posttreatment

185plusmn018

034plusmn010

232plusmn069

232plusmn073

058plusmn031

110plusmn037

080plusmn028

706plusmn057

Calcium

grou

pPriortotre

atment

182plusmn010lowast

042plusmn023

262plusmn10

1212plusmn033lowast

053plusmn011lowast

100plusmn034lowast

081plusmn025lowast

714plusmn077lowast

Posttreatment

190plusmn032

035plusmn019

258plusmn065

244plusmn041

061plusmn020

124plusmn032

O091plusmn033

752plusmn071

O

PEMFgrou

pPriortotre

atment

188plusmn016

053plusmn027

225plusmn097

227plusmn040lowast

053plusmn012lowast

095plusmn026lowast

086plusmn032lowast

709plusmn080lowast

Posttreatment

204plusmn021

O046plusmn023

273plusmn068

233plusmn039

063plusmn018

O10

2plusmn027

099plusmn034

768plusmn064

O

Com

paredwith

thec

ontro

lgroup

before

treatmentlowast

119875lt005com

paredwith

theG

IOPgrou

paft

ertre

atment

119875lt005com

paredwith

before

treatment

O119875lt005


Beforetreatment

Aftertreatment

OPG

RA

NKL

mRN

A

1000

2000

3000

ALP

(UL

)

10

20

30

TRA

P (U

L)

10

20

30

40

expr

essio

n

OPG

RA

NKL

pro

tein

expr

essio

n

05

10

15

20

25

10

20

30

40

(mm

olL

)

10

20

30

40(m

mol

L)



Beforetreatment

Aftertreatment





lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(G) (H)

(F)

(D)

(E)

(C)

(A) (B)

(A) (B)

(A) (C)

(D)(B)(a)

(b)

(c)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

∘

∘

∘

∘

∘

∘

lowast∘ lowast∘

00 00

0000

00 00
















4 Discussion















05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n


OPG RANKL Runx2


Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000



Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Beforetreatment

Aftertreatment

OPG

RA

NKL

mRN

A

1000

2000

3000

ALP

(UL

)

10

20

30

TRA

P (U

L)

10

20

30

40

expr

essio

n

OPG

RA

NKL

pro

tein

expr

essio

n

05

10

15

20

25

10

20

30

40

(mm

olL

)

10

20

30

40(m

mol

L)



Beforetreatment

Aftertreatment





lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(G) (H)

(F)

(D)

(E)

(C)

(A) (B)

(A) (B)

(A) (C)

(D)(B)(a)

(b)

(c)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

∘

∘

∘

∘

∘

∘

lowast∘ lowast∘

00 00

0000

00 00
















4 Discussion















05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n


OPG RANKL Runx2


Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000



Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























4 Discussion















05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n


OPG RANKL Runx2


Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000



Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

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H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

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p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

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ium

PEM

F

Con

trol

grou

p

grou

p

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p

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p

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PEM

F

Con

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p

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p

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p

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Con

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Con

trol

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p

GIO

P

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Con

trol

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grou

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P

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Con

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Con

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Con

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ium

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Con

trol

grou

p

grou

p

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p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























05

10

15

20

Relat

ive g

ene e

xpre

ssio

n

05

10

15

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

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ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

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ssio

n

10

20

30

Relat

ive g

ene e

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ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

Relat

ive g

ene e

xpre

ssio

n

10

20

30

40

50

Relat

ive g

ene e

xpre

ssio

n

10

20

30

Relat

ive g

ene e

xpre

ssio

n


OPG RANKL Runx2


Axin2 Dkk-1 SOST

lowast

lowast

∘

∘

lowast∘

lowast∘

00 00 00

00 0000

00 00 00

000000



Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

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p

(kDa)

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P

Calc

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PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

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ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

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PEM

F

Con

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grou

p

grou

p

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p

grou

pG

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PEM

F

Con

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grou

p

grou

p

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p

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P

Calc

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PEM

F

Con

trol

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p

grou

p

grou

p

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Con

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PEM

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Con

trol

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grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Wnt10b 43

LRP5 178

90

GAPDH 36

OPG 60

RANKL 30

Runx2 55

36

57

45

FABP4 15

36

Axin2 94

Dkk-1 34

SOST 25

36

lowast

lowast

G

IOP

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

120573-catenin

PPAR-120574

CEBP120572

10

15

20

Wnt

10b

GA

PDH

pro

tein

10

15

20

OPG

GA

PDH

pro

tein

10

15

20

PPA

R-120574

GA

PDH

pro

tein

05

10

15

20

Axi

n2G

APD

H p

rote

in

10

15

20

Dkk

-1G

APD

H p

rote

in

10

15

20

CEB

P120572G

APD

H p

rote

in

10

15

20

RAN

KLG

APD

H p

rote

in

10

15

20

LRP5

GA

PDH

pro

tein

10

15

20

120573-c

aten

inG

APD

H p

rote

in

10

15

20

Runx

2G

APD

H p

rote

in

10

15

20

FABP

4G

APD

H p

rote

in

10

15

20

SOST

GA

PDH

pro

tein

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GAPDH

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

(kDa)

(kDa)

(kDa)

(kDa)

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

IOP

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ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

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ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

pG

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ium

PEM

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Con

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p

grou

p

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p

grou

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GIO

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PEM

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Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

GIO

P

Calc

ium

PEM

F

Con

trol

grou

p

grou

p

grou

p

grou

p

lowast∘lowast∘

lowast∘

∘ ∘

lowast

lowast

lowast

lowast∘

lowast∘

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

05

00

















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article effect of pulsed electromagnetic field on...

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