Research Article Tetramethylpyrazine Enhances...

Research ArticleTetramethylpyrazine Enhances Vascularization andPrevents Osteonecrosis in Steroid-Treated Rats

Yini Jiang12 Chunfang Liu1 Weiheng Chen3 Hui Wang1 Chao Wang1 and Na Lin1

1 Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences No 16 NanxiaojieDongzhimennei Beijing 100700 China2School of Pharmacy Jiangxi University of Traditional Chinese Medicine Nanchang 330004 China3Wangjing Hospital China Academy of Chinese Medical Sciences Beijing 100102 China

Correspondence should be addressed to Na Lin linna888163com

Received 2 December 2014 Revised 20 January 2015 Accepted 20 January 2015

Academic Editor Magali Cucchiarini

Copyright copy 2015 Yini Jiang et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Steroid-induced osteonecrosis of the femoral head (steroid-induced ONFH) is an avascular necrosis disease of bone Tetram-ethylpyrazine (TMP) with significant vascular protective properties has been widely used for the treatments of ischemic neuraldisorders and cardiovascular diseases However its role in the treatment of steroid-induced ONFH has not been evaluated In thisstudy our results showed that TMP significantly decreased the ratio of empty lacuna adipose tissue area and adipocyte perimeterin steroid-induced ONFH rats histopathologically TMP also reduced the levels of serum lipid dramatically by haematologicalexamination According to the micro-CT quantification TMP could improve the microstructure of the trabecular bone andincreases bone mineral density in steroid-induced ONFH rats Moreover TMP significantly increased the vessel volume vesselsurface percentage of vessel volume and vessel thickness of the femoral heads by micro-CT Interestingly the downregulation ofVEGF and FLK1 proteins in the sera and necrotic femoral heads could be reversed by TMP treatment and this was true for theirmRNA expressions in femoral heads In conclusion these findings suggest for the first time that TMPmay prevent steroid-inducedONFH and also enhance femoral head vascularization by inhibiting the effect of steroid on VEGFFLK1 signal pathway

1 Introduction

Steroid-induced osteonecrosis of the femoral head (steroid-inducedONFH) represents a degenerative bone disease char-acterized by the increase of intraosseous pressure because oflipid metabolism disturbance such as elevating of adipogene-sis and fat cell hypertrophy in the bonemarrow subsequentlyleading to disturbances of coagulation-fibrinolysis system inthe femoral head and finally resulting in bone ischemia [1] Itis a common complication with disabling effect for patientsafter high-dose corticosteroid treatment [2] Previous studieshave demonstrated that steroids decrease skeletal angiogene-sis vascularity and hydration [3] reduce femoral head bloodflow and have a vasoconstrictive effect on lateral epiphysealarteries of the femoral head which could lead to femoral headischemia and subsequent necrosis [4 5] The pathogenesisand aetiology of steroid-induced ONFH have not been fullyelucidated According to the recent study increased osteo-cytes apoptosis the impairment of bone cell survival and

bone formation promotion of osteoclastic resorption andadipocytic differentiation in bonemicroenvironments as wellas ischemia may be the assumed causes for this disease [6 7]Among these ischemia is a feature of the necrotic lesion[8] The current surgical treatments for this disease aimed athip preservation have attempted to induce revascularizationor transposition of the necrotic area [9ndash11] However thesetreatments were limited in their ability to support subchon-dral bone and were not necessarily successful Therefore it isextremely necessary to develop novel and efficient agents forthe treatment of this disease

Tetramethylpyrazine (TMP relative molecular mass2085) is one of the most important active ingredients ofthe traditional Chinese herbal medicine Ligusticum wallichiiFranchat (ChuanXiong) [12 13] It has beenwidely used espe-cially in the treatments of ischemic neural disorders and cardi-ovascular diseases such as ischemic stroke and pulmonaryhypertension secondary to chronic obstructive pulmonarydiseases for a long time in China [14ndash16] TMP has been

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 315850 12 pageshttpdxdoiorg1011552015315850

2 BioMed Research International

demonstrated to inhibit platelet aggregation dilate bloodvessels depress blood viscosity improve microcirculationincrease coronary and cerebral blood flow promote vas-cular recanalization and endotheliocyte proliferation andmigration and scavenge reactive oxygen species [14 17ndash22]On this basis we hypothesize that TMP might possess atherapeutic effect on steroid-induced ONFH an ischemicnecrosis disease of bone Thus the aim of the current studywas to verify whether TMP possesses any preventive effectson the osteonecrotic changes repair and revascularizationprocesses in steroid-induced ONFH rats Moreover theunderlying pharmacological mechanisms of revasculariza-tion are investigated

2 Materials and Methods

The study was approved by Research Ethics Committee ofChina Academy of Chinese Medical Sciences in accordancewith the National Institutes of Health Guidelines for the Careand Use of Laboratory Animals All animals were treated inaccordance with the guidelines and regulations for the useand care of animals of theCenter for LaboratoryAnimalCareChina Academy of Chinese Medical Sciences

21 Animals Eighty-five 12-week-old male Wistar rats (Catnumber SCXK-(Jun) 2007-004) weighting 300ndash320 g wereobtained from Experimental Animal Centre of Academy ofMilitary Medical Sciences (Beijing China) All rats weremaintained in a room equipped with an air-filtering systemand the cages and water were sterilized

22 Groups and Treatment After one week feeding adap-tation the animals were accurately weighed and randomlydivided into four groups control (119899 = 20) model (steroid-induced ONFH rats 119899 = 25) TMP 20mgkg (steroid-induced ONFH rats treated with 20mgkg TMP 119899 = 20)and TMP 40mgkg (steroid-induced ONFH rats treatedwith 40mgkg TMP 119899 = 20) Steroid-induced ONFH ratmodels were constructed according to the previous studies[23 24] Briefly rats were injected subcutaneously at the backby methylprednisolone acetate (MPSL Pfizer Manufactur-ing BeLgium NV Puurs Belgium) (21mgkg) daily for sixweeks to induce osteonecrosis One hour after the MPSLinjection per day rats in TMP 20mgkg and TMP 40mgkggroups respectively received ligustrazine hydrochloride(Tianjin Pharmaceutical Jiaozuo Co Ltd Jiaozuo China)by intramuscular injection at the hip with the dosages of20mgkgday and 40mgkgday for six weeks In the controland model groups the rats received distilled water Theanimals were fed a standard diet and allowed free activity

23 Tissue Sample Preparation Rats from each group werekilled six weeks after theMPSL injectionThe rats were anaes-thetized with an intravenous injection of trichloroacetalde-hyde hydrate (03mLkg Sinopharm Chemical Reagent CoLtd China) and were then killed by exsanguination via anaorta The left sides of femora were obtained and fixed forthree days in 4paraformaldehyde (pH74) and then decalci-fied with 10 EDTA for one month Samples were sectioned

along the coronal plane for the proximal one-third and cutalong the axial plane in the distal part (condyle) Finally thespecimens were embedded in paraffin cut into 5120583msectionsand stained with haematoxylin and eosin The right sideswere stored at minus80∘C for western blot and real-time PCR test

24 Evaluation of Steroid-Induced ONFH The osteonecroticchanges and repair processes in steroid-treated rats wereobserved by the histopathological examination using a lightmicroscope six weeks after the MPSL injection The slideswere evaluated in a blinded fashion by three independentobservers The evaluation criteria for osteonecrosis werebased on the report of Yamamoto et al [25] Osteonecro-sis was judged to be present when there was necrosis ofmedullary hematopoietic cells or fat cells or there were emptylacunae or condensed nuclei in osteocytesThe ratio of emptylacunae (empty lacunaethe total number of osteocytes) wascalculated for each femoral head using a coronal section takenat themaximal femoral width A computerized image analysisprogram (image pro 60) was used for this calculation

25 Micro-CT A micro-CT (GE Healthcare BiosciencesPiscataway NJ USA) was used to detect the change of theexcised femoral head samplesThe following parameters werecalculated bone volume (BV) tissue volume (TV) trabecularbone pattern factor (TbPf) structure model index (SMI)trabecular thickness (TbTh) trabecular number (TbN) tra-becular separation (TbSp) and bonemineral density (BMD)After micro-CT the decalcified samples were embedded inparaffin and cut into 5120583msections along the coronal plane forthe proximal parts Sections were stained with hematoxylinand eosin (HampE) for evaluation of ON and repair process

26 Quantification and Three-Dimensional Visualization ofVessel Networks Femoral head blood vascularization insteroid-treated rats was measured using the micro-CT-basedmicroangiography six weeks after theMPSL injection accord-ing to the previous studies [26 27] Briefly rats from eachgroup (119899 = 10) were anaesthetized as described above and thethoracic and abdomen cavities of the animals were openedA hypodermic needle with disposable infusion device wasinserted in the ventriculus sinister with ligation of that proxi-mal to the aorta ascendens The vasculature was flushed with500mL 50UmL heparinized saline at 37∘C and at maximumflow via a disposable infusion device As soon as the outflowfrom an incision of the auricula dextra was limpidness500mL 4 paraformaldehyde solution was pumped into thevasculature to fix the tissues and blood vessels The vascula-ture was then injected with Microfil based on the manufac-turerrsquos protocol (Microfil MV-122 Flow Tech Carver MAUSA) Animals were then stored overnight at 4∘C to ensurepolymerization of the contrast agent before microangiogra-phy Bilateral femoral samples were then harvested fixed in4 paraformaldehyde and decalcified in 10 EDTA Afterthat the femoral shaft was fixed in a polymethylmethacrylatesample tube with its long axis perpendicular to the bottom ofthe tube in preparation for micro-CT scanningThe scan wasperpendicular to the shaft and was initiated from a reference

BioMed Research International 3

line 10mm away from the bottom with an entire scan lengthof 10mm

27 Hematological Examination In order to detect thehyperlipidemia-improving effects of TMP the blood sampleswere collected from the abdominal aorta six weeks afterthe MPSL injection The serum levels of total cholesterol(TC) triglycerides (TG) low-density lipoprotein (LDL)high-density lipoprotein (HDL) apolipoprotein A1 (ApoA1)and apolipoprotein B (ApoB) were determined by automaticbiochemical analyzer (AU 1000 Olympus Japan)

28 Immunohistochemical Staining Paraffin sections (5 120583m)of tissue from the femoral head tissues were mountedon poly-L-lysine-coated slides The paraffin sections weredewaxed by routine method and incubated for 10min with3H2O2The sections were placed in a 37∘C 01 trypsinase

for 5ndash30min for antigen retrieval Each sectionwas incubatedwith normal goat serum for 20min at room temperatureand then with primary antibody against rat VEGF (rabbitantibody dilution 1 50 Abcam Cambridge MA UK) FLK1(rabbit antibody dilution 1 50 Cell Signaling Boston USA)overnight at 4∘C After incubation with Polymer Helper for20min at 37∘C sections were reacted with poly-HRP anti-rabbit IgG for 20min at 37∘CThe sections were then stainedwith 33-diaminobenzidine (Sigma St Louis MO USA) andcounterstained with hematoxylin Specimens were examinedusing a Leica image analyzer and analyzed by computerimage analysis (Leica Microsystem Wetzlar Gmbh WetzlarGermany) in a blindedmanner To localize and identify areaswith positively stained cells 10 random digital images perspecimen of the femoral head tissues were recorded andquantitative analysis was performed according to the colorcell separation The results are expressed as the mean regionof interest representing the percentage of area covered withpositively stained cells per image at a magnification of 400x

29 RNA Isolation and Real-Time PCR The whole femoralhead tissues were dissected from rats six weeks after theMPSL injection snap-frozen in liquid nitrogen ground intopowder and homogenized The RNA isolation and real-time PCR assay were carried out following the protocol ofour previous study [28] Briefly total RNA was extractedwith TRIzol reagent (Invitrogen Carlsbad CA USA) fromthe tissue homogenates according to the manufacturerrsquosinstructions The total RNA (3mg) was reverse-transcribedto cDNA using the QuantiTect Reverse Transcription Kit(Thermo Fisher Scientific Inc CA USA) according to theinstruction manual The specific transcripts were quantifiedby quantitative real-time PCR using QuantiTect SYBR GreenPCR Kit (Takara Bio Inc Tokyo Japan) and analyzed withABI 7500 real-time PCR system (Applied Biosystems Fostercity CA USA) Gene-specific primers were used for VEGF(51015840-TTCATGGATGTCTATCAGCG-31015840 as forward and 51015840-GCTCATCTCTCCTATGTGCT-31015840 as reverse) FLK1 (51015840-TAGTATTCCGTTCGCAAG-31015840 as forward and 51015840-TAC-AACTTTCTCCCTCGT-31015840 as reverse) and 120573-actin (51015840-ACCCTAAGGCCAACCGTGAAAAG-31015840 as forward and 51015840-CATGAGGTAGTCTGTCAGGT-31015840 as reverse) The mRNA

levels of VEGF and FLK1 were normalized to 120573-actin mRNAlevel PCR was performed as 40 cycles at 94∘C for 15 s 55∘Cfor 30 s and 72∘C for 30 sThe relative mRNA expression wascalculated with comparative CT method

210 Western Blot Analysis The protein expression levelsof VEGF FLK1 and GAPDH in the femoral head tissuesobtained from rats in different groups were detected by west-ern blot analysis The protocol and semiquantitative analysiswere carried out following the protocol of our previous study[26] The following antibodies were used VEGF antibody(rabbit antibody dilution 1 50 Santa Cruz BiotechnologyInc Santa Cruz CA USA) FLK1 (rabbit antibody dilution1 50 Santa Cruz) and GAPDH antibody (internal controlrabbit polyclonal antibody dilution 1 200 Santa Cruz)

211 Statistical Analysis The software of SPSS version 130for Windows (SPSS Inc Chicago IL USA) was used forstatistical analysis The results were expressed as 119883 plusmn 119904For comparisons of means among multiple groups one-wayANOVA followed by LSD test was performed Differenceswere considered statistically significant when 119875 lt 005

3 Results

31 TMP Prevents Histopathological Changes in Steroid-Induced ONFH Rats The osteonecrotic changes and repairprocesses of rats in each group were histopathologicallyobserved to evaluate the effect of TMP treatment on steroid-induced ONFH Compared with the control group there wasan accumulation of bone marrow cell debris found in ONFHlesions in the model group while TMP treatment coulddramatically attenuate this change in steroid-induced ONFHrats (Figure 1(a)) In addition the ratio of empty lacunae inthe bone trabeculae of the model group was significantlymore than that of control group but was decreased bythe treatment of TMP (Figure 1(b)) Moreover the adiposetissue area and adipocyte perimeter in the bone marrowwhich were dramatically increased in steroid-inducedONFHrats were both significantly reduced by the TMP treatment(Figures 1(c) and 1(d)) The incidence of osteonecrosis was2325 in the model group (92) 1320 (65) in the TMP20mgkg group and 920 (45) in the TMP 40mgkggroup while no osteonecrosis was observed in the controlgroup Next apparent adverse effects including weight lossalterations of physical appearance and behavior changeswerenot noted in rats treated with TMP (results not shown)Taken together these observations indicate that systemicadministration of TMP in rats prevents steroid-inducedONFH without serious side effects

32 TMP Suppresses Hyperlipidemia in Steroid-InducedONFH Rats In the lipid system the blood chemistry datashowed that steroid hormone administration (model group)induced marked hyperlipidemia such as significantly ele-vated TG (Figure 2(a)) TC (Figure 2(b)) LDL (Figure 2(c))ApoA1 (Figure 2(e)) and ApoB (Figure 2(f)) levels but sig-nificantly decreased HDL levels (Figure 2(d)) More interest-ingly doses of 20sim40mgkg TMP could improve hyperlipi-demia by decreasing TG (Figure 2(a)) TC (Figure 2(b)) LDL


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Figure 1 Tetramethylpyrazine (TMP) enhances osteogenesis and reverses bone marrow adipogenesis (a) Histological features of the normalbone in normal rat and the osteonecrotic bone in steroid-induced ONFH rats with or without TMP administration Statistical analysis on thedifferences of the ratio of empty lacuna (b) the adipose tissue area (c) and the adipocyte perimeter (d) in the control model TMP 20mgkgand TMP 40mgkg groups Data are represented as the mean plusmn SD (119899 = 20 for control 119899 = 25 for model 119899 = 20 for TMP 20mgkg and40mgkg resp) 119875 lt 0001 compared with the control group lowast119875 lt 005 and lowastlowast119875 lt 001 respectively compared with the model groupThe arrow heads indicate adipose tissue and the arrows indicate empty lacuna

(Figure 2(c)) ApoA1 (Figure 2(e)) and ApoB (Figure 2(f))levels and increasing HDL levels (Figure 2(d))

33 TMP Inhibits the Microstructure Destruction of theTrabecular Bone and Increases BMD in Steroid-InducedONFHRats Micro-CT scanwas performed to validate the efficiency

of TMP in steroid-inducedONFHrats Two-dimensional andthree-dimensional reconstruction images from the micro-CT scanning data revealed the degree of microstructuredestruction of femoral head in different groups As shownin Figures 3(a) and 3(b) compared with model group dosesof 20mgkg and 40mgkg TMP both markedly reduced


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Figure 2 Tetramethylpyrazine (TMP) improves hyperlipidemia in steroid-induced ONFH ratsThe steroid hormone administration (modelgroup) inducedmarked hyperlipidemia such as significantly elevated TG (a) TC (b) LDL (c) ApoA1 (e) andApoB (f) levels but significantlydecreased HDL levels (d) More interestingly doses of 20sim40mgkg TMP could significantly reduce hyperlipidemia by decreasing TG (a)TC (b) LDL (c) ApoA1 (e) and ApoB (f) levels and increasing HDL levels (d) Data are represented as the mean plusmn SD (119899 = 10 for control119899 = 15 for model 119899 = 10 for TMP 20mgkg TMP 40mgkg resp) 119875 lt 005 and

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Figure 3 Tetramethylpyrazine (TMP) inhibits the microstructure destruction of the femoral head (a) and (b) respectively refer to two-dimensional and three-dimensional pictures of the normal bone in normal rats and the osteonecrotic bone in steroid-induced ONFH ratswith or without TMP treatment Statistical analysis on the differences of the BVTV (c) TbPf (d) TbTh (e) TbN (f) Tbsp (g) and BMD(h) in the control model TMP 20mgkg and TMP 40mgkg groups Data are represented as the mean plusmn SD (119899 = 10 for control 119899 = 15for model 119899 = 10 for TMP 20mgkg and TMP 40mgkg resp) 119875 lt 001 compared with the control group lowast119875 lt 005 and lowastlowast119875 lt 001respectively compared with the model group


the extent of microstructure destruction of femoral headAs shown in Figures 3(c)ndash3(g) microstructural parametersBVTV TbTh TbPf and TbN were significantly reducedwhile TbSp and SMI were significantly increased in rats withsteroid-induced ONFH when compared with controls TMPtreatment protected rats from steroid-induced effects on thelevels of the above microstructural parameters To determinewhether TMP treatment increases the bone mass of rats withsteroid-induced ONFH BMD values were calculated Asshown in Figure 3(h) the rats with steroid-induced ONFHshowed markedly reduced BMD in the femoral head com-paredwith the control ratsTheBMDvalues in the rats treatedwith 20ndash40mgkg TMP were increased in a dose-dependentmanner compared to those without drug treatment

34 TMP Enhances Femoral Head Vascularization in Steroid-Induced ONFH Rats The blood vessel microarchitectureof each group was reconstructed in three dimensions forpresentation Compared with the control both the numberand the thickness of vessels in the necrotic lesion of femoralhead of steroid-induced ONFH rats were markedly reducedand the vasculatures were not visible while the sample inthe TMP 20mgkg group showed lightly increasing capillaryvessels and the sample in the TMP 40mgkg groups showedintensive vascular architecture (Figure 4(a)) QuantitativelyTMP increased vessel volume (Figure 4(b)) vessel thickness(Figure 4(c)) percentage of vessel volume (Figure 4(d)) andvessel surface (Figure 4(e)) of the femoral heads of steroid-induced ONFH rats

35 TMP Inhibits the Effect of Steroid on VEGFFLK1 SignalPathway in the Sera and Femoral Heads of Steroid-InducedONFH Rats We detected the changes of VEGF and FLK1expression at both protein and mRNA levels in the seraand femoral heads of steroid-induced ONFH rats with orwithout TMP treatment As shown in Figures 5(a) and 5(b)the serum VEGF and FLK1 levels in steroid-induced ONFHrats were significantly lower than those in control rats Afterthe TMP treatment the serum VEGF and FLK1 levels weredramatically elevated as compared to the model As shown inFigures 5(c)ndash5(e) light amounts of VEGF positive stainingwere present in endothelial cells osteoblasts osteocytesand bone marrow haematopoietic cells and FLK1 positivestaining in endothelial cells of the femoral heads frommodelrats while there was evident rise of VEGF and FLK1 inthe femoral heads from TMP-treated rats In addition TMPsignificantly increased the protein expression of VEGF andFLK1 which were downregulated in the femoral heads ofsteroid-induced ONFH rats (Figures 6(a) and 6(b)) In linewith the findings of western blot analysis VEGF and FLK1mRNA expression levels detected by quantitative real-timeRT-PCR were also reversed by doses of 20sim40mgkg TMPtreatments significantly (Figures 6(c) and 6(d))

4 Discussion

Steroid treatment is one of the most comment risk fac-tors associated with osteonecrosis [6] Prolonged steroiduse produces a hyperlipidemic state in most patients and

subsequently results in abnormal coagulopathy and bonemarrow fat-cell packing leading to microvascular occlusionand high intraosseous pressure all of which put them at riskfor osteonecrosis [29] Therefore many researchers postu-late that a microvascular enhancing agent may prevent theconditions associated with the development of osteonecrosisTMP has increasingly gained attention due to its significantvascular protective properties A number of studies on theeffects of TMP on ischemic neural disorders and cardio-vascular diseases have been documented [14ndash16] Howeverits role in the treatment of steroid-induced ONFH has notbeen evaluated In the current study we investigate the effectof TMP on steroid-induced ONFH and the main findingsare as the following four points (1) TMP prevents steroid-induced ONFH by reducing the osteonecrotic changes andthe bone marrow adipogenesis of steroid-induced ONFHrats (2) TMP inhibits the microstructure destruction of thetrabecular bone and increases BMD in the femoral headof steroid-induced ONFH rats (3) TMP enhances femoralhead neovascularization and improves hyperlipidemic stateof steroid-induced ONFH rats (4) TMP inhibits the effect ofsteroid on VEGFFLK1 signal pathway in ONFH rats

In the present study the steroid-induced osteonecrosismodel was established in rats by three injections of high-doseMPSL as previously reported [23 24] The histopathologyof osteonecrosis generated in this model was characterizedby empty lacunae accompanied by surrounding narrow cellnecrosis and occupation of adipocytes which was similarto osteonecrosis in steroid-treated patients However thereare some differences in the osteonecrosis observed in rats ascompared to humans For example osteonecrosis often leadsto femoral head collapse in humans but not in rats as theepiphyseal line of the femur is permanent in adult rats but notin adult humans [30] In the present study the incidence ofosteonecrosis (in theMPSL group) was 92 and no rats diedduring the experimental period indicating that this ratmodelis safe and effective Thus this model is useful for steroidinduced osteonecrosis studies

In our study TMP treatment could significantly reducethe ration of empty lacunae and the area of bonemarrowoccu-pied by adipocytes suggesting the improvement on necrosisof femoral head by TMP administration Apart from the areaof bonemarrow occupied by adipocytes TMP also decreasedthe serummarkers of adipogenesis in steroid-inducedONFHrats demonstrating the global effect of TMP in both systemicand local lipid metabolism in rats In addition the TMPtreatment resulted in the decreased bone destruction in thefemoral head and these changes were associated with higherBVTV andTbN TMP also improved the trabecularmicroar-chitecture partially by restoring trabecular connectivitythrough increasing TbTh while reducing TbSp which isconsistent with the increase in BMD suggesting that TMPcould prevent the loss of bone mass induced by the excessivesteroid treatment

Since the impeded blood flow through the femoral headis implicated in the aetiopathogenesis of steroid-inducedONFH our study utilized a novel technique using micro-CT-based microangiography to visualize and quantify new



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Figure 4 Tetramethylpyrazine (TMP) enhances femoral head neovascularization (a) Representative images of micro-CT reconstructed 3-dimensional microangiography of proximal femur from control model TMP 20mgkg and TMP 40mgkg groups Statistical analysis onthe differences of vessel volume (b) vessel thickness (c) percentage of vessel volume (d) and vessel surface (e) in the femoral heads of steroid-induced ONFH rats in different groups Data are represented as the mean plusmn SD (119899 = 10) 119875 lt 0001 compared with the control grouplowastlowast

119875 lt 001 and lowastlowastlowast119875 lt 0001 respectively compared with the model group

blood vessel formation and vascularization in rat femoralhead Recent studies have demonstrated that this techniqueis quantitative and effective for assessing vascularization [2627] Consistent with the improvement of the TMP treatmenton the microstructure of the trabecular bone and BMD theTMP-treated groups showed a significant increase in bloodvessel volume vessel surface percentage of vessel volumeand vessel thickness suggesting this administration may alsoincrease vascularization of the femoral heads in rat modelThese findings imply that TMP treatment might produce an

environment conducive to prevention bone destruction bygenerating a blood supply

The local blood supply and vascularization of the femoralhead are generally considered to be involved in the occur-rence of ONFH [31] Glucocorticoids could decrease boneangiogenesis by suppression VEGF production of osteoblastsand osteocytes as well as by interference withVEGF action [332] Surgical procedures that aim to enhance revasculariza-tion of the necrotic area by applying angiogenic growth factor(mainlyVEGF) have shown viable results demonstrating that


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Figure 5 Tetramethylpyrazine (TMP) upregulates the VEGF and FLK1 expression in the sera and femoral heads of steroid-induced ONFHrats by ELISA and immnohistochemistryTheprotein levels ofVEGF (a) and FLK1 (b) in the sera of steroid-inducedONFHrats in the differentgroups were detected by ELISA Statistical analysis of immunohistochemistry on the positive rate of VEGF (c) and FLK1 (d) expression inthe femoral heads of steroid-induced ONFH rats from different groups The positive expressions of VEGF and FLK1 in the femoral heads ofsteroid-induced ONFH rats from control model TMP 20mgkg and TMP 40mgkg groups were detected by immnohistochemistry (e)Thearrows and arrow heads indicate VEGF and FLK1 positive expression cells respectively Data are represented as the mean plusmn SD (119899 = 10 forcontrol 119899 = 15 for model 119899 = 10 for TMP treatment groups resp) 119875 lt 001 compared with the control group lowast119875 lt 005 and lowastlowast119875 lt 001respectively compared with the model group

VEGF and blood supply may play a role in the developmentof osteonecrosis [33 34] VEGF as an important mediatorof angiogenesis stimulates endothelial cells proliferationpromotes neovascularization increases vascular permeability[35 36] and plays a critical role in vascularization andvessel function [33] During the process of angiogenesis

VEGFFLK1 is the key signaling system to regulate the prolif-eration andmigration of endothelial cells forming the basis ofany vessel In the current study we found the downregulationof VEGF and FLK1 protein and gene expression in thesera and the necrotic femoral head which were reversedby the treatment of TMP In this respect we speculate that


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Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)

Figure 6 Tetramethylpyrazine (TMP) upregulates VEGF and FLK1 protein and gene expression in the femoral heads of steroid-inducedONFH rats by western blot and real-time RT-PCR VEGF and FLK1 expressions at both protein and mRNA levels in the femoral heads ofsteroid-induced ONFH rats with or without TMP treatment were detected by western blot ((a) and (b)) and quantitative real-time RT-PCR((c) and (d)) respectively Data are represented as the mean plusmn SD (119899 = 10 for control 119899 = 15 for model 119899 = 10 for TMP treatment groupsresp) 119875 lt 005 and

119875 lt 001 compared with the control group respectively lowast119875 lt 005 compared with the model group

TMP might prevent the effect of steroid on VEGFFLK1signal pathway which has been considered as a potentialtarget for enhancing revascularization in steroid-inducedONFH patients Furthermore because VEGF is essentialfor bone formation [37] it may act as a key regulator thatcouples angiogenesis bone formation and repair of necroticsubchondral bone in femoral heads treated with TMP

In conclusion our data offer the convincing evidencefor the first time that TMP may prevent steroid-inducedONFH and also enhance femoral head neovascularization byinhibiting the effect of steroid onVEGFFLK1 signal pathwayThese findings highlight the improvement of TMP on theblood supply and neovascularization during the progressionof steroid-induced ONFH

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Yini Jiang andChunfang Liu contributed equally to this work

Acknowledgments

This study was supported by grants from the National MajorScientific and Technological Special Project for ldquoSignificantNewDrugsCreationrdquo (no 2013ZX09301307) and theNationalNatural Science Foundation of China (no 81173417)


References

[1] X-S Wang Q-Y Zhuang X-S Weng J Lin J Jin and W-WQian ldquoEtiological and clinical analysis of osteonecrosis of thefemoral head in Chinese patientsrdquo Chinese Medical Journal vol126 no 2 pp 290ndash295 2013

[2] M A Mont and D S Hungerford ldquoNon-traumatic avascularnecrosis of the femoral headrdquo The Journal of Bone and JointSurgerymdashAmerican Volume vol 77 no 3 pp 459ndash474 1995

[3] R S Weinstein C Wan Q Liu et al ldquoEndogenous glucocorti-coids decrease skeletal angiogenesis vascularity hydration andstrength in agedmicerdquoAging Cell vol 9 no 2 pp 147ndash161 2010

[4] W Drescher T Schneider C Becker et al ldquoSelective reduc-tion of bone blood flow by short-term treatment with high-dose methylprednisolone An experimental study in pigsrdquo TheJournal of Bone amp Joint SurgerymdashBritish Volume vol 83 no 2pp 274ndash277 2001

[5] W Drescher H Li A Lundgaard C Bunger and E-S HansenldquoEndothelin-1-induced femoral head epiphyseal artery con-striction is enhanced by long-term corticosteroid treatmentrdquoThe Journal of Bone and Joint SurgerymdashAmerican Volume vol88 no 3 pp 173ndash179 2006

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

[7] T Mikami T Ichiseki A Kaneuji et al ldquoPrevention of steroid-induced osteonecrosis by intravenous administration of vita-min e in a rabbit modelrdquo Journal of Orthopaedic Science vol15 no 5 pp 674ndash677 2010

[8] Y Assouline-Dayan C Chang A Greenspan Y Shoenfeld andM E Gershwin ldquoPathogenesis and natural history of osteone-crosisrdquo Seminars in Arthritis and Rheumatism vol 32 no 2 pp94ndash124 2002

[9] S Biswal S Hazra H H Yun C Y Hur and W YShon ldquoTranstrochanteric rotational osteotomy for nontrau-matic osteonecrosis of the femoral head in young adultsrdquoClinical Orthopaedics and Related Research vol 467 no 6 pp1529ndash1537 2009

[10] A V Korompilias M G Lykissas A E Beris J R Urbaniakand P N Soucacos ldquoVascularised fibular graft in the manage-ment of femoral head osteonecrosis twenty years laterrdquo TheJournal of Bone amp Joint Surgery Series B vol 91 no 3 pp 287ndash293 2009

[11] M A Mont G A Marulanda T M Seyler J F Plate and RE Delanois ldquoCore decompression and nonvascularized bonegrafting for the treatment of early stage osteonecrosis of thefemoral headrdquo Instructional Course Lectures vol 56 pp 213ndash220 2007

[12] L Chun-Sheng Y Hsiao-Meng H Yun-Hsiang P Chun andS Chi-Fen ldquoRadix salviae miltiorrhizae and Rhizoma ligusticiwallichii in coronary heart diseaserdquo Chinese Medical Journalvol 4 no 1 pp 43ndash46 1978

[13] Y-P Liu H Cao G-R Han H Fushimi and K KomatsuldquomatK and its nucleotide sequencing of crude drug chuanxiongand phylogenetic relationship between their species fromChinaand Japanrdquo Yaoxue Xuebao vol 37 no 1 pp 63ndash68 2002

[14] S-Y Li Y-H Jia W-G Sun et al ldquoStabilization of mitochon-drial function by tetramethylpyrazine protects against kainate-induced oxidative lesions in the rat hippocampusrdquo Free RadicalBiology and Medicine vol 48 no 4 pp 597ndash608 2010

[15] W-M Li H-T Liu X-Y Li et al ldquoThe effect of tetram-ethylpyrazine on hydrogen peroxide-induced oxidative damage

in human umbilical vein endothelial cellsrdquo Basic amp ClinicalPharmacology amp Toxicology vol 106 no 1 pp 45ndash52 2010

[16] S-L Liao T-K Kao W-Y Chen et al ldquoTetramethylpyrazinereduces ischemic brain injury in ratsrdquo Neuroscience Letters vol372 no 1-2 pp 40ndash45 2004

[17] L Zhang M Deng and S Zhou ldquoTetramethylpyrazine inhibitshypoxia-induced pulmonary vascular leakage in rats via theROS-HIF-VEGF pathwayrdquo Pharmacology vol 87 no 5-6 pp265ndash273 2011

[18] S Gao Z-W Chen H Zheng and X-L Chen ldquoLigustrazineattenuates acute myocardium injury after thermal traumardquoBurns vol 33 no 3 pp 321ndash327 2007

[19] Y Kang M Hu Y Zhu X Gao and M-W Wang ldquoAntiox-idative effect of the herbal remedy Qin Huo Yi Hao and itsactive component tetramethylpyrazine on high glucose-treatedendothelial cellsrdquo Life Sciences vol 84 no 13-14 pp 428ndash4362009

[20] M Zhang F Gao F Teng and C Zhang ldquoTetramethylpyrazinepromotes the proliferation and migration of brain endothelialcellsrdquoMolecular Medicine Reports vol 10 no 1 pp 29ndash32 2014

[21] C Y Kwan E E Daniel and M C Chen ldquoInhibition ofvasoconstriction by tetramethylpyrazine does it act by blockingthe voltage-dependent Ca channelrdquo Journal of CardiovascularPharmacology vol 15 no 1 pp 157ndash162 1990

[22] W Peng D Hucks R M Priest Y M Kan and J P T WardldquoLigustrazine-induced endothelium-dependent relaxation inpulmonary arteries via an NO-mediated and exogenous L-arginine-dependent mechanismrdquo British Journal of Pharmacol-ogy vol 119 no 5 pp 1063ndash1071 1996

[23] A Bitto F Polito B Burnett et al ldquoProtective effect of genisteinaglycone on the development of osteonecrosis of the femoralhead and secondary osteoporosis induced by methylpred-nisolone in ratsrdquo Journal of Endocrinology vol 201 no 3 pp321ndash328 2009

[24] N Han Z Yan C-A Guo et al ldquoEffects of p-glycoprotein onsteroid-induced osteonecrosis of the femoral headrdquo CalcifiedTissue International vol 87 no 3 pp 246ndash253 2010

[25] T Yamamoto T Irisa Y Sugioka and K Sueishi ldquoEffectsof pulse methylprednisolone on bone and marrow tissuescorticosteroid-induced osteonecrosis in rabbitsrdquo Arthritis ampRheumatism vol 40 no 11 pp 2055ndash2064 1997

[26] Y Sun Y Feng C Zhang et al ldquoBeneficial effect of autolo-gous transplantation of endothelial progenitor cells on steroid-induced femoral head osteonecrosis in rabbitsrdquo Cell Transplan-tation vol 20 no 2 pp 233ndash243 2011

[27] Y Sun Y Feng and C Zhang ldquoThe effect of bone marrowmononuclear cells on vascularization and bone regeneration insteroid-induced osteonecrosis of the femoral headrdquo Joint BoneSpine vol 76 no 6 pp 685ndash690 2009

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

[29] T Kabata K Shimanuki K Shimanuki and H TsuchiyaldquoOsteonecrosis of the femoral head and glaucoma caused bytopical corticosteroid applicationrdquo Modern Rheumatology vol21 no 6 pp 706ndash709 2011

[30] 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


[31] M A Kerachian E J Harvey D Cournoyer T Y K Chowand C Seguin ldquoAvascular necrosis of the femoral head vascularhypothesesrdquo Endothelium vol 13 no 4 pp 237ndash244 2006

[32] W Drescher G Schlieper J Floege and F Eitner ldquoSteroid-related osteonecrosismdashan updaterdquo Nephrology Dialysis Trans-plantation vol 26 no 9 pp 2728ndash2731 2011

[33] S G Ball C A Shuttleworth and C M Kielty ldquoMesenchy-mal stem cells and neovascularization role of platelet-derivedgrowth factor receptors Angiogenesis Review Seriesrdquo Journalof Cellular and Molecular Medicine vol 11 no 5 pp 1012ndash10302007

[34] K Katsube A T Bishop R D Simari S Yla-Herttuala and PF Friedrich ldquoVascular endothelial growth factor (VEGF) genetransfer enhances surgical revascularization of necrotic bonerdquoJournal of Orthopaedic Research vol 23 no 2 pp 469ndash4742005

[35] W G Roberts and G E Palade ldquoIncreased microvascularpermeability and endothelial fenestration induced by vascularendothelial growth factorrdquo Journal of Cell Science vol 108 no6 pp 2369ndash2379 1995

[36] H Winet ldquoThe role of microvasculature in normal and per-turbed bone healing as revealed by intravital microscopyrdquo Bonevol 19 no 1 pp 39Sndash57S 1996

[37] H-P Gerber T H Vu A M Ryan J Kowalski Z Werb andN Ferrara ldquoVEGF couples hypertrophic cartilage remodelingossification and angiogenesis during endochondral bone for-mationrdquo Nature Medicine vol 5 no 6 pp 623ndash628 1999

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


demonstrated to inhibit platelet aggregation dilate bloodvessels depress blood viscosity improve microcirculationincrease coronary and cerebral blood flow promote vas-cular recanalization and endotheliocyte proliferation andmigration and scavenge reactive oxygen species [14 17ndash22]On this basis we hypothesize that TMP might possess atherapeutic effect on steroid-induced ONFH an ischemicnecrosis disease of bone Thus the aim of the current studywas to verify whether TMP possesses any preventive effectson the osteonecrotic changes repair and revascularizationprocesses in steroid-induced ONFH rats Moreover theunderlying pharmacological mechanisms of revasculariza-tion are investigated

2 Materials and Methods

The study was approved by Research Ethics Committee ofChina Academy of Chinese Medical Sciences in accordancewith the National Institutes of Health Guidelines for the Careand Use of Laboratory Animals All animals were treated inaccordance with the guidelines and regulations for the useand care of animals of theCenter for LaboratoryAnimalCareChina Academy of Chinese Medical Sciences

21 Animals Eighty-five 12-week-old male Wistar rats (Catnumber SCXK-(Jun) 2007-004) weighting 300ndash320 g wereobtained from Experimental Animal Centre of Academy ofMilitary Medical Sciences (Beijing China) All rats weremaintained in a room equipped with an air-filtering systemand the cages and water were sterilized

22 Groups and Treatment After one week feeding adap-tation the animals were accurately weighed and randomlydivided into four groups control (119899 = 20) model (steroid-induced ONFH rats 119899 = 25) TMP 20mgkg (steroid-induced ONFH rats treated with 20mgkg TMP 119899 = 20)and TMP 40mgkg (steroid-induced ONFH rats treatedwith 40mgkg TMP 119899 = 20) Steroid-induced ONFH ratmodels were constructed according to the previous studies[23 24] Briefly rats were injected subcutaneously at the backby methylprednisolone acetate (MPSL Pfizer Manufactur-ing BeLgium NV Puurs Belgium) (21mgkg) daily for sixweeks to induce osteonecrosis One hour after the MPSLinjection per day rats in TMP 20mgkg and TMP 40mgkggroups respectively received ligustrazine hydrochloride(Tianjin Pharmaceutical Jiaozuo Co Ltd Jiaozuo China)by intramuscular injection at the hip with the dosages of20mgkgday and 40mgkgday for six weeks In the controland model groups the rats received distilled water Theanimals were fed a standard diet and allowed free activity

23 Tissue Sample Preparation Rats from each group werekilled six weeks after theMPSL injectionThe rats were anaes-thetized with an intravenous injection of trichloroacetalde-hyde hydrate (03mLkg Sinopharm Chemical Reagent CoLtd China) and were then killed by exsanguination via anaorta The left sides of femora were obtained and fixed forthree days in 4paraformaldehyde (pH74) and then decalci-fied with 10 EDTA for one month Samples were sectioned

along the coronal plane for the proximal one-third and cutalong the axial plane in the distal part (condyle) Finally thespecimens were embedded in paraffin cut into 5120583msectionsand stained with haematoxylin and eosin The right sideswere stored at minus80∘C for western blot and real-time PCR test

24 Evaluation of Steroid-Induced ONFH The osteonecroticchanges and repair processes in steroid-treated rats wereobserved by the histopathological examination using a lightmicroscope six weeks after the MPSL injection The slideswere evaluated in a blinded fashion by three independentobservers The evaluation criteria for osteonecrosis werebased on the report of Yamamoto et al [25] Osteonecro-sis was judged to be present when there was necrosis ofmedullary hematopoietic cells or fat cells or there were emptylacunae or condensed nuclei in osteocytesThe ratio of emptylacunae (empty lacunaethe total number of osteocytes) wascalculated for each femoral head using a coronal section takenat themaximal femoral width A computerized image analysisprogram (image pro 60) was used for this calculation

25 Micro-CT A micro-CT (GE Healthcare BiosciencesPiscataway NJ USA) was used to detect the change of theexcised femoral head samplesThe following parameters werecalculated bone volume (BV) tissue volume (TV) trabecularbone pattern factor (TbPf) structure model index (SMI)trabecular thickness (TbTh) trabecular number (TbN) tra-becular separation (TbSp) and bonemineral density (BMD)After micro-CT the decalcified samples were embedded inparaffin and cut into 5120583msections along the coronal plane forthe proximal parts Sections were stained with hematoxylinand eosin (HampE) for evaluation of ON and repair process

26 Quantification and Three-Dimensional Visualization ofVessel Networks Femoral head blood vascularization insteroid-treated rats was measured using the micro-CT-basedmicroangiography six weeks after theMPSL injection accord-ing to the previous studies [26 27] Briefly rats from eachgroup (119899 = 10) were anaesthetized as described above and thethoracic and abdomen cavities of the animals were openedA hypodermic needle with disposable infusion device wasinserted in the ventriculus sinister with ligation of that proxi-mal to the aorta ascendens The vasculature was flushed with500mL 50UmL heparinized saline at 37∘C and at maximumflow via a disposable infusion device As soon as the outflowfrom an incision of the auricula dextra was limpidness500mL 4 paraformaldehyde solution was pumped into thevasculature to fix the tissues and blood vessels The vascula-ture was then injected with Microfil based on the manufac-turerrsquos protocol (Microfil MV-122 Flow Tech Carver MAUSA) Animals were then stored overnight at 4∘C to ensurepolymerization of the contrast agent before microangiogra-phy Bilateral femoral samples were then harvested fixed in4 paraformaldehyde and decalcified in 10 EDTA Afterthat the femoral shaft was fixed in a polymethylmethacrylatesample tube with its long axis perpendicular to the bottom ofthe tube in preparation for micro-CT scanningThe scan wasperpendicular to the shaft and was initiated from a reference










3 Results





50120583m

100120583m

(a)


10

20

30

40

50

Ratio

of e

mpt

y la

cuna

()

20mgkg 40mgkg

lowastlowast

(b)

Control Model0

100

200

300

400

500

600


lowastlowast

lowast

Adip

ose t

issue

area

(120583m2)

(c)

0

20

40

60

80


lowastlowast

lowast

Adip

ocyt

e per

imet

er (120583

m)

(d)






0

02

04

06

08


TG (m

mol

L)

20mgkg 40mgkg

lowastlowast

lowast

(a)

0

05

1

15

2

25

Control Model

TC (m

mol

L)


lowastlowast

(b)

0

02

04

06

08

1

Control Model

LDL

(mm

olL

)


lowastlowast

lowast

(c)

0

02

04

06

08

Control Model

HD

L (m

mol

L)


lowast

(d)

Control Model0

05

1

15

2


lowast

lowast

ApoA

1(g

L)

(e)

0

05

1

15

2

25


lowastlowast

ApoB

(gL

)

(f)






(a)


(b)

0

20

40

60

80

100

120


lowastlowast

lowastlowast

BVT

V (

)

(c)

Control Model0

10

20

30

40

50


lowastlowast

lowast

TbP

f (1

mm

)

(d)

Control Model0

01

02

03

04


lowastlowast

lowastTbTh

(mm

)

(e)

0

2

4

6

8

10

12


lowast

TbN

(1m

m)

(f)

Control Model0

004

008

012

016

02



TbS

p (m

m)

(g)

0

200

400

600

800

1000


lowastlowast

lowastlowast

BMD

(gm

m3)

(h)






4 Discussion








(a)

0

01

02

03

Control Model

Vess

el v

olum

e (m

m3)

lowastlowastlowast

lowastlowast


(b)

0

20

40

60

80

Control Model

Vess

el th

ickn

ess (120583

m)

lowastlowastlowast

lowastlowast


(c)

0

005

01

015

02

Control Model

Pres

ent v

esse

l vol

ume (

)

lowastlowastlowast

lowastlowast


(d)

Control Model0

4

8

12

16

Vess

el su

rface

(120583m2)

lowastlowastlowast

lowastlowast


(e)







Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










3 Results





50120583m

100120583m

(a)


10

20

30

40

50

Ratio

of e

mpt

y la

cuna

()

20mgkg 40mgkg

lowastlowast

(b)

Control Model0

100

200

300

400

500

600


lowastlowast

lowast

Adip

ose t

issue

area

(120583m2)

(c)

0

20

40

60

80


lowastlowast

lowast

Adip

ocyt

e per

imet

er (120583

m)

(d)






0

02

04

06

08


TG (m

mol

L)

20mgkg 40mgkg

lowastlowast

lowast

(a)

0

05

1

15

2

25

Control Model

TC (m

mol

L)


lowastlowast

(b)

0

02

04

06

08

1

Control Model

LDL

(mm

olL

)


lowastlowast

lowast

(c)

0

02

04

06

08

Control Model

HD

L (m

mol

L)


lowast

(d)

Control Model0

05

1

15

2


lowast

lowast

ApoA

1(g

L)

(e)

0

05

1

15

2

25


lowastlowast

ApoB

(gL

)

(f)






(a)


(b)

0

20

40

60

80

100

120


lowastlowast

lowastlowast

BVT

V (

)

(c)

Control Model0

10

20

30

40

50


lowastlowast

lowast

TbP

f (1

mm

)

(d)

Control Model0

01

02

03

04


lowastlowast

lowastTbTh

(mm

)

(e)

0

2

4

6

8

10

12


lowast

TbN

(1m

m)

(f)

Control Model0

004

008

012

016

02



TbS

p (m

m)

(g)

0

200

400

600

800

1000


lowastlowast

lowastlowast

BMD

(gm

m3)

(h)






4 Discussion








(a)

0

01

02

03

Control Model

Vess

el v

olum

e (m

m3)

lowastlowastlowast

lowastlowast


(b)

0

20

40

60

80

Control Model

Vess

el th

ickn

ess (120583

m)

lowastlowastlowast

lowastlowast


(c)

0

005

01

015

02

Control Model

Pres

ent v

esse

l vol

ume (

)

lowastlowastlowast

lowastlowast


(d)

Control Model0

4

8

12

16

Vess

el su

rface

(120583m2)

lowastlowastlowast

lowastlowast


(e)







Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



50120583m

100120583m

(a)


10

20

30

40

50

Ratio

of e

mpt

y la

cuna

()

20mgkg 40mgkg

lowastlowast

(b)

Control Model0

100

200

300

400

500

600


lowastlowast

lowast

Adip

ose t

issue

area

(120583m2)

(c)

0

20

40

60

80


lowastlowast

lowast

Adip

ocyt

e per

imet

er (120583

m)

(d)






0

02

04

06

08


TG (m

mol

L)

20mgkg 40mgkg

lowastlowast

lowast

(a)

0

05

1

15

2

25

Control Model

TC (m

mol

L)


lowastlowast

(b)

0

02

04

06

08

1

Control Model

LDL

(mm

olL

)


lowastlowast

lowast

(c)

0

02

04

06

08

Control Model

HD

L (m

mol

L)


lowast

(d)

Control Model0

05

1

15

2


lowast

lowast

ApoA

1(g

L)

(e)

0

05

1

15

2

25


lowastlowast

ApoB

(gL

)

(f)






(a)


(b)

0

20

40

60

80

100

120


lowastlowast

lowastlowast

BVT

V (

)

(c)

Control Model0

10

20

30

40

50


lowastlowast

lowast

TbP

f (1

mm

)

(d)

Control Model0

01

02

03

04


lowastlowast

lowastTbTh

(mm

)

(e)

0

2

4

6

8

10

12


lowast

TbN

(1m

m)

(f)

Control Model0

004

008

012

016

02



TbS

p (m

m)

(g)

0

200

400

600

800

1000


lowastlowast

lowastlowast

BMD

(gm

m3)

(h)






4 Discussion








(a)

0

01

02

03

Control Model

Vess

el v

olum

e (m

m3)

lowastlowastlowast

lowastlowast


(b)

0

20

40

60

80

Control Model

Vess

el th

ickn

ess (120583

m)

lowastlowastlowast

lowastlowast


(c)

0

005

01

015

02

Control Model

Pres

ent v

esse

l vol

ume (

)

lowastlowastlowast

lowastlowast


(d)

Control Model0

4

8

12

16

Vess

el su

rface

(120583m2)

lowastlowastlowast

lowastlowast


(e)







Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

02

04

06

08


TG (m

mol

L)

20mgkg 40mgkg

lowastlowast

lowast

(a)

0

05

1

15

2

25

Control Model

TC (m

mol

L)


lowastlowast

(b)

0

02

04

06

08

1

Control Model

LDL

(mm

olL

)


lowastlowast

lowast

(c)

0

02

04

06

08

Control Model

HD

L (m

mol

L)


lowast

(d)

Control Model0

05

1

15

2


lowast

lowast

ApoA

1(g

L)

(e)

0

05

1

15

2

25


lowastlowast

ApoB

(gL

)

(f)






(a)


(b)

0

20

40

60

80

100

120


lowastlowast

lowastlowast

BVT

V (

)

(c)

Control Model0

10

20

30

40

50


lowastlowast

lowast

TbP

f (1

mm

)

(d)

Control Model0

01

02

03

04


lowastlowast

lowastTbTh

(mm

)

(e)

0

2

4

6

8

10

12


lowast

TbN

(1m

m)

(f)

Control Model0

004

008

012

016

02



TbS

p (m

m)

(g)

0

200

400

600

800

1000


lowastlowast

lowastlowast

BMD

(gm

m3)

(h)






4 Discussion








(a)

0

01

02

03

Control Model

Vess

el v

olum

e (m

m3)

lowastlowastlowast

lowastlowast


(b)

0

20

40

60

80

Control Model

Vess

el th

ickn

ess (120583

m)

lowastlowastlowast

lowastlowast


(c)

0

005

01

015

02

Control Model

Pres

ent v

esse

l vol

ume (

)

lowastlowastlowast

lowastlowast


(d)

Control Model0

4

8

12

16

Vess

el su

rface

(120583m2)

lowastlowastlowast

lowastlowast


(e)







Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



(a)


(b)

0

20

40

60

80

100

120


lowastlowast

lowastlowast

BVT

V (

)

(c)

Control Model0

10

20

30

40

50


lowastlowast

lowast

TbP

f (1

mm

)

(d)

Control Model0

01

02

03

04


lowastlowast

lowastTbTh

(mm

)

(e)

0

2

4

6

8

10

12


lowast

TbN

(1m

m)

(f)

Control Model0

004

008

012

016

02



TbS

p (m

m)

(g)

0

200

400

600

800

1000


lowastlowast

lowastlowast

BMD

(gm

m3)

(h)






4 Discussion








(a)

0

01

02

03

Control Model

Vess

el v

olum

e (m

m3)

lowastlowastlowast

lowastlowast


(b)

0

20

40

60

80

Control Model

Vess

el th

ickn

ess (120583

m)

lowastlowastlowast

lowastlowast


(c)

0

005

01

015

02

Control Model

Pres

ent v

esse

l vol

ume (

)

lowastlowastlowast

lowastlowast


(d)

Control Model0

4

8

12

16

Vess

el su

rface

(120583m2)

lowastlowastlowast

lowastlowast


(e)







Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





4 Discussion








(a)

0

01

02

03

Control Model

Vess

el v

olum

e (m

m3)

lowastlowastlowast

lowastlowast


(b)

0

20

40

60

80

Control Model

Vess

el th

ickn

ess (120583

m)

lowastlowastlowast

lowastlowast


(c)

0

005

01

015

02

Control Model

Pres

ent v

esse

l vol

ume (

)

lowastlowastlowast

lowastlowast


(d)

Control Model0

4

8

12

16

Vess

el su

rface

(120583m2)

lowastlowastlowast

lowastlowast


(e)







Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



(a)

0

01

02

03

Control Model

Vess

el v

olum

e (m

m3)

lowastlowastlowast

lowastlowast


(b)

0

20

40

60

80

Control Model

Vess

el th

ickn

ess (120583

m)

lowastlowastlowast

lowastlowast


(c)

0

005

01

015

02

Control Model

Pres

ent v

esse

l vol

ume (

)

lowastlowastlowast

lowastlowast


(d)

Control Model0

4

8

12

16

Vess

el su

rface

(120583m2)

lowastlowastlowast

lowastlowast


(e)







Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control Model0

20

40

60

80

100

lowastlowast

lowast


VEG

F in

seru

m (p

gm

L)

(a)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


FLK1

in se

rum

(pg

mL)

(b)

0

20

40

60

80

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f VEG

F ex

pres

sion

()

(c)

0

20

40

60

80

100

Control Model

lowastlowast

lowast


Posit

ive r

ate o

f FLK

1ex

pres

sion

()

(d)

Control Model

VEGF

FLK1


100120583m

100120583m

(e)





0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

05

1

15

2

25

VEG

F pr

otei

nG

APD

H p

rote

in

VEGF

GAPDH


lowast

lowast

(a)

0

05

1

15

2

25

GAPDH

FLK1

FLK1

pro

tein

GA

PDH

pro

tein

Control Model

lowast

lowast


(b)

Control Model0

04

08

12


lowast

lowast

VEG

F m

RNA

120573-a

ctin

mRN

A

(c)

Control Model0

04

08

12

lowast

lowast


FLK1

mRN

A120573

-act

in m

RNA

(d)









Acknowledgments



References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Tetramethylpyrazine Enhances...

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Transcript of Research Article Tetramethylpyrazine Enhances...