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Journal of Ethnopharmacology 131 (2010) 70–77

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

rynaria fortunei J. Sm. promotes osteoblast maturation by inducingifferentiation-related gene expression and protecting against oxidativetress-induced apoptotic insults

ai-Yuan Hunga,b,1, Ta-Liang Chenc,1, Mei-Hsiu Liaod, Wei-Pin Hob,er-Zen Liue, Wu-Chang Chuangf, Ruei-Ming Chenb,d,∗

Department of Orthopedic Surgery, Yuan’s General Hospital, Kaohsiung, TaiwanCell Physiology and Molecular Image Research Center, Taipei Medical University-Wan Fang Hospital, Taipei, TaiwanDepartment of Anesthesiology, Taipei Medical University Hospital, Taipei, TaiwanGraduate Institute of Medical Sciences, Taipei Medical University, Taipei, TaiwanGraduate Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei, TaiwanBrion Research Institute of Taiwan, Taipei, Taiwan

r t i c l e i n f o

rticle history:eceived 24 December 2009eceived in revised form 24 May 2010ccepted 31 May 2010vailable online 8 June 2010

eywords:rynaria fortunei J. Sm.steoblast maturationene expressionnti-apoptosis

a b s t r a c t

Aim of the study: Drynaria fortunei J. Sm. is one variety of the traditional Chinese medical herb Gusuibu. Thisstudy was aimed to evaluate the effects of water extracts of Kunze on regulation of osteoblast maturationand its possible mechanisms.Materials and methods: Primary osteoblasts prepared from neonatal rat calvarias were exposed to thewater extracts of Kunze (WEK), and the cytotoxicity was assayed. Osteoblast maturation was evaluatedby analyzing cell mineralization. RT-PCR was executed to determine the effects of WEK on regulation ofosteoblast differentiation-related gene expression. Nitrosative stress and apoptotic cells were quantifiedusing flow cytometry.Results: Exposure of rat calvarial osteoblasts to WEK did not affect cell viability, but significantly promotedosteoblast mineralization. WEK induced osteoprogenitor proliferation-related insulin-like growth factor-1 mRNA, but did not affect collagen type 1 mRNA expression. Treatment with WEK likewise induced

the expression of matrix maturation-related bone morphogenetic protein (BMP)-2 and BMP-6 mRNA.Consequently, WEK enhanced the levels of mineralization-related alkaline phosphatase, ostepontin, andosteocalcin mRNA in osteoblasts. In addition, exposure of osteoblasts to WEK alleviated nitrosative stress-caused apoptotic insults.Conclusions: This study shows that WEK can promote osteoblast maturation by regulating bone

ene e

differentiation-related ginsults.

. Introduction

Bone plays a critical role in skeletal support, tissue protection,nd bodily movement. The structure of bone is maintained through

Abbreviations: ALP, alkaline phosphatase; BMP, bone morphogenetic protein;MEM, Dulbecco’s modified Eagle’s medium; IGF-1, insulin-like growth factor-1;TT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OCN, osteo-

alcin; OPN, osteopontin; PBS, phosphate-based saline; ROS, reactive oxygenpecies; RT-PCR, reverse-transcription polymerase chain reaction; WEK, waterxtracts of Kunze.∗ Corresponding author at: Graduate Institute of Medical Sciences, College ofedicine, Taipei Medical University, 250, Wu-Xing St., Taipei 110, Taiwan.

el.: +886 2 27361661x3222; fax: +886 2 86621119.E-mail address: rmchen@tmu.edu.tw (R.-M. Chen).

1 These authors contributed equally to this work.

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.05.063

xpression and defending against nitrosative stress-induced apoptotic

© 2010 Elsevier Ireland Ltd. All rights reserved.

a dynamic balance, termed bone remodeling, which is a combina-tion of bone formation and resorption (Seeman and Delmas, 2006).Osteoblasts are central components in bone formation (Ducy etal., 2000). During osteogenesis, osteoblasts are differentiated fromosteoprogenitors that are originally derived from stromal stem cells(Aubin, 1998). Stem and primitive osteoprogenitors contribute toreplacement of osteoblasts in bone turnover or fracture healing.Sequential differentiation of these precursor cells is a mechanismthat accomplishes osteoblast maturation and bone nodule for-mation (Aubin, 1998). A complicated network of differentiationproteins is involved in the regulation of bone maturation (Stein et

al., 1996; Vandenput and Ohlsson, 2009). Thus, comprehending theprocess of osteogenic differentiation is helpful to develop innova-tive biomaterials for preventing or treating bone-related diseasessuch as osteoporosis and bone fracture.The succession of osteoblastdevelopment includes osteoprogenitor proliferation, matrix matu-

T.-Y. Hung et al. / Journal of Ethnopharmacology 131 (2010) 70–77 71

F e (WEr /ml ofu

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ig. 1. Voucher specimen of Kunze (A) and the effects of the water extracts of Kunzat calvaria were exposed to 1, 10, and 100 �g/ml of WEK for 6 days (B) or to 10 �gsing a light microscope, 100×.

ation, and cell mineralization (Aubin, 1998; Giustina et al., 2008).hese progressive events are tightly regulated by sequential induc-ion of osteoblast-associated genes and closely associated with

evelopment of bone cell function. Insulin-like growth factor (IGF-) is essential to regulate normal longitudinal bone growth andone mass, and its deficiency leads to osteoporosis with high risk ofone fracture (Giustina et al., 2008). IGF-1 can stimulate osteopro-

K) on morphologies of rat calvarial osteoblasts. Osteoblasts prepared from neonatalWEK for 1, 3, and 6 days (C). Cell morphologies were observed and photographed

genitor proliferation and differentiation and in conjunction withbone morphogenetic protein (BMP)-2-stimulated matrix mineral-ization (Jia and Heersche, 2000; Koch et al., 2005). BMPs, which

are multi-functional growth factors, play important roles in thedevelopment of bone and cartilage (Wu et al., 2007). EndogenousBMP-2 and BMP-6 acting together play pivotal roles in matrixmaturation and bone formation (Kugimiya et al., 2005). Alkaline

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2 T.-Y. Hung et al. / Journal of Eth

hosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) arearly osteoblast markers participating in control of osteoblast func-ion and bone extracellular matrix mineralization (van Leeuwen etl., 2001). BMPs have been reported to trigger Smad-signaling toegulate mineralization-related gene expression (Yamachika et al.,009; Trzeciakiewicz et al., 2010). Regulation of these osteoblast-ssociated genes is closely related to osteogenic differentiation andaturation.Bone fracture is a commonly occurring result of traffic acci-

ents. During the progression of bone fracture healing, osteogenicifferentiation and maturation occurs sequentially and finallytimulates bone formation (Shapiro, 2008). Inflammation-inducedxidative/nitrosative stress usually impairs bone healing (Yeler etl., 2005). Oxidative and nitrosative stress has been shown to inhibitsteogenic differentiation of bone marrow stromal cells, osteo-rogenitor, and calvarial osteoblasts (Shouhed et al., 2005). Ourrevious studies have further showed that nitric oxide-induceditrosative stress reduced ALP activity and induced apoptotic

nsults to rat calvarial osteoblasts through a mitochondrial-ependent pathway (Chen et al., 2005; Chang et al., 2006).itrosative stress can suppress c-Jun N-terminal kinase 1/2-edicated activation of activator protein-1 and simultaneously

nhibits anti-apoptotic bcl-2 gene expression (Ho et al., 2009).eanwhile, reducing the levels of oxidative/nitrosative stress with

n antioxidant, N-acetylcystein, stimulates osteoblast differentia-ion of mouse calvarial cells and osteogenesis in mouse calvarialells in parallel (Jun et al., 2008). Thus, discovering effective naturalroducts with osteogenesis-promoting and antioxidant propertiesay support the treatment of some bone diseases.Drynaria fortunei J. Sm. (Korean name; Kunze) (Pteridophyta)

s a variety of the traditional Chinese herb Gusuibu, which isrequently used by Chinese people for the prevention or treat-

ent of bone-related diseases. Our previous study has shown thatater extracts of Kunze can protect rat calvarial osteoblasts fromydrogen peroxide-induced insults (Liu et al., 2001). Wang et al.2008) reported that the components of Kunze exhibited prolif-rative activity in osteoblast-like UMR106 cells. In addition, theavonoid fractions of Kunze can prevent nephrotoxicity and pro-otes kidney primary epithelial tubular cell regeneration (Long et

l., 2005). Crude extracts from Kunze were also shown to have sys-emic effects on bone formation in mice (Wong and Rabie, 2006).

eanwhile, the effects of Kunze on the regulation of osteogenicifferentiation-related gene expression and osteoblast maturationre still unknown. Therefore, this study was aimed to evaluate theffects of Kunze on osteoblast maturation and possible explanatoryechanisms for them, in terms of osteoblast-related gene regula-

ion and antioxidant protection.

. Materials and methods

.1. Preparation of water extracts of Kunze (WEK)

The Kunze (Fig. 1A) used in this study was grown in Chengdu,ichuan Province, China. The herb was provided from Brionesearch Institute, Sun Ten Group (Taipei, Taiwan), and identi-ed by the institutional experts according to macroscopic andicroscopic approaches. In addition, the chemical and physical

haracteristics of Kunze are routinely analyzed, and the productas been commercialized (CAS No. 6005, Sun Ten Pharmaceuticalo., Sun Ten Group). Dry pieces of Kunze were ground into a fine

owder. WEK were prepared by decocting 50 g herb powder with00 ml water for 1 h, as previously described (Liu et al., 2001). Afterltration, the filtrate was frozen at −70 ◦C and then concentrated byreeze Dryer FD24 (Kingmech, Taiwan) into a dry powder. WEK wastored at room temperature and protected from light and moisture.

rmacology 131 (2010) 70–77

2.2. Preparation of rat osteoblasts and drug treatment

Rat osteoblasts were prepared from 3-day-old Wistar rat cal-varia according to a previously described method (Chen et al.,2005). All procedures were performed according to the NationalInstitutes of Health Guidelines for the Use of Laboratory Animalsand approved by the Institutional Animal Care and Use Commit-tee of Taipei Medical University, Taipei, Taiwan. Osteoblasts wereseeded in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL,Grand Island, NY, USA) supplemented with 10% heat-inactivatedfetal bovine serum, l-glutamine, penicillin (100 IU/ml), and strep-tomycin (100 �g/ml) in 75-cm2 flasks at 37 ◦C in a humidifiedatmosphere of 5% CO2. Osteoblasts were grown to confluence priorto drug treatment. Only the first passage of rat osteoblasts was usedin this study. WEK was dissolved in deionized distilled water andfiltrated through 0.25 mm filters. Osteoblasts were treated with dif-ferent concentrations of water extracts for various time intervalsin independent experiments.

2.3. Assays of cell viability

Toxicity of WEK was evaluated by observing cell morphologiesusing a light microscope and assaying cell viability by a colorimet-ric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, as previously described in the literature (Ho etal., 2009). After drug treatment, morphologies of osteoblastswere observed and photographed using an inverted microscope.Osteoblasts (2 × 104 cells per well) were seeded in 96-well tissueculture plates overnight. After WEK treatment, osteoblasts werecultured with new medium containing 0.5 mg/ml MTT for a further3 h. The blue formazan products in the osteoblasts were dissolvedin DMSO and spectrophotometrically measured at a wavelength of550 nm.

2.4. Assays of osteoblast mineralization

Osteoblast maturation was determined by evaluating cellmineralization using the von Kossa and Alizarin red S dye-staining protocols (Cooper et al., 1998; Reseland et al., 2001).Osteoblasts were treated with 10 �g/ml of WEK or a combinationof 10 nM dexamethasone, 100 �g/ml ascorbic acid, and 10 mM �-glycerophosphate for 21 days. After drug treatment, osteoblastswere washed with ice-cold phosphate-based saline (PBS) buffer(0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4) andthen fixed in ice-cold 10% formalin for 20 min. For the von Kossaprotocol, mineralized matrix was detected by treating fixed cellswith 5% silver nitrate for 30 min, followed by subsequent washeswith 5% sodium carbonate in 10% formalin for 1 min and 5% sodiumthiosulfate for 5 min. The reaction was stopped by washing the cellstwice with deionized water. For the Alizarin red S dye-stainingprotocol, the fixed osteoblasts were rinsed thoroughly and thenincubated in 1% alcian blue pH 2.5 (Fisher Scientific, Pittsburgh,PA) for 12 h. The sections were then incubated in Alizarin red S(Fisher Scientific, Pittsburgh, PA) for 8 min, dehydrated briefly inxylene and coverslipped in Permount (Fisher Scientific, Pittsburgh,PA). Mineralized nodules were visualized and counted under aninverted microscope. Each experiment was performed in duplicatewells and repeated three times.

2.5. Reverse-transcription polymerase chain reaction (RT-PCR)assay

Messenger (m)RNAs from rat osteoblasts exposed to WEKwere prepared for RT-PCR of IGF-1, collagen type I, BMP-2,BMP-6, ALP, OPN, OCN, and �-actin mRNA. Oligonicleotideprimers were designed and synthesized by Clontech Labo-

nopharmacology 131 (2010) 70–77 73

roaaCTaTfG3CfCwbaooC

2

ac2wWAPtJ

2

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Table 1Concentration- and time-dependent effects of the water extracts of Kunze (WEK)on viability of rat osteoblasts.

Cell viability(OD values at550 nm)

Cell viability(OD values at550 nm)

0 �g/ml 1.59 ± 0.08 0 day 1.62 ± 0.101 �g/ml 1.51 ± 0.10 1 day 1.74 ± 0.1210 �g/ml 1.49 ± 0.08 3 days 1.48 ± 0.11100 �g/ml 1.52 ± 0.09 6 days 1.73 ± 0.13

FcA

T.-Y. Hung et al. / Journal of Eth

atories (Palo Alto, CA, USA). The oligonucleotide sequencesf the upstream and downstream primers for these mRNAnalyses were, respectively, 5′-CGGGGCTGAGCTGGTGGAC-3′

nd 5′-CTGGGCCCGGATGGAACG-3′ for IGF-1; 5′-TTGTCCTCATGGCTGTGAAAC-3′ and 5′-TATTGCTGGTGCTCC-GGCTTC-3 for collagen type I; 5′-GCAGAGCTCCAGATTTTTCG-3′

nd 5′-TTAAGACGCTTCCGCTGTTT-3′ for BMP-2; 5′-AGGATGGGG-GTCAGAGGGAG A-3′ and 5′-GTTGTGCTGCGGTGTCACCA-3′

or BMP-6; 5′-CCTTGAAAAATGCCCTGAAA-3′ and 5′-CTTGGA-AGAGCCACAAAGG-3′ for ALP, 5′-GTGAACTCGGATGAATCTGACG-′ and 5′-CTTGTCCTCATGGCTGTGAAAC-3′ for OPN; 5′-ATGAGGA-CCTCTCTCTGCTC-3′ and 5′-GTGGTGCCATAATGCGCTTG-3′

or OCN; and 5′-GTGGGCCGCTCTAGGCACCAA-3′ and 5′-TCTTTGATGTCACGCACGATTTC-3′ for �-actin. The PCR productsere loaded onto a 1.8% agarose gel containing 0.1 �g/ml ethidium

romide and electrophoretically separated. DNA bands were visu-lized and photographed under UV-light exposure. The intensitiesf the DNA bands in the agarose gel were quantified with the aidf the UVIDOCMW version 99.03 digital imaging system (Uvtec,ambridge, UK).

.6. Quantification of nitrosative stress

Nitrosative stress levels were quantified by measuring themounts of intracellular reactive oxygen species (ROS) using a flowytometer, according to a previously described method (Ho et al.,009). Briefly, 1 × 105 rat calvarial osteoblasts were cultured in 12-ell tissue culture clusters overnight, and then co-treated withEK and 2,7-dichlorofluorescin diacetate, an ROS-sensitive dye.

fter drug treatment, osteoblasts were harvested and suspended inBS buffer. The relative fluorescent intensities in cells were quan-ified with a flow cytometer (FACS Calibur, Becton Dickinson, Sanose, CA, USA).

.7. Analysis of apoptotic cells

Apoptotic cells were determined using propidium iodide toetect DNA fragments in nuclei, according to a previously describedethod (Chang et al., 2006). After drug treatment, the medium con-

aining the floating cells was collected, and the attached cells wereashed and trypsinized. The floating and trypsinized osteoblasts

ig. 2. Effects of the water extracts of Kunze (WEK) on osteoblast mineralization. Osteombination of dexamethasone (Dex), ascorbic acid (AA), and �-glycerophosphate (GPlizarin red S-staining protocols (B), and photographed by a light microscope, 100×.

Osteoblasts prepared from neonatal rat calvaria were exposed to 1, 10, and100 �g/ml of WEK for 6 days or to 10 �g/ml of WEK for 1, 3, and 6 days. Cell viabilitywas assayed using a colorimetric method. Each value represents the mean ± SEMfor n = 6.

were collected in the same centrifuge tubes. After centrifugationand washing, the cell pellets were fixed in cold 80% ethanol. Thefixed cells were stained with propidium iodide. Apoptotic cellswere quantified by detecting the proportion of rat osteoblastsarrested at sub-G1 phase using flow cytometry (Becton Dickinson).

2.8. Statistical analyses

The statistical significance of differences between the controland drug-treated groups was evaluated using Student’s t-test, anddifferences were considered statistically significant at p values of<0.05. Differences between drug-treated groups were consideredsignificant when the p value of Duncan’s multiple-range test was<0.05. Statistical analysis between groups over time was carried outby two-way ANOVA.

3. Results

3.1. Toxicity of WEK to rat osteoblasts

Cell morphologies and viability were analyzed to determinethe toxicity of WEK to rat calvarial osteoblasts (Fig. 1B and C and

Table 1). Exposure of rat osteoblasts to 1, 10, and 100 �g/ml of WEKfor 6 days did not affect cell morphologies (Fig. 1B and C). Addi-tionally, analysis of cell viability further showed that exposure to10 �g/ml of WEK for 1, 2, and 6 days did not lead to death of ratosteoblasts (Table 1).

oblasts prepared from neonatal calvarias were exposed to 10 �g/ml of WEK or a) for 21 days. Mineralized nodules were stained using the von Kossa (A) and the

74 T.-Y. Hung et al. / Journal of Ethnopharmacology 131 (2010) 70–77

Fig. 3. Effects of the water extracts of Kunze (WEK) on osteoprogenitorproliferation-related IGF-1 and collagen type I mRNA expressions. Osteoblasts pre-pared from neonatal calvarias were exposed to 1, 10, and 100 �g/ml of WEK for 3days. Messenger (m)RNA was prepared for RT-PCR analyses of IGF-1 and collagentype I mRNA (A, top two panels). Amounts of �-actin mRNA were analyzed as theinternal standards (bottom panel). These DNA bands were quantified and analyzedstatistically (B and C). Each value represents the mean ± SEM for n = 6. The symbol *i

3

bKc(1sdo

Fig. 4. Effects of the water extracts of Kunze (WEK) on extracellular matrixmaturation-related BMP-2 and BMP-6 mRNA expression. Osteoblasts prepared fromneonatal calvarias were exposed to 1, 10, and 100 �g/ml of WEK for 3 days. Mes-senger (m)RNA was prepared for RT-PCR analyses of BMP-2 and BMP-6 mRNA (A,

(Fig. 3A, top panel, lane 1). After treatment with 1, 10, and 100 �g/mlof WEK, amounts of IGF-1 mRNA were apparently induced (lanes

ndicates that the values significantly differ from the respective control, p < 0.05.

.2. WEK promotes osteoblast mineralization

Effects of WEK on osteoblast maturation were determinedy analyzing cell mineralization (Fig. 2). Staining by the vonossa and Alizarin red S dye-staining protocols revealed that inontrol osteoblasts, there was no mineralized nodule observedFig. 2A and B, left panels). Meanwhile, exposure of osteoblasts to0 �g/ml WEK (middle panels) and a combination of dexametha-

one, ascorbic acid, and �-glycerophosphate (right panels) for 21ays significantly stimulated the formation of mineralized nodulesf osteoblasts.

top two panels). Amounts of �-actin mRNA were analyzed as the internal standards(bottom panel). These DNA bands were quantified and analyzed statistically (B andC). Each value represents the mean ± SEM for n = 6. The symbol * indicates that thevalues differ significantly from the respective control, p < 0.05.

3.3. WEK stimulates osteoprogenitor proliferation-related geneexpression

The levels of IGF-1 and collagen type I mRNA were determinedto evaluate the effects of WEK on regulation of the osteoprogenitorproliferation-related gene expressions during osteoblast differen-tiation (Fig. 3). In control osteoblasts, IGF-1 mRNA was detected

2–4). Levels of �-actin were analyzed as the internal standards(bottom panel). These DNA bands were quantified and statisticallyanalyzed (Fig. 3B). WEK at 1, 10, and 100 �g/ml induced IGF-1

T.-Y. Hung et al. / Journal of Ethnopharmacology 131 (2010) 70–77 75

Fig. 5. Effects of the water extracts of Kunze (WEK) and the active components on mineralization-related ALP, OPN, or OCN mRNA expression. Osteoblasts prepared fromn sengert ttom pw mRNAm the r

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eonatal calvarias were exposed to 1, 10, and 100 �g/ml of WEK for 3 days. Meswo panels). Amounts of �-actin mRNA were analyzed as the internal standards (boere treated with 10 �M naringin for 3 days. RT-PCR analyses of OCN and �-actinean ± SEM for n = 6. The symbol * indicates that the values differ significantly from

RNA expression by 2.1-fold, 76%, and 2-fold, respectively. How-ver, treatment of rat osteoblasts with WEK did not alter the levelsf collagen type I mRNA expression (Fig. 3A, middle panel and C).

.4. WEK induces matrix maturation-related gene expression

Effects of WEK on matrix maturation-related BMP-2 and BMP-mRNA expression were evaluated by RT-PCR analyses (Fig. 4).

n control osteoblasts, BMP-2 mRNA was detected (Fig. 4A, topanel, lane 1). After exposure to 1 �g/ml WEK, amounts of BMP-2RNA were enhanced (lane 2). WEK at 10 and 100 �g/ml obvi-

usly elevated the expression of BMP-2 mRNA in osteoblasts (lanesand 4). Similarly, exposure of rat osteoblasts to WEK induced the

xpression of BMP-6 mRNA (Fig. 4A, middle panel, lanes 2–4) in aoncentration-dependent manner. Levels of �-actin were analyzeds the internal standards (bottom panel). These DNA bands wereuantified and analyzed statistically (Fig. 4B and C). WEK at 1, 10,nd 100 �g/ml caused significant 52%, 89%, and 2.2-fold increasesn the levels of BMP-2 mRNA (Fig. 4B). The expression of BMP-6

RNA in rat calvarial osteoblasts was induced 2.1-fold, 84%, and.7-fold following administration of 1, 10, and 100 �g/ml of WEK,espectively (Fig. 4C).

.5. WEK stimulates mineralization-related gene expression

To further determine the mechanisms of WEK-stimulatedsteoblast maturation, the expression of mineralization-relatedLP, OPN, and OCN mRNA was determined (Fig. 5). Exposure of

at osteoblasts to 10 and 100 �g/ml of WEK obviously augmentedhe amounts of ALP mRNA (Fig. 5A, top panel, lanes 3 and 4). WEKt 1, 10, and 100 �g/ml induced the expression of OPN and OCNRNA (middle two panels). Levels of �-actin were analyzed as the

nternal standards (bottom panel). These DNA bands were quanti-

(m)RNA was prepared for RT-PCR analyses of ALP, OPN, and OCN mRNA (A, topanel). These DNA bands were quantified and analyzed statistically (B). Osteoblastswere validated (C), and the bands were quantified (D). Each value represents the

espective control, p < 0.05.

fied and analyzed statistically (Fig. 5B). Treatment with WEK at 10and 100 �g/ml caused significant 33% and 2.1-fold increases in theexpression of ALP mRNA, respectively. The levels of OPN mRNAin rat calvarial osteoblasts were increased by 98%, 2.3-fold, and2.2-fold following treatment with 1, 10, and 100 �g/ml of WEK,respectively. Significantly, exposure to 1, 10, and 100 �g/ml WEKresulted in 3.1-, 2.7-, and 2.1-fold induction of OCN mRNA (Fig. 5B).Exposure of osteoblasts to 10 �M naringin, a component of WEK,obviously induced OCN mRNA expression by 2.1-fold, respectively(Fig. 5C and D).

3.6. WEK protects osteoblasts from nitrosative stress-inducedapoptotic insults

Effects of WEK on nitrosative stress-induced insults to rat cal-varial osteoblasts were determined by analyzing intracellular ROSlevels, cell viability, and apoptotic cells (Fig. 6). Treatment of ratcalvarial osteoblasts with SNP increased the levels of cellular nitricoxide (data not shown) and nitrosative stress 8.5-fold. Exposureto WEK did not affect the basal levels of intracellular ROS, butcaused a significant 60% decrease in SNP-stimulated nitrosativestress (Fig. 6A). In parallel with the augmentation of nitrosativestress, exposure to SNP led to a significant 78% decrease in viabilityof osteoblasts (Fig. 6B). WEK did not affect cell viability, but signif-icantly lowered SNP-caused osteoblast death by 36%. SNP induced67% of osteoblasts undergoing apoptosis (Fig. 6C). Treatment of ratcalvarial osteoblasts with WEK significantly reduced SNP-causedcell apoptosis by 32%.

4. Discussion

Kunze promotes osteogenic differentiation without affectingcell viability. This study showed that exposure of rat calvarial

7 nopharmacology 131 (2010) 70–77

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Fig. 6. Effects of the water extracts of Kunze (WEK) on sodium nitroprusside (SNP)-induced alterations in the levels of nitrosative stress, cell viability, and apoptoticcells. Osteoblasts prepared from neonatal calvarias were exposed to 2 mM SNP,10 �g/ml WEK, and a combination of SNP and WEK for 24 h. Levels of nitrosativestress were quantified using a flow cytometer (A). Cell viability was assayed accord-

6 T.-Y. Hung et al. / Journal of Eth

steoblasts to 1, 10, and 100 �g/ml of the water extracts of Kunzeor 1, 3, and 6 days did not alter cell morphologies. Assay of the

itochondrial NADH dehydrogenase activity further demonstratedunze did not affect osteoblast viability. Thus, our present results

ndicate that the water extracts of Kunze at concentrations of100 �g/ml do not cause cytotoxicity to osteoblasts. Meanwhile,nder such a treatment, Kunze stimulated osteoblast mineraliza-ion. Wang et al. (2008) extracted the components of Kunze andhowed them to exhibit the proliferative activity of osteoblast-likeMR106 cells. In this study, we further demonstrate that extractsf Kunze can promote the maturation of primary and normal ratalvarial osteoblasts. Bone is dynamically balanced by bone forma-ion and bone resorption (Ducy et al., 2000; Seeman and Delmas,006). Osteoblast maturation is a key step in the process of boneevelopment (Aubin, 1998). Therefore, Kunze may promote boneormation by stimulating osteoblast maturation, rather than by itsytotoxicity.

Kunze selectively induces IGF-1 expression and possibly stim-lates osteoprogenitor proliferation. After exposure to waterxtracts of Kunze, the levels of IGF-1 mRNA were augmented inconcentration-dependent manner. Meanwhile, Kunze did not

ffect collagen type I mRNA expression. IGF-1 has been reportedo trigger osteoprogenitor proliferation and differentiation (Jia andeersche, 2000). Our present data show that treatment with Kunzerobably enhances osteoprogenitor proliferation due to its effectsn selective induction of IGF-1 expression. IGF-1 deficiency haseen shown to put patients with osteoporosis at a high risk ofone fracture (Giustina et al., 2008). During osteogenesis, osteo-rogenitor differentiated from stromal stem cells are transformedo osteoblast precursors (Aubin, 1998). The present study showshat Kunze can stimulate osteoblast mineralization. Thus, Kunzeromotes osteoblast maturation, possibly by improving osteopro-enitor proliferation.

Regulation of BMP-2 and BMP-6 expression is involved inunze-induced osteoblast maturation. Treatment of rat calvar-

al osteoblasts with water extracts of Kunze induced BMP-2 andMP-6 mRNA expression in a concentration-dependent manner.imilarly, the expression of IGF-1 mRNA was induced by Kunze.GF-1 has been reported to induce early osteoblast gene expres-ion, including BMP2, in human mesenchymal stem cells (Koch etl., 2005). Thus, one of the possible mechanisms that could explainhe Kunze-involved regulation of BMP-2 may be that this tradi-ional Chinese herb induces IGF-1. Endogenous BMP-2 and BMP-6ave been reported to contribute to matrix maturation and bone

ormation (Kugimiya et al., 2005). A recent study further showedhat, in a murine model of osteopenia and in human osteoblasts,MP-6 exerts its osteoinductive effect through IGF-I and epider-al growth factor-1 pathways (Grasser et al., 2007). Therefore,

unze may stimulate the matrix maturation by increasing BMP-2nd BMP-6 expression, and consequently induces osteoblast mat-ration.

Kunze-induced osteoblast maturation involves regulation ofLP, OPN, and OCN expression. During osteogenic differentiation,MPs have been shown to induce the expression of ALP, OPN, andCN genes via activation of transcription factors Smads and Runx2

Yamachika et al., 2009; Trzeciakiewicz et al., 2010). Smads andunx2 can be detected in osteoblasts (Grasser et al., 2007; Ho et al.,009). Thus, the increases in BMPs caused by Kunze may engage

n the regulation of ALP, OPN, and OCN mRNA expression. ALP,PN, and OCN are all early osteoblast markers that participate inontrol of osteoblast function and bone extracellular matrix min-

ralization (van Leeuwen et al., 2001). This study shows that theater extracts of Kunze can enhance osteoblast maturation possi-

ly through IGF-1-BMP-mediated induction of ALP, OPN, and OCNene expression. Wang et al. (2008) reported that there are elevenavonoids extracted from Kunze. Our results by analysis of high

ing to a colorimetric method (B). Apoptotic cells were determined using flowcytometry (C). Each value represents the mean ± SEM for n = 6. The symbols * and# indicate that the values differ significantly from the respective control and SNP-treated groups, p < 0.05.

performance liquid chromatography revealed that naringin, one of

flavonoids, is the major component of the water extracts of Kunze,and it could induce OCN mRNA expression. Thus, naringin may beone of active components in the extracts of Kunze.

Kunze can alleviate nitrosative stress-induced apoptotic insults.Exposure of rat calvarial osteoblasts to SNP, a nitric oxide donor,

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T.-Y. Hung et al. / Journal of Eth

ncreased the levels of nitric oxide and nitrosative stress andimultaneously caused cell death. Meanwhile, treatment with thextracts of Kunze lowered SNP-induced nitrosative stress and cellnjury. At the same time, the nitrosative stress-caused increase inhrinkage of osteoblasts and the fractions of cells arrested at sub-G1hase were attenuated following administration of Kunze. Shrink-

ng morphologies and arresting at sug-G1 phase are two typicalharacteristics of cells undergoing apoptosis (Chen et al., 2005; Leet al., 2009). Thus, Kunze can protect rat calvarial osteoblasts fromitrosative stress-induced insults via an anti-apoptotic mechanism.

n osteoblast-like MC3T3-E1 cells, flavonoids are shown to preventxidative stress-induced dysfunction and damage (Lee and Choi,008). Extracts of Kunze contain a variety of flavonoids (Wang etl., 2008). Our previous study also showed that hydrogen peroxide-aused nitrosative stress induced apoptosis of osteoblasts, butater extracts of Kunze attenuated such damage (Liu et al., 2001).

his study further demonstrates that the nitrosative stress-inducedpoptotic insults to rat calvarial osteoblasts can be alleviated byunze. During the healing of bone fractures, inflammation-inducedtress can lessen osteogenic differentiation by hurting stromal stemells, osteoprogenitors, and osteoblasts (Shouhed et al., 2005). Ourresent study shows furthermore that Kunze can defend againstitrosative stress-induced insults to osteoblasts and promotessteoblast maturation.

In conclusion, this study has shown that water extracts of Kunzeid not affect morphologies and viability of rat calvarial osteoblasts.eanwhile, Kunze promoted osteoblast mineralization. Kunze

electively induced osteoprogenitor proliferation-related IGF-1RNA expression without affecting collagen type I expres-

ion. The levels of extracellular matrix maturation-associatedMP-2 and BMP-6 mRNA were sequentially enhanced followingunze treatment. Consequently, Kunze increased the expressionf mineralization-related ALP, OPN, and OCN mRNA. In addi-ion, exposure of rat calvarial osteoblasts to water extracts ofunze lowered SNP-enlarged intracellular ROS levels and simul-

aneously prevented nitrosative stress-induced insults via annti-apoptotic mechanism. Therefore, our present results sug-est that Kunze can promote osteoblast maturation by regulatingsteogenic differentiation-related gene expression and protect-ng against nitrosative stress-induced apoptotic insults. An in vivotudy is being carried out in our lab to evaluate the protectiveffects of water extracts of Kunze on bone formation using femurractured animals as the experimental models. Additionally, theffective components water-extracted from Kunze are also beingetermined using liquid chromatography–mass spectrometry.

cknowledgments

This study was supported by the Yuan’s General Hospital98YGH-TMU-07) and the National Science Council (NSC96-628-B-038-005-MY3), Taipei, Taiwan. The authors express theirratitude to Ms. Yi-Ling Lin for her technical support and data col-ection for these experiments.

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