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Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling
European Journal of Pharmacology 721 (2013) 225–230
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling Xiaozhou Ying a,n,1, Liaojun Sun a,1, Xiaowei Chen b, Huazi Xu a, Xiaoshan Guo a, Hua Chen a, Jianjun Hong a, Shaowen Cheng c, Lei Peng c a
Department of Orthopedic Surgery, The Second Affiliated Hospital of Wenzhou Medical College, 109 Xue Yuan xi Road, Wenzhou 325000, China Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China c Trauma Center of the Affiliated Hospital of Hainan Medical College, Haikou 570100, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 April 2013 Received in revised form 31 August 2013 Accepted 11 September 2013 Available online 25 September 2013
Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. & 2013 Elsevier B.V. All rights reserved.
Keywords: Silibinin Human bone marrow stromal cells (hBMSCs) Osteogenic RUNX2 Bone morphogenetic protein
1. Introduction Bone formation is characterized by proliferation and formation of a properly laid-out collagenous extracellular matrix. Once matrix synthesis begins, osteoblast marker genes are activated in a clear temporal sequence; alkaline phosphatase and the parathyroid hormone (PTH)/PTH-related protein receptor are induced at early times while osteopontin and osteocalcin appear somewhat later (Wang et al., 1999). Once these marker genes are induced, mineralization of the collagenous extracellular matrix follows. Bone mass is controlled by continuous bone remodeling through osteoblastic bone formation and osteoclastic bone resorption. The balance between bone formation and bone resorption must be delicately maintained to ensure the integrity of the skeletal system. An imbalance brought about by increased bone resorption over bone
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Corresponding author. Tel.: þ 86 13676497718. E-mail address: [email protected] (X. Ying). 1 Contributed equally to this work.
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.09.031
formation can lead to most adult skeletal diseases including osteoporosis (Lee et al., 2008; Rodan and Martin, 2000). Until recently, however, most therapies for skeletal disorders are focused mainly on the resorption side and far less attention has been paid to promoting bone formation (Rodan and Martin, 2000). Effective therapeutic strategy is urgently needed to have satisfactory bone building (anabolic) agents that stimulate new bone formation and correct the imbalance of trabecular microarchitecture characteristic of established osteoporosis (Berg et al., 2003; Ducy et al., 2000). Bone marrow stromal cells (BMSCs) are the precursor cells of osteoblast lineage, they play an important role in bone modeling and remodeling, where they give rise to the essential osteoblasts for bone formation (Pittenger et al., 1999). They also serve to maintain a balance between bone formation and resorption (Manolagas, 2000), agents which regulate bone formation act by increasing the proliferation and inducing differentiation of the BMSCs. Silibinin is the major active constituent of the natural compound silymarin, the isomeric mixture of flavonolignans extracted from milk thistle (Silybum marianum) consisting of silibinin A and B, isosilibinin A and B, silicristin, and silidianin. Several animal
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studies have shown that silymarin and silibinin have hepatoprotective activity against toxins and oxidative attack, in which these compounds act as a bioactive antioxidant (Al-Anati et al., 2009; Ha et al., 2010). Silibinin exerts anti-inflammatory and antifibrogenic effects on human hepatic stellate cells isolated from human liver (Trappoliere et al., 2009). In addition, silibinin possesses anti-cancer effects against various human carcinoma cells (Singh and Agarwal, 2005). Recently, Kim et al. (2011, 2012) reported that silibinin has a potential to enhance osteoblastogenesis in murine MC3T3-E1 pre-osteoblastic cells. Some studies also found that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with receptor activator of nuclear factor-kB ligand (RANKL) or tumor necrosis factor-α (TNF-α) (Kavitha et al., 2012; Kim et al., 2009, 2011, 2013). Although a number of studies have established the various role of silibinin in both in vitro and in vivo models, the effect of silibinin on osteogenic differentiation of BMSC has yet to be revealed. In the present study, we found that silibinin promotes osteogenic differentiation and mineralization via BMP signaling pathways accompanied by upregulation of RUNX2 and Osterix.
2. Materials and methods 2.1. Isolation and culture of hBMSCs Bone marrow (5 ml) aspirates were obtained from the posterior iliac crest of three healthy volunteers, aged 35–60 years (2 males and 1 female). Full ethical consent was obtained from all patients and the study was granted ethical approval by the Medical Ethical Committee of the Second Affiliated Hospital, Wenzhou Medical College. Cells from each of the three donors were cultured independently and experiments performed in triplicates. Bone marrow mononuclear cells (BMMNC) were prepared as previously described (Gronthos and Simmons, 1995). The bone marrow was washed with growth culture medium (DMEM, Gibco) supplemented with 10% (V/ V) fetal bovine serum (FBS, Gibco), 1% (V/V) penicillin and streptomycin (Gibco). The mixture of bone marrow and medium was gently added to the 50% Percoll solution (Sigma) and centrifuged at 3000 rpm for 30 min. The cell suspension was obtained between the layer of Percoll and the supernatant liquid layer. Cells were plated and then incubated in a humidified atmosphere of 5% CO2 at 37 1C. They were passaged every 3–4 days using 0.25% (w/v) trypsin-EDTA solution (Gibco) and the third passage cells were used in our experiments. To examine the effects of silibinin on terminal differentiation, the hBMSCs were cultured in an osteogenic medium [growth culture medium supplemented with 10 8 M dexamethasone (Sigma), 50 μg/ml ascorbic acid (Sigma) and 5 mM β-glycerol phosphate (Sigma)] at an initial density of 1 104 cells/cm2 with 0, 1, 10 or 20 μmol/l silibinin (Sigma). Silibinin was dissolved in dimethyl sulfoxide for live culture with cells; its final culture concentration was 0.5%. The medium was changed every four days during osteogenic differentiation.
2.3. Alkaline phosphatase (ALP) assay ALP staining was performed at day seven after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. Cells which were cultured in osteogenic medium were rinsed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde. ALP substrate mixture (ALP staining kit, Sigma) was then added and incubated for ten minutes. The ALP activity was determined at 405 nm using p-nitrophenyl phosphate (pNPP) (Sigma) as the substrate, and the total protein contents were determined with the bicinchoninic acid (BCA) method, which was previously described in the literature (Sun et al., 2006). 2.4. Calcium deposit analysis To assess cell-mediated calcium deposition, hBMSCs which were cultured in osteogenic medium were visualized by von Kossa staining, which was performed at day 21 after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. Cells in the well plates were fixed in 4% paraformaldehyde, then stained with 1% silver nitrate, placed under a UV lamp for 20 min and rinsed with distilled water before treatment with 5% sodium thiosulfate for two minutes. Von Kossa-positive (black) deposits were observed after alcohol washing. Pixel values were measured using imaging software (Sante DICOM viewer, Santesoft) as previously described (Hakki et al., 2010; Yoo et al., 2011). Measured pixel values were normalized versus image background pixel values: Normalized pixel value¼ pixel value of Von Kossa staining/pixel value of background image. 2.5. Real-time polymerase chain reaction (PCR) assay Real-time PCR was used to detect the expression of several osteogenic differentiation related marker genes (COL-I, ALP, OCN, Osterix, BMP-2, RUNX2.) at 4th and 7th day respectively. Total RNA was extracted using TriZol (Invitrogen) according to the manufacturer's instructions and quantified. Its concentration was determined spectrophotometrically at 260 nm (HP 8452A Diode Array Spectrophotometer). First strand complementary DNAs (cDNAs) were synthesized from 0.3 mg of the isolated RNA by oligo (deoxythymidine) using DyNamoTM cDNA Synthesis Kit (Fermentas), and used as templates for real-time PCR. The expression of mRNAs was determined quantitatively using DyNamo SYBR1 Green qPCR kit (Takara, Japan).The PCR was performed on a final volume of 25 ml containing 2 ml cDNA, 7.5 pmol of each primer, 1 ml ROX reference dye and 12.5 ml of SYBR Green Master mix (TIANGEN),with ABI Prism 7300 (Applied Biosystems, Foster City, CA, USA). The samples underwent 40 cycles consisting of the following steps: initial denaturation at 95 1C for 5 min, followed by a set cycle of denaturation at 94 1C for 10 s, different annealing temperatures for each pair of primers (ranging between 53 and 62 1C) for 10 s, extension at 72 1C for 27 s and a final elongation at 72 1C for 5 min. Fold increment of any assayed gene was calculated by normalizing its expression level to that of the glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene, which was used as an internal control. Each gene analysis was performed in triplicate. Primer's sequences of the targeted genes were listed in Table 1.
2.2. Cell viability by cell counting kit-8 (CCK-8) assay
2.6. Reporter gene assays
For the cell viability assays, hBMSCs were cultured in 96-well plates at 1 104 cells per well with growth culture medium. Twentyfour hours later, cells were switched to silibinin containing media for up to 14 days. The viability of hBMSCs was determined by the CCK-8 (Kumamoto, Japan) and measured by microplate reader scanning (ELx800,BioTek) at 450 nm as previously described elsewhere (Liu et al., 2010).
hBMSCs were seeded onto 48-well plates and allowed to reach approximately 80% confluency. The cells were then transfected with 0.1 μg of reporter plasmids containing BMP-responsive elements (12xGCCG-Luc) (Kusanagi et al., 2000) or Runx2-responsive elements (6xOSE2-Luc) (Ducy and Karsenty, 1995) and 0.01 μg of control reporter plasmids encoding TK-Renilla luciferase (Promega, Madison, WI) using Lipofectamine LTX (Invitrogen) according to the
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Table 1 Primers for real-time PCR. Gene
Primer sequences (5'-3')
COL-I
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
ALP OCN RUNX2 BMP-2 Osterix GAPDH
GAG AGC ATG ACC GAT GGA T ATG TTT TGG TGG TTC AGG AGG CGC TGT GTC AAC TCC ACC T CCA GAA GGT TCT GTT AAC TTG GTG CAG CCT TTG TGT CCA AG GTC AGC CAA CTC GTC ACA GT GCG TCA ACA CCA TCA TTC TG CAG ACC AGC AGC ACT CCA TC CAG AAA CGA GTG GGA AAA CAA C ATT CGG TGA TGG AAA CTG CTA T GA GAC TCA ACA GCC CTG GGA AA GGG TGG GTA GTC ATT GGC ATA G CCT CAA GAT CAT CAG CAA T CCA TCC ACA GTC TTC TGG GT
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manner. At 10 μmol/l silibinin, ALP activity was increased approximately 1.6 folds as compared with control, while at 20 μmol/l silibinin, the activity was increased about 2.3 folds (Fig. 2).
3.3. Quantitation of the deposition of calcium Compared to the control group, increased calcium depositions were observed in 10 or 20 μmol/l silibinin treatment groups (Po0.05). The quantitative intensity of von Kossa staining was consistent with its image patterns (Fig. 3).
Fig. 1. The viability of hBMSCs was analyzed at 1, 4, 7, and 14 days, respectively. The data are represented as mean 7 standard deviation.
manufacturer's instructions. After 8 h, the cells were cultured with or without silibinin for 60 h. After the culture, the cells were lysed in Passive Lysis Buffer (Promega). Dual luciferase assays were performed using a Dual Luciferase Reporter Assay System (Promega) and a Glomax 96 Microplate Luminometer (Promega). All measurements were carried out on triplicate samples.
Fig. 2. Alkaline phosphatas(ALP) staining was performed at 7th day after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. The ALP activity of hBMSCs was measured with the pNPP assay. The data are represented as mean7 standard deviation.nP o0.05.
2.7. Statistical analysis All data are expressed as the mean7S.D. Statistical analyses of the significance of differences among values were carried out by one-way ANOVA with a post hoc Dunnett's test or Student's t-test. Values of Po0.05 were considered to indicate statistical significance.
3. Results 3.1. Cell viability The viability of hBMSCs was analyzed at days 1, 4, 7 and 14, respectively. By CCK-8 assay (Fig. 1), no cytotoxic effect of silibinin on cell viability was noted within the concentration range of 1, 10 or 20 μmol/l silibinin (P 40.05). 3.2. ALP activity The ALP activity of hBMSCs was measured in the presence of 0, 1, 10 or 20 μmol/l silibinin by pNPP assay at day 7. Silibinin significantly augmented ALP activity on 7 days in dose-dependent
Fig. 3. Von Kossa staining was performed at 21th day after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. Pixel values were calculated for comparison. The data are represented as mean 7standard deviation.nPo 0.05.
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3.4. Real-time polymerase chain reaction (PCR) assay Real-time PCR (Fig. 4) was used to detect the expression of several osteogenic differentiation-related marker genes when the hBMSCs were cultured in osteogenic medium supplemented with silibinin for 4 and 7 days. The expression of ALP (Fig. 4A) and OCN (Fig. 4B) was significantly increased in hBMSCs cultured with 10 or 20 μmol/l silibinin at 4th and 7th day in dose-dependent manners. COL-I is an early osteoblastic marker, and as shown in Fig. 4C, upon
incubation of cells with 10 or 20 μmol/l silibinin, the expression of COL-I was increased significantly compared to the control at day four (Po0.05). At day seven, it was only increased in the 10 μmol/l silibinin treatment group (P o0.05), and there was no difference at the concentrations of 1 μmol/l silibinin compared to the control at 4th and 7th day respectively. The level of BMP-2 mRNA expression was increased about 1.5 and 2.8-folds at 20 μmol/l silibinin treatment group after 4 and 7 days respectively (Fig. 4D). The level of RUNX2 mRNA expression was increased
Fig. 4. Real-time PCR reactions were performed using primers for osteoblastic genes, including COL-I, ALP, OCN, Osterix, BMP-2 and RUNX2 for 4 and 7 days. Each bar represents the mean 7 standard deviation. nP o0.05.
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about 1.7 and 2.2-fold in the 10 or 20 μmol/l silibinin treatment groups at day four (Fig. 4E). The expression of osterix (Fig. 4F) was significantly increased in hBMSCs cultured with 10 or 20 μmol/l silibinin at 4th and 7th day in dose-dependent manners.
3.5. Involvement of BMP and RUNX2 signaling pathways in the effects of silibinin on osteoblast differentiation To clarify the possible participation of BMP signaling pathways in the effects of silibinin on osteoblast differentiation, hBMSCs were cultured with silibinin in the presence of BMP inhibitors, and the ALP activity was measured. The BMP antagonist noggin and type I BMP receptor kinase inhibitors dorsomorphin and LDN-193289 partly abolished the silibinin-promoted ALP activity (Fig. 5A). To confirm the possible involvement of BMP- and Runx2dependent signaling in the effects of silibinin, luciferase assays were performed using a BMP-responsive reporter (12xGCCG-Luc), which has Smad-binding motifs, and a RUNX2-responsive reporter (6xOSE2-Luc), which has RUNX2-binding motifs. Both reporters were significantly activated in response to silibinin (Fig. 5B).
Fig. 5. Effects of silibinin on BMP-related signaling pathways. (A) hBMSCs were cultured with the BMP inhibitors noggin (Nog; 100 ng/ml), dorsomorphin (Dor; 0.5 μM) or LDN-193189 (LDN; 0.2 μM) in the presence of silibinin (20 μmol/l) for 7 days. After the culture, the cells were fixed and the ALP activity was measured. The data represent the means 7S.D. of three or more determinations. nPo 0.05 vs. the silibinin-treated cells without an inhibitor. (B) hBMSCs were transiently transfected with a BMP-responsive reporter (12xGCCG-Luc) or a Runx2-responsive reporter (6xOSE2-Luc) and treated with silibinin (20 μmol/l) for 60 h. After the culture, the cells were lysed and the luciferase activities were measured. The data represent the means 7 S.D. of three or more determinations nPo 0.05 vs. the control cells.
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4. Discussion To our knowledge, we have shown for the first time that silibinin can promote osteogenic differentiation of hBMSCs via the induction of BMPs and RUNX2. In our study, we found that the proliferation of hBMSCs was no different from control group when added silibinin within the concentration range of 1, 10 or 20 μmol/l silibinin, indicating that silibinin had no cytotoxic effect on cells at these concentrations. This result is similar to the data of Kim et al. (2012) that MC3T3-E1 cell proliferation was minimally increased by 1–20 μmol/l silibinin during 15-day differentiation in osteogenic medium (P4 0.05). ALP, a cell membrane-associated enzyme, appeared early during osteoblast differentiation and was the most widely recognized marker of osteoblastic differentiation (Serigano et al., 2010; Zou et al., 2008). ALP activity correlates with matrix formation in osteoblasts prior to the initiation of mineralization (Gerstenfeld et al., 1990). In this study, we discovered that silibinin significantly augmented ALP activity on 7 days, which was consistent with the mRNA expression of ALP. Our results were similar to Kim et al. (2012) that they found ALP activity of MC3T3-E1 was increased significantly in the presence of silibinin. OCN was involved in controlling the mineralization process, appeared at a late stage of osteogenic differentiation and was characterized by mature cells of the osteoblastic lineage (Hauschka et al., 1989; Sun et al., 2010). COL-I is the most abundant protein in bone matrix; it constitutes about 90% of the organic bone matrix and high levels of COL-I mRNA expression would be observed during proliferation (Owen et al., 1990). In this study, we found that silibinin can enhance osteoblastic differentiation by increasing the level of OCN and COL-I. Kim et al. (Kim et al., 2012) got similar results in MC3T3-E1 cells. Also our results suggested that silibinin stimulated osteogenic differentiation of hBMSCs not only at the early stage, but also at the maturation stage. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of hBMSCs. During osteogenic differentiation, RUNX2, BMPs, transforming growth factor-β (TGF-β), osterix, β-catenin, and Wnt signaling play essential roles in the commitment of mesenchymal cells to the osteoblastic lineage (Komori, 2006; Wang et al., 2010). BMPs are the most potent inducer cytokines of osteoblastogenesis (Yamaguchi et al., 2000). Through binding to type I and type II serine/threonine kinase receptors, BMPs activate the transcription factor Smad, which translocates into the nucleus and modulates the expression of many target genes (Miyazono et al., 2005). BMPs were also reported to activate these transcription factors for osteoblast differentiation (Marie, 2008). Among many other BMPs, BMP-2 induces differentiation of preosteoblasts into mature osteoblasts by regulating signals that stimulate a specific transcriptional program required for bone formation (Sakou, 1998). RUNX2 and osterix are known to be essential transcription factors for osteoblast differentiation. Gene knockout mice for Runx2 and osterix exhibit a complete defect in bone formation (Ducy et al., 1997; Nakashima et al., 2002). It was reported that Runx2 null mice had no bone tissue, osteoblasts or osteoclasts. RUNX2 is also active in mature osteoblasts, and when active RUNX2 levels have been reduced, decreased expression of the genes encoding the main bone matrix proteins including bone sialoprotein (BSP), OCN, osteopontin (OPN) and COL-I were observed (Bronckers et al., 2001). In our study, the BMP antagonist noggin and type I BMP receptor kinase inhibitors dorsomorphin and LDN-193289 partly abolished the silibinin promoted ALP activity. In addition, the results of luciferase assays found that both BMPresponsive reporter (12xGCCG-Luc), which has Smad-binding motifs, and a Runx2-responsive reporter (6xOSE2-Luc), which has RUNX2-binding motifs were significantly activated in response to silibinin. Thus, silibinin may promote osteoblast differentiation
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through the induction of BMPs, and subsequent activation of BMPSmad and RUNX2 signaling. This may be one of the mechanisms applied in osteogenic differentiation of hBMSCs. Further studies are required to realize other signaling pathways of the regulation of osteogenic differentiation of hBMSCs. Also there are some limitations of these kinds of in vitro studies (they cannot always mimic in vivo conditions), so the results still need further validation in vivo. Bone remodeling is tightly regulated by two processes: bone formation by osteoblasts and bone resorption by osteoclasts. The balance between both processes is important for maintaining bone density. When bone resorption exceeds bone formation, an imbalance of skeletal turnover causes bone resorption diseases such as osteoporosis, Paget's disease, and periodontal disease. Recently, some studies found that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL or TNF-α (Kavitha et al., 2012; Kim et al., 2009, 2011, 2013). Accordingly, silibinin may works as a BMP modulator and is an osteoprotective agent inhibiting osteoclastic bone resorption. Taken all together, it was the first time to report that silibinin can increase osteogenic effect by stimulating osteogenic biomarkers of ALP, COL-I and OCN expression during the differentiation phase in human hBMSCs. The osteoblastogenic activity of silibinin was mediated by triggering BMP-2-responsive and RUNX2 signaling. Although further studies are required to clarify the in vivo actions and mechanisms, silibinin may become a superior drug candidate for the treatment of osteoporosis. Acknowledgments The authors thank all the staff in the Laboratory of Orthopedic Research Institute and Scientific Research Center of Second Affiliated Hospital of Wenzhou Medical College. This work was supported by grants from the Wenzhou Technology Project (Y20090288), Hainan Provincial Health Department (2011-34). References Al-Anati, L., Essid, E., Reinehr, R., Petzinger, E., 2009. Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells. Molecular Nutrition and Food Research 53, 460–466. Berg, C., Neumeyer, K., Kirkpatrick, P., 2003. Teriparatide. Nature Reviews Drug Discovery 2, 257–258. Bronckers, A.L., Engelse, M.A., Cavender, A., Gaikwad, J., D’Souza, R.N., 2001. Cellspecific patterns of Cbfa1 mRNA and protein expression in postnatal murine dental tissues. Mechanisms of Development 101, 255–258. Ducy, P., Karsenty, G., 1995. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Molecular and Cellular Biology 15, 1858–1869. Ducy, P., Schinke, T., Karsenty, G., 2000. The osteoblast: a sophisticated fibroblast under central surveillance. Science 289, 1501–1504. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L., Karsenty, G., 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, 747–754. Gerstenfeld, L.C., Gotoh, Y., McKee, M.D., Nanci, A., Landis, W.J., Glimcher, M.J., 1990. Expression and ultrastructural immunolocalization of a major 66 kDa phosphoprotein synthesized by chicken osteoblasts during mineralization in vitro. Anatomical Record 228, 93–103. Gronthos, S., Simmons, P.J., 1995. The growth factor requirements of STRO-1positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 85, 929–940. Ha, H.L., Shin, H.J., Feitelson, M.A., Yu, D.Y., 2010. Oxidative stress and antioxidants in hepatic pathogenesis. World Journal of Gastroenterology 16, 6035–6043. Hakki, S.S., Bozkurt, B.S., Hakki, E.E., 2010. Boron regulates mineralized tissueassociated proteins in osteoblasts (MC3T3-E1). Journal of Trace Elements in Medicine and Biology 24, 243–250. Hauschka, P.V., Lian, J.B., Cole, D.E., Gundberg, C.M., 1989. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiological Reviews 69, 990–1047. Kavitha, C.V., Deep, G., Gangar, S.C., Jain, A.K., Agarwal, C., Agarwal, R., 2012. Silibinin inhibits prostate cancer cells- and RANKL-induced osteoclastogenesis
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling Xiaozhou Ying a,n,1, Liaojun Sun a,1, Xiaowei Chen b, Huazi Xu a, Xiaoshan Guo a, Hua Chen a, Jianjun Hong a, Shaowen Cheng c, Lei Peng c a
Department of Orthopedic Surgery, The Second Affiliated Hospital of Wenzhou Medical College, 109 Xue Yuan xi Road, Wenzhou 325000, China Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China c Trauma Center of the Affiliated Hospital of Hainan Medical College, Haikou 570100, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 April 2013 Received in revised form 31 August 2013 Accepted 11 September 2013 Available online 25 September 2013
Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. & 2013 Elsevier B.V. All rights reserved.
Keywords: Silibinin Human bone marrow stromal cells (hBMSCs) Osteogenic RUNX2 Bone morphogenetic protein
1. Introduction Bone formation is characterized by proliferation and formation of a properly laid-out collagenous extracellular matrix. Once matrix synthesis begins, osteoblast marker genes are activated in a clear temporal sequence; alkaline phosphatase and the parathyroid hormone (PTH)/PTH-related protein receptor are induced at early times while osteopontin and osteocalcin appear somewhat later (Wang et al., 1999). Once these marker genes are induced, mineralization of the collagenous extracellular matrix follows. Bone mass is controlled by continuous bone remodeling through osteoblastic bone formation and osteoclastic bone resorption. The balance between bone formation and bone resorption must be delicately maintained to ensure the integrity of the skeletal system. An imbalance brought about by increased bone resorption over bone
n
Corresponding author. Tel.: þ 86 13676497718. E-mail address: [email protected] (X. Ying). 1 Contributed equally to this work.
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.09.031
formation can lead to most adult skeletal diseases including osteoporosis (Lee et al., 2008; Rodan and Martin, 2000). Until recently, however, most therapies for skeletal disorders are focused mainly on the resorption side and far less attention has been paid to promoting bone formation (Rodan and Martin, 2000). Effective therapeutic strategy is urgently needed to have satisfactory bone building (anabolic) agents that stimulate new bone formation and correct the imbalance of trabecular microarchitecture characteristic of established osteoporosis (Berg et al., 2003; Ducy et al., 2000). Bone marrow stromal cells (BMSCs) are the precursor cells of osteoblast lineage, they play an important role in bone modeling and remodeling, where they give rise to the essential osteoblasts for bone formation (Pittenger et al., 1999). They also serve to maintain a balance between bone formation and resorption (Manolagas, 2000), agents which regulate bone formation act by increasing the proliferation and inducing differentiation of the BMSCs. Silibinin is the major active constituent of the natural compound silymarin, the isomeric mixture of flavonolignans extracted from milk thistle (Silybum marianum) consisting of silibinin A and B, isosilibinin A and B, silicristin, and silidianin. Several animal
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studies have shown that silymarin and silibinin have hepatoprotective activity against toxins and oxidative attack, in which these compounds act as a bioactive antioxidant (Al-Anati et al., 2009; Ha et al., 2010). Silibinin exerts anti-inflammatory and antifibrogenic effects on human hepatic stellate cells isolated from human liver (Trappoliere et al., 2009). In addition, silibinin possesses anti-cancer effects against various human carcinoma cells (Singh and Agarwal, 2005). Recently, Kim et al. (2011, 2012) reported that silibinin has a potential to enhance osteoblastogenesis in murine MC3T3-E1 pre-osteoblastic cells. Some studies also found that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with receptor activator of nuclear factor-kB ligand (RANKL) or tumor necrosis factor-α (TNF-α) (Kavitha et al., 2012; Kim et al., 2009, 2011, 2013). Although a number of studies have established the various role of silibinin in both in vitro and in vivo models, the effect of silibinin on osteogenic differentiation of BMSC has yet to be revealed. In the present study, we found that silibinin promotes osteogenic differentiation and mineralization via BMP signaling pathways accompanied by upregulation of RUNX2 and Osterix.
2. Materials and methods 2.1. Isolation and culture of hBMSCs Bone marrow (5 ml) aspirates were obtained from the posterior iliac crest of three healthy volunteers, aged 35–60 years (2 males and 1 female). Full ethical consent was obtained from all patients and the study was granted ethical approval by the Medical Ethical Committee of the Second Affiliated Hospital, Wenzhou Medical College. Cells from each of the three donors were cultured independently and experiments performed in triplicates. Bone marrow mononuclear cells (BMMNC) were prepared as previously described (Gronthos and Simmons, 1995). The bone marrow was washed with growth culture medium (DMEM, Gibco) supplemented with 10% (V/ V) fetal bovine serum (FBS, Gibco), 1% (V/V) penicillin and streptomycin (Gibco). The mixture of bone marrow and medium was gently added to the 50% Percoll solution (Sigma) and centrifuged at 3000 rpm for 30 min. The cell suspension was obtained between the layer of Percoll and the supernatant liquid layer. Cells were plated and then incubated in a humidified atmosphere of 5% CO2 at 37 1C. They were passaged every 3–4 days using 0.25% (w/v) trypsin-EDTA solution (Gibco) and the third passage cells were used in our experiments. To examine the effects of silibinin on terminal differentiation, the hBMSCs were cultured in an osteogenic medium [growth culture medium supplemented with 10 8 M dexamethasone (Sigma), 50 μg/ml ascorbic acid (Sigma) and 5 mM β-glycerol phosphate (Sigma)] at an initial density of 1 104 cells/cm2 with 0, 1, 10 or 20 μmol/l silibinin (Sigma). Silibinin was dissolved in dimethyl sulfoxide for live culture with cells; its final culture concentration was 0.5%. The medium was changed every four days during osteogenic differentiation.
2.3. Alkaline phosphatase (ALP) assay ALP staining was performed at day seven after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. Cells which were cultured in osteogenic medium were rinsed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde. ALP substrate mixture (ALP staining kit, Sigma) was then added and incubated for ten minutes. The ALP activity was determined at 405 nm using p-nitrophenyl phosphate (pNPP) (Sigma) as the substrate, and the total protein contents were determined with the bicinchoninic acid (BCA) method, which was previously described in the literature (Sun et al., 2006). 2.4. Calcium deposit analysis To assess cell-mediated calcium deposition, hBMSCs which were cultured in osteogenic medium were visualized by von Kossa staining, which was performed at day 21 after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. Cells in the well plates were fixed in 4% paraformaldehyde, then stained with 1% silver nitrate, placed under a UV lamp for 20 min and rinsed with distilled water before treatment with 5% sodium thiosulfate for two minutes. Von Kossa-positive (black) deposits were observed after alcohol washing. Pixel values were measured using imaging software (Sante DICOM viewer, Santesoft) as previously described (Hakki et al., 2010; Yoo et al., 2011). Measured pixel values were normalized versus image background pixel values: Normalized pixel value¼ pixel value of Von Kossa staining/pixel value of background image. 2.5. Real-time polymerase chain reaction (PCR) assay Real-time PCR was used to detect the expression of several osteogenic differentiation related marker genes (COL-I, ALP, OCN, Osterix, BMP-2, RUNX2.) at 4th and 7th day respectively. Total RNA was extracted using TriZol (Invitrogen) according to the manufacturer's instructions and quantified. Its concentration was determined spectrophotometrically at 260 nm (HP 8452A Diode Array Spectrophotometer). First strand complementary DNAs (cDNAs) were synthesized from 0.3 mg of the isolated RNA by oligo (deoxythymidine) using DyNamoTM cDNA Synthesis Kit (Fermentas), and used as templates for real-time PCR. The expression of mRNAs was determined quantitatively using DyNamo SYBR1 Green qPCR kit (Takara, Japan).The PCR was performed on a final volume of 25 ml containing 2 ml cDNA, 7.5 pmol of each primer, 1 ml ROX reference dye and 12.5 ml of SYBR Green Master mix (TIANGEN),with ABI Prism 7300 (Applied Biosystems, Foster City, CA, USA). The samples underwent 40 cycles consisting of the following steps: initial denaturation at 95 1C for 5 min, followed by a set cycle of denaturation at 94 1C for 10 s, different annealing temperatures for each pair of primers (ranging between 53 and 62 1C) for 10 s, extension at 72 1C for 27 s and a final elongation at 72 1C for 5 min. Fold increment of any assayed gene was calculated by normalizing its expression level to that of the glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene, which was used as an internal control. Each gene analysis was performed in triplicate. Primer's sequences of the targeted genes were listed in Table 1.
2.2. Cell viability by cell counting kit-8 (CCK-8) assay
2.6. Reporter gene assays
For the cell viability assays, hBMSCs were cultured in 96-well plates at 1 104 cells per well with growth culture medium. Twentyfour hours later, cells were switched to silibinin containing media for up to 14 days. The viability of hBMSCs was determined by the CCK-8 (Kumamoto, Japan) and measured by microplate reader scanning (ELx800,BioTek) at 450 nm as previously described elsewhere (Liu et al., 2010).
hBMSCs were seeded onto 48-well plates and allowed to reach approximately 80% confluency. The cells were then transfected with 0.1 μg of reporter plasmids containing BMP-responsive elements (12xGCCG-Luc) (Kusanagi et al., 2000) or Runx2-responsive elements (6xOSE2-Luc) (Ducy and Karsenty, 1995) and 0.01 μg of control reporter plasmids encoding TK-Renilla luciferase (Promega, Madison, WI) using Lipofectamine LTX (Invitrogen) according to the
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Table 1 Primers for real-time PCR. Gene
Primer sequences (5'-3')
COL-I
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
ALP OCN RUNX2 BMP-2 Osterix GAPDH
GAG AGC ATG ACC GAT GGA T ATG TTT TGG TGG TTC AGG AGG CGC TGT GTC AAC TCC ACC T CCA GAA GGT TCT GTT AAC TTG GTG CAG CCT TTG TGT CCA AG GTC AGC CAA CTC GTC ACA GT GCG TCA ACA CCA TCA TTC TG CAG ACC AGC AGC ACT CCA TC CAG AAA CGA GTG GGA AAA CAA C ATT CGG TGA TGG AAA CTG CTA T GA GAC TCA ACA GCC CTG GGA AA GGG TGG GTA GTC ATT GGC ATA G CCT CAA GAT CAT CAG CAA T CCA TCC ACA GTC TTC TGG GT
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manner. At 10 μmol/l silibinin, ALP activity was increased approximately 1.6 folds as compared with control, while at 20 μmol/l silibinin, the activity was increased about 2.3 folds (Fig. 2).
3.3. Quantitation of the deposition of calcium Compared to the control group, increased calcium depositions were observed in 10 or 20 μmol/l silibinin treatment groups (Po0.05). The quantitative intensity of von Kossa staining was consistent with its image patterns (Fig. 3).
Fig. 1. The viability of hBMSCs was analyzed at 1, 4, 7, and 14 days, respectively. The data are represented as mean 7 standard deviation.
manufacturer's instructions. After 8 h, the cells were cultured with or without silibinin for 60 h. After the culture, the cells were lysed in Passive Lysis Buffer (Promega). Dual luciferase assays were performed using a Dual Luciferase Reporter Assay System (Promega) and a Glomax 96 Microplate Luminometer (Promega). All measurements were carried out on triplicate samples.
Fig. 2. Alkaline phosphatas(ALP) staining was performed at 7th day after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. The ALP activity of hBMSCs was measured with the pNPP assay. The data are represented as mean7 standard deviation.nP o0.05.
2.7. Statistical analysis All data are expressed as the mean7S.D. Statistical analyses of the significance of differences among values were carried out by one-way ANOVA with a post hoc Dunnett's test or Student's t-test. Values of Po0.05 were considered to indicate statistical significance.
3. Results 3.1. Cell viability The viability of hBMSCs was analyzed at days 1, 4, 7 and 14, respectively. By CCK-8 assay (Fig. 1), no cytotoxic effect of silibinin on cell viability was noted within the concentration range of 1, 10 or 20 μmol/l silibinin (P 40.05). 3.2. ALP activity The ALP activity of hBMSCs was measured in the presence of 0, 1, 10 or 20 μmol/l silibinin by pNPP assay at day 7. Silibinin significantly augmented ALP activity on 7 days in dose-dependent
Fig. 3. Von Kossa staining was performed at 21th day after cells were cultured in 24-well tissue culture plates at a cell density of 1 104. Pixel values were calculated for comparison. The data are represented as mean 7standard deviation.nPo 0.05.
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3.4. Real-time polymerase chain reaction (PCR) assay Real-time PCR (Fig. 4) was used to detect the expression of several osteogenic differentiation-related marker genes when the hBMSCs were cultured in osteogenic medium supplemented with silibinin for 4 and 7 days. The expression of ALP (Fig. 4A) and OCN (Fig. 4B) was significantly increased in hBMSCs cultured with 10 or 20 μmol/l silibinin at 4th and 7th day in dose-dependent manners. COL-I is an early osteoblastic marker, and as shown in Fig. 4C, upon
incubation of cells with 10 or 20 μmol/l silibinin, the expression of COL-I was increased significantly compared to the control at day four (Po0.05). At day seven, it was only increased in the 10 μmol/l silibinin treatment group (P o0.05), and there was no difference at the concentrations of 1 μmol/l silibinin compared to the control at 4th and 7th day respectively. The level of BMP-2 mRNA expression was increased about 1.5 and 2.8-folds at 20 μmol/l silibinin treatment group after 4 and 7 days respectively (Fig. 4D). The level of RUNX2 mRNA expression was increased
Fig. 4. Real-time PCR reactions were performed using primers for osteoblastic genes, including COL-I, ALP, OCN, Osterix, BMP-2 and RUNX2 for 4 and 7 days. Each bar represents the mean 7 standard deviation. nP o0.05.
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about 1.7 and 2.2-fold in the 10 or 20 μmol/l silibinin treatment groups at day four (Fig. 4E). The expression of osterix (Fig. 4F) was significantly increased in hBMSCs cultured with 10 or 20 μmol/l silibinin at 4th and 7th day in dose-dependent manners.
3.5. Involvement of BMP and RUNX2 signaling pathways in the effects of silibinin on osteoblast differentiation To clarify the possible participation of BMP signaling pathways in the effects of silibinin on osteoblast differentiation, hBMSCs were cultured with silibinin in the presence of BMP inhibitors, and the ALP activity was measured. The BMP antagonist noggin and type I BMP receptor kinase inhibitors dorsomorphin and LDN-193289 partly abolished the silibinin-promoted ALP activity (Fig. 5A). To confirm the possible involvement of BMP- and Runx2dependent signaling in the effects of silibinin, luciferase assays were performed using a BMP-responsive reporter (12xGCCG-Luc), which has Smad-binding motifs, and a RUNX2-responsive reporter (6xOSE2-Luc), which has RUNX2-binding motifs. Both reporters were significantly activated in response to silibinin (Fig. 5B).
Fig. 5. Effects of silibinin on BMP-related signaling pathways. (A) hBMSCs were cultured with the BMP inhibitors noggin (Nog; 100 ng/ml), dorsomorphin (Dor; 0.5 μM) or LDN-193189 (LDN; 0.2 μM) in the presence of silibinin (20 μmol/l) for 7 days. After the culture, the cells were fixed and the ALP activity was measured. The data represent the means 7S.D. of three or more determinations. nPo 0.05 vs. the silibinin-treated cells without an inhibitor. (B) hBMSCs were transiently transfected with a BMP-responsive reporter (12xGCCG-Luc) or a Runx2-responsive reporter (6xOSE2-Luc) and treated with silibinin (20 μmol/l) for 60 h. After the culture, the cells were lysed and the luciferase activities were measured. The data represent the means 7 S.D. of three or more determinations nPo 0.05 vs. the control cells.
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4. Discussion To our knowledge, we have shown for the first time that silibinin can promote osteogenic differentiation of hBMSCs via the induction of BMPs and RUNX2. In our study, we found that the proliferation of hBMSCs was no different from control group when added silibinin within the concentration range of 1, 10 or 20 μmol/l silibinin, indicating that silibinin had no cytotoxic effect on cells at these concentrations. This result is similar to the data of Kim et al. (2012) that MC3T3-E1 cell proliferation was minimally increased by 1–20 μmol/l silibinin during 15-day differentiation in osteogenic medium (P4 0.05). ALP, a cell membrane-associated enzyme, appeared early during osteoblast differentiation and was the most widely recognized marker of osteoblastic differentiation (Serigano et al., 2010; Zou et al., 2008). ALP activity correlates with matrix formation in osteoblasts prior to the initiation of mineralization (Gerstenfeld et al., 1990). In this study, we discovered that silibinin significantly augmented ALP activity on 7 days, which was consistent with the mRNA expression of ALP. Our results were similar to Kim et al. (2012) that they found ALP activity of MC3T3-E1 was increased significantly in the presence of silibinin. OCN was involved in controlling the mineralization process, appeared at a late stage of osteogenic differentiation and was characterized by mature cells of the osteoblastic lineage (Hauschka et al., 1989; Sun et al., 2010). COL-I is the most abundant protein in bone matrix; it constitutes about 90% of the organic bone matrix and high levels of COL-I mRNA expression would be observed during proliferation (Owen et al., 1990). In this study, we found that silibinin can enhance osteoblastic differentiation by increasing the level of OCN and COL-I. Kim et al. (Kim et al., 2012) got similar results in MC3T3-E1 cells. Also our results suggested that silibinin stimulated osteogenic differentiation of hBMSCs not only at the early stage, but also at the maturation stage. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of hBMSCs. During osteogenic differentiation, RUNX2, BMPs, transforming growth factor-β (TGF-β), osterix, β-catenin, and Wnt signaling play essential roles in the commitment of mesenchymal cells to the osteoblastic lineage (Komori, 2006; Wang et al., 2010). BMPs are the most potent inducer cytokines of osteoblastogenesis (Yamaguchi et al., 2000). Through binding to type I and type II serine/threonine kinase receptors, BMPs activate the transcription factor Smad, which translocates into the nucleus and modulates the expression of many target genes (Miyazono et al., 2005). BMPs were also reported to activate these transcription factors for osteoblast differentiation (Marie, 2008). Among many other BMPs, BMP-2 induces differentiation of preosteoblasts into mature osteoblasts by regulating signals that stimulate a specific transcriptional program required for bone formation (Sakou, 1998). RUNX2 and osterix are known to be essential transcription factors for osteoblast differentiation. Gene knockout mice for Runx2 and osterix exhibit a complete defect in bone formation (Ducy et al., 1997; Nakashima et al., 2002). It was reported that Runx2 null mice had no bone tissue, osteoblasts or osteoclasts. RUNX2 is also active in mature osteoblasts, and when active RUNX2 levels have been reduced, decreased expression of the genes encoding the main bone matrix proteins including bone sialoprotein (BSP), OCN, osteopontin (OPN) and COL-I were observed (Bronckers et al., 2001). In our study, the BMP antagonist noggin and type I BMP receptor kinase inhibitors dorsomorphin and LDN-193289 partly abolished the silibinin promoted ALP activity. In addition, the results of luciferase assays found that both BMPresponsive reporter (12xGCCG-Luc), which has Smad-binding motifs, and a Runx2-responsive reporter (6xOSE2-Luc), which has RUNX2-binding motifs were significantly activated in response to silibinin. Thus, silibinin may promote osteoblast differentiation
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through the induction of BMPs, and subsequent activation of BMPSmad and RUNX2 signaling. This may be one of the mechanisms applied in osteogenic differentiation of hBMSCs. Further studies are required to realize other signaling pathways of the regulation of osteogenic differentiation of hBMSCs. Also there are some limitations of these kinds of in vitro studies (they cannot always mimic in vivo conditions), so the results still need further validation in vivo. Bone remodeling is tightly regulated by two processes: bone formation by osteoblasts and bone resorption by osteoclasts. The balance between both processes is important for maintaining bone density. When bone resorption exceeds bone formation, an imbalance of skeletal turnover causes bone resorption diseases such as osteoporosis, Paget's disease, and periodontal disease. Recently, some studies found that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL or TNF-α (Kavitha et al., 2012; Kim et al., 2009, 2011, 2013). Accordingly, silibinin may works as a BMP modulator and is an osteoprotective agent inhibiting osteoclastic bone resorption. Taken all together, it was the first time to report that silibinin can increase osteogenic effect by stimulating osteogenic biomarkers of ALP, COL-I and OCN expression during the differentiation phase in human hBMSCs. The osteoblastogenic activity of silibinin was mediated by triggering BMP-2-responsive and RUNX2 signaling. Although further studies are required to clarify the in vivo actions and mechanisms, silibinin may become a superior drug candidate for the treatment of osteoporosis. Acknowledgments The authors thank all the staff in the Laboratory of Orthopedic Research Institute and Scientific Research Center of Second Affiliated Hospital of Wenzhou Medical College. This work was supported by grants from the Wenzhou Technology Project (Y20090288), Hainan Provincial Health Department (2011-34). References Al-Anati, L., Essid, E., Reinehr, R., Petzinger, E., 2009. Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells. Molecular Nutrition and Food Research 53, 460–466. Berg, C., Neumeyer, K., Kirkpatrick, P., 2003. Teriparatide. Nature Reviews Drug Discovery 2, 257–258. Bronckers, A.L., Engelse, M.A., Cavender, A., Gaikwad, J., D’Souza, R.N., 2001. Cellspecific patterns of Cbfa1 mRNA and protein expression in postnatal murine dental tissues. Mechanisms of Development 101, 255–258. Ducy, P., Karsenty, G., 1995. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Molecular and Cellular Biology 15, 1858–1869. Ducy, P., Schinke, T., Karsenty, G., 2000. The osteoblast: a sophisticated fibroblast under central surveillance. Science 289, 1501–1504. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L., Karsenty, G., 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, 747–754. Gerstenfeld, L.C., Gotoh, Y., McKee, M.D., Nanci, A., Landis, W.J., Glimcher, M.J., 1990. Expression and ultrastructural immunolocalization of a major 66 kDa phosphoprotein synthesized by chicken osteoblasts during mineralization in vitro. Anatomical Record 228, 93–103. Gronthos, S., Simmons, P.J., 1995. The growth factor requirements of STRO-1positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 85, 929–940. Ha, H.L., Shin, H.J., Feitelson, M.A., Yu, D.Y., 2010. Oxidative stress and antioxidants in hepatic pathogenesis. World Journal of Gastroenterology 16, 6035–6043. Hakki, S.S., Bozkurt, B.S., Hakki, E.E., 2010. Boron regulates mineralized tissueassociated proteins in osteoblasts (MC3T3-E1). Journal of Trace Elements in Medicine and Biology 24, 243–250. Hauschka, P.V., Lian, J.B., Cole, D.E., Gundberg, C.M., 1989. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiological Reviews 69, 990–1047. Kavitha, C.V., Deep, G., Gangar, S.C., Jain, A.K., Agarwal, C., Agarwal, R., 2012. Silibinin inhibits prostate cancer cells- and RANKL-induced osteoclastogenesis
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