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Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-kB regulated HIF-1[alpha] and [PGE.sub.2] synthesis.


Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the detail molecular mechanisms of icariin on lipopolysaccharide (LPS)-induced osteolysis. Our hypothesis is that icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-kB regulated HIF-1[alpha] and [PGE.sub.2] synthesis.

After treatment with icariin. the activity of osteoclasts differentiation maker, tatrate resistances acid phosphatease (TRAP), significantly decreased at the concentration of [10.sup.-8] M. Icariin ([10.sup.-8] M) reduced the size of LPS-induced osteoclasts formation, and diminished their TRAP and acid phosphatease (ACP) activity without inhibition of cell viability. Icariin also inhibited LPS-induced bone resorption and interleukin-6 (IL-6). and tumor necrosis factor-[alpha] (TNF-[alpha]) expression. The gene expression of osteoprotegerin (OPG) was up-regulated, while receptor activator of NF-kB ligand (RANKL) was down-regulated. Icariin also inhibited the synthesis of cyclo-oxygenase type-2 (COX-2) and prostaglandin [E.sub.2] ([PGE.sub.2]). In addition, icariin had a dominant repression effect on LPS-induced hypoxia inducible factor-1[alpha] (HIF-1[alpha]) expression of osteoclasts. On osteoclasts, icariin suppresses LPS-mediated activation of the p38 and JNK; while on the osteoblasts, icariin reduced the LPS-induced activation of ERK1/2 and I-kappa-B-alpha (I[kappa]B[alpha]), but increased the activation of p38.

In conclusion, we demonstrated that icariin has an in vitro inhibitory effects on osteoclasts differentiation that can prevent inflammatory bone loss. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway.

[C] 2010 Elsevier GmbH. All rights reserved.




Epimedium pubescens



Gene expression





The human bone is a highly dynamic organ that maintains its homeostasis through a delicate balance between the bone-forming osteoblasts (bone formation) and the bone-eroding osteoclasts (bone resorption). The dynamic balance between these two cells types results in bone remodeling. Increased osteoclast activity induces erosion of trabecular bone and fragile bones. Conversely, increased osteoblast activity increases bone density, which is associated with bone deformity and osteopetrosis (Boyle et al. 2003; Teitebaum 2000).

Osteoclasts are multinucleated giant cells that differentiated from cells of hematopoietic monocyte-macrophage linage under the presence of two critical factors: the receptor activator of NF-kB ligand (RANKL) and the macrophage/monocyte colony-stimulating factor (M-CSF). Both factors (the RANKL and M-CSF) are produced by osteoblasts or stromal cells (Boyle et al. 2003). Osteoprotegerin (OPG), a soluble decoy receptor for RANKL, produced by osteoblasts can inhibit osteoclasts formation by blocking RANKL binding to RANK (Hsu et al. 1999). RANKL represents the osteoblasts-derived factor required for osteoclasts formation, whereas OPG blocks these effects and prevents bone resorption in the various microenviron-ments (Teitebaum 2000). Many factors, such as lipopolysaccharide (LPS), 1,25-dihydroxyvitamin [D.sub.3] (Vit [D.sub.3]), and pro-inflammatory cytokines, can increase osteoclasts formation via up-regulating the expression of RANKL and/or down-regulating OPG in osteoblasts or stromal cells (Lorenzo et al. 2008).

LPS, a bacteria-derived cell wall product, has long been recognized as a key factor implicated in the development of bone loss (Smith et al. 2006). LPS plays an important role in bone resorption by initiating a local host response that involves recruitment of inflammatory cells, production of prostanoids [such as prostaglandin [E.sub.2] ([PGE.sub.2])], synthesis of cytokines [such as interleukin-6 (IL-6) and tumor necrosis factor-[alpha] (TNF-[alpha])), and activation of osteoclasts formation and differentiation (Islam et al. 2007). LPS induces production of pro-inflammatory cytokines on osteoclasts through the NF-[kappa]B pathway and the three major mitogen activated protein kinases (MAPKs): extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK), and p38 (Hotokezaka et al. 2007; Kirkwood et al. 2007; Rogers et al. 2007). In macrophages, LPS induces hypoxia-inducible factor-la (HIF-1[alpha]) protein accumulation under normoxic condition, and it has demonstrated that HIF-1[alpha] plays an essential role in the inflammatory response (Nishi et al. 2008; Tacchini et al. 2008). However, the biological effect of LPS on HIF-1[alpha] protein during inflammation of osteoclasts and/or osteoblasts has not been reported.

Many plant-derived natural products have been used in traditional medicine for the treatment of various diseases. Herba Epimedii is a traditional Chinese herbal medicine, which has been commonly used as tonic, aphrodisiac and anti-rheumatic in China for thousands of years. Its physical and functional characteristics have been thoroughly documented in the Chinese pharmacopoeia 2005. It was reported as an effective enhancer of bone healing (Qin et al. 2005) that could be prescribed for treating osteoporosis. It is known to increase the overall mineral content, therefore, to promote bone formation and to increase lumbar bone mineral density (BMD) (Zhang et al. 2009). Several compounds derived from natural products have been recently reported to possess inhibitory effects on osteoclast differentiation and function, leading to decreased bone loss in vivo. Zhang et al. reported that the flavonoids of Herba Epimedii (HEF) could concurrently improve osteogenic differentiation and inhibit the osteoclast differentiation of human bone marrow-derived MSCs (BM-MSCs) (Zhang et al. 2009).

Icariin ([C.sub.33][H.sub.40][O.sub.15]; molecular weight: 676.67), the main active flavonoid glucoside isolated from Epimedium pubescens, was found to have a therapeutic effect on osteoporosis by ovariectomy rat models and postmenopausal women (Zhang et al. 2007). Further studies demonstrated that icariin also suppressed mice osteoclast differentiation (Huang et al. 2007), but the detailed molecular mechanisms underlying these effects remain unclear. Recently, we found that icariin might exert its osteogenic effects through induction of bone morphogenic protein-2 (BMP-2) and NO synthesis, subsequently regulate Core binding factor A1/runt-related transcription factor 2 (Cbfa 1/Runx2), OPG. and RANKL genes expression (Hsieh et al. 2009). In the present study, we further examined the detailed molecular mechanisms of the effect of icariin on LPS-induced osteoysis by primary co-culture models obtained from adult female mice. Our hypothesis is that icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-[kappa]B regulated HIF-1[alpha] and [PGE.sub.2] synthesis.

Materials and methods

Chemicals and reagents

Icariin was obtained from Biotic Chemical Co., Ltd., Taipei, Taiwan. Icariin stock solutions were prepared in dimethyl sulfoxide (DMSO, Sigma Chemical, St. Louis. MO. USA) and stored at -20[degrees]C. The final concentration of DMSO used in the culture was 0.01%. Lipopolysaccharide (LPS, Sigma Chemical, St. Louis, MO, USA) was obtained from Escherichia coli 026:B6.

Cell culture: osteoblasts and osteoclasts co-culture

This study was reviewed and received prior approval of the Taipei Medical University Animal Research Committee (Affidvait of Approval of Animal Use Protocol Taipei Medical University, LAC-96-0098). For the osteoclasts co-culture cell. 8-month old female Imprinting Control Region (ICR) mice were killed by [CO.sub.2] asphyxia, then the femur and tibia bones were dissected asepti-cally. The marrow cells were flushed out with a-Minimum Essential Medium ([alpha]-MEM, GibcoBRL; Grand Island, NY, USA). The bones (without marrow) were cut into pieces (less than 1 mm in diameter), digested with 0.2% collagenase for 1 h to harvest the mature osteoblast cells. For the osteoclast co-culture cells model, the cells were seeded into 6-wells plates at a density of osteoblasts 2 x [10.sup.5] cells and bone marrow cells 1.4 x [10.sup.7] cells/well in [alpha]-MEM supplemented with 10% fetal bovine serum (FBS, Sigma Chemical, St. Louis, MO, USA), antibiotics (100U/ml of penicillin G and streptomycin 100 [micro]g/ml. GibcoBRL; Grand Island, NY, USA). 28 nM ascorbic acid 2-phosphate (Sigma Chemical, St. Louis, MO. USA), and lOnM 1[alpha], 25[(OH).sub.2] Vit [D.sub.3] (Sigma Chemical, St. Louis, MO, USA) in a humidified atmosphere with 5% [CO.sub.2] and 95% air at 37 [degrees]C

Osteoclasts validation and viability

Tartrate-resistant acid phosphatase (TRAP) stain

Cells were treated with different concentration of icariin in 90% [alpha]-MEM with 1% FBS, 28 nM ascorbic acid 2-phosphate, 10nM 1[alpha], 25[(OH).sub.2] Vit [D.sub.3], and 1 [micro]g/ml LPS with/or without icariin. After 6 days incubation, cells were fixed in 4% formaldehyde in PBS for 20min, stained for TRAP activity using a commercialized kit (Sigma Chemical, St. Louis, MO, USA) according to the manufacturer's instruction. After incubation at room temperature for 1 h, cells were washed with distilled water for three times. Red color-TRAP-positive cells were observed and photographed for further analysis.

Acid phosphatase (ACP) and TRAP activity

TRAP is a maker of osteoclast differentiation, the effects of icariin on osteoclasts differentiation can be observed by the changes in TRAP activity. In this study, osteoclasts co-culture was treated with different concentration of icariin in [alpha]-MEM with 1% FBS, 28 nM ascorbic acid 2-phosphate, 10 nM 1[alpha], 25[(OH).sub.2] Vit [D.sub.3], and 1 [micro]g/ml LPS with/or without icariin. The treatments were exchanged every 3 days. The ACP and TRAP assays for osteoclasts differentiation were performed in 3rd, 6th, and 9th day of culture. Briefly, cells were lysis by 0.2% Trion-X 100 in PBS, the collections was measured with a commercially available ACP and TRAP assays kit (Acid Phosphatase Liquicolor: Human Gesellschaft; Germany) by ELISA reader (Spectra max 340, molecular Devices; CA, USA) at wavelength of 405 nm. The measured ACP/TRAP activities were then normalized with their specific protein titer.

3-[4,5-Dimethylthiazol]-2,5-diphenylterazolium bromide assay (MTT assay)

Co-culture cells were treated with [10.sup.-8] M icariin in a-MEM with 1 % FBS, 28 nM ascorbic acid 2-phosphate. 10 nM 1[alpha], 25[(OH).sub.2] Vit [D.sub.3], and 1 [micro]g/ml LPS with/or without icariin. The MTT (Sigma Co., St. Louis. MO. USA) assay for cell viability was performed on 6th day of culture. During the experiment, the treatments (including medium and medication) were changed every 3 days and that fresh icariin was added at each medium change. The mitochondrial activity of the cells after icariin treatments was determined by colorimetric assay. Briefly, at the end of each time interval, the supernatant was removed, and 25 [micro]l per well of 2.5 mg/ml MTT solution was added and the wells were incubated at 37 [degrees]C for 3.5 h to allow the formation of formazan crystal. Then, the supernatant was removed and acid-ethanol (200 [micro]l of 0.04 N HC1 in ethanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After a few minutes at room temperature to ensure that all crystals were totally dissolved, the plates were read on ELISA reader at wavelength of 570 nm.

In vitro bone resorption assay

The co-culture cells were seeded on BD Biocoat[TM] Osteologic[TM] Multitest Slides (BD Biosciences. San Jose, CA, USA) at a density of 2 x [10.sup.4] osteoblasts cells and 1.4 x [10.sup.6] bone marrow cells in each well. After 6 days pre-incubation, osteoclast cells were treated with icariin or LPS in a-MEM with 1% FBS, 28 nM ascorbic acid 2-phosphate and 10 nM 1[alpha], 25[(OH).sub.2] Vit [D.sub.3]. At the end of test, Osteologic[TM] slides were bleached to remove cells and then stained using the von Kossa stain to visualize the remaining mineral substrate, then washed three times with distilled water. Resorbed areas showed as clear areas against the contrasting brown to black background. The area of resorption was measured by image analysis using Multi Gauge 3.0 analysis software (FUJIFILM, Tokyo, Japan) (Voronov et al.2005).

Gene expression and signal pathways

RNA extraction, cDNA synthesis, and quantitative real-time PCR

In this study, RNA isolation was performed at the pre-set time point (1st, 3rd, and 6th day) after icariin or LPS addition. The internal control gene used was [beta]-microglobulin. Briefly, osteoclasts co-culture were washed with PBS and then total RNA was extracted with TRIZOL (Invitrogen Corporation, Carlsbad. CA. USA). For cDNA synthesis, total RNA (2 [micro]g) was used to generate cDNA in each sample by using Superscript II reverse transcriptase (Invitrogen Corporation, Carlsbad. CA. USA) with 0.5 [micro]g oligo(dT) primers. The [OD.sub.260] of prepared total RNA was measured with spectrophotometer and then used to determine RNA concentration. One hundred nanograms of total RNA from each sample was applied to 1% agarose gels to determine the purity and quantity of RNA by ethid-ium bromide stain. The intensity of 28S rRNA bands, determined by densitometry, was used to control RNA amounts used in realtime PCR analysis. One microliter of cDNA was amplified in each PCR reaction mixture containing 200 nM of sense and antisense primers of selected genes. The primers of internal standard gene ([beta]-microglobulin)and the genes being analyzed were listed in Table 1. PCR amplification was performed on Light Cycler FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany). The amplification was performed in a Roche LightCycler 2.0 instrument under the following condition: initial denaturation at 95 [degrees]C for 10 min, 40 cycles of amplification at 95 [degrees]C for 5 s, annealing at 55 [degrees]C for 5 s. and then extension at 72 [degrees]C for 8 s.
Table 1
Primers sequences for reverse transcription of semi-quantitative real
time PCR.

       Gene name          Primer (sense primer) (antisense  Gene PCR
  (Gene Bank number)                  primer)                product

[beta]-microglobulin      5'-TTCAGTGTGAGCCAGGATATAGAAA-3'     153
(NM.009735.3)             5'-GAACCCGAACATACTGAACTGCr-3'

OPG (U_94331)             5'-GCTGAGTGTTTTGGTGGACAGTT-3'       101

RANKL (AF_053713)         5'-ACATCGGGAAGCGTACCTACA-3'         102

IL-6 (X54542)             5'-CAAGTCGGAGGCrTAAAC-3'            101

TNF-[alpha] (NM013693.1)  5'-TCTCTACCTT-GTTGCCTCCTCTTTT-3'    150

Measurement of IL-6, TNF-[alpha] and prostaglandin [E.sub.2] ([PCE.sub.2]) by enzyme-linked immunosorbent assay (ELISA) and western blotting analysis

Osteoclasts co-culture was treated with a-MEM with 1% FBS. 28 nM ascorbic acid 2-phosphate. 10 nM 1[alpha], 25[(OH).sub.2] Vit [D.sub.3]. and 1 [micro]g/ml LPS with/or without icariin for 3 days. The super-natants were collected and the level of IL-6. TNF-[alpha] and [PGE.sub.2] was determined using an ELISA kit (for IL-6 and TNF-[alpha]: Bender MedSystems. Vienna. Austria; for [PGE.sub.2]: R&D Systems. Inc., Minneapolis. MN, USA). Cells were lysed in radioimmunoprecip-itation assay buffer (RIPA buffer. Cell Signaling Technology, Inc., Danvers. MA. USA) (20 mM Tris-HCl. pH 7.5; 150 mM NaCl. 1 mM [Na.sub.2]EDTA. 1 mM EGTA, 1% NP-40.1% sodium deoxycholate. 2.5 mM sodium pyrophosphate, 1 mM [beta]-glycerophosphate. 1 mM [Na.sub.3] [VO.sub.4]. 1 [micro]g/ml leupeptin, and 1 mM PMSF), and protein concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories. CA. USA) as recommended by the manufacturer. Then, 30-50 [micro]g of protein was loaded from each sample in 8 and 10% poly-acrylamide gels. Proteins were separated electrophoretically and transferred to nitrocellulose membranes (Millipore, Bedford. MA. USA) using the Bio-Rad MiniGel system with 400 mA for 1.5 h on ice. For immunoblotting the membranes were blocked with 5% non-fat dried milk in Tris-buffered saline (25 mM Tris base. pH 7.5 containing 137 mM NaCl) with 0.1% Tween-20 (TBST) for 1 h. Immunostaining was performed with rabbit anti-phospho-p38, anti-phospho-ERK1/2. anti-phospho-JNK, anti-phospho-I[kappa]Ba. [beta]-actin (Cell Signaling Technology Inc., Danvers. MA, USA), COX-2 (Epitomics, Burlingame, CA, USA) and HIF-1[alpha] (Santa Cruz Biotechnology Inc., Santa Cruz, USA) antibodies diluted 1:500 or 1:1000 at 4[degrees]C overnight After washing with Tris-buffered saline Tween-20 (TBST), the blots were incubated with secondary horseradish peroxidase (HRP) conjugated antibody (Cell Signaling Technology Inc., Danvers. MA. USA) diluted 1:2000 for 1 h at room temperature. Blots were washed, the signal determined by enhanced chemilu-minescence reagents (Millipore, Bedford, MA. USA). All analyzed were using Luminescence Imaging System (LAS-4000mini, FUJI-FILM. Tokyo. Japan). Quantification of protein expression was accomplished by using Multi Gauge 3.0 analysis software (FUJIFILM, Tokyo, Japan). Results were expressed as the ratio between target protein and [beta]-actin bands.

Statistical analysis

Results were expressed as mean [+ or -] standard deviation of these experiments and statistically analyzed by two-way ANOVA. Statistical significance by Dunnett's test was set at p < 0.05 between the means of the control and test groups. Each experiment was performed more than three times, and results from one representative experiment were shown.


Low concentration icariin inhibits LPS-induced osteoclastogenesis, but not cell viability

The effect of icariin on the ACP and TRAP activity of osteoclasts co-culture was examined at icariin concentrations of 0, [10.sup.-9], [10.sup.-8], [10.sup.-7], and [10.sup.-6] M for 3 days of culture (n = 5. Fig. 1A and B). The results showed that ACP and TRAP activities were significantly affected by icariin treatment (p < 0.05). We chose [10.sup.-8] M icariin for the further study because maximal inhibition of osteoclasts co-culture activity was observed at this concentration. In the presence of [10.sup.-8] M icariin, the ACP and TRAP activity was significantly decreased during the experimental culture period (n = 6. Fig. 1C and D).


Lipopolysaccharide can enhance ACP/TRAP synthesis, while icariin can inhibit LPS-induced osteoclastogenesis through suppressing the synthesis of ACP and TRAP (n =4, Fig. 2A and B). The icariin effect of LPS-induced osteoclastogenesis was quantified by bone resorption area assays. The results show that icariin can significantly reduce the LPS-induced bone resorption (n = 8, Fig. 2C).To determine whether the reduction in osteoclastogenesis by icariin could be due to decreased osteoclasts viability, MTT assay for cell viability was performed. The results showed that icariin did not show any cytotoxicity at tested concentrations (i.e., [10.sup.-8] M icariin; n = 6. Fig. 2D); this indicated that the suppression of LPS-induced osteoclastogenesis by icariin was not due to toxic effect of icariin on osteoclasts co-culture.


Icariin scavenge LPS-induced RANKL up-regulation and OPC down-regulation

In this test, total mRNA was extracted after osteoclasts co-culture was treated with [10.sup.-8] M icariin for 1, 3, 6 days, then quantitative real time-PCR was performed on RANKL and OPG. The starting RNA was normalized to the internal control ([beta]-microglobulin). The results indicated that icariin (in the absence of LPS) significantly down-regulated RANKL expression and up-regulated OPG expression; while LPS significantly down-regulated OPG expression and up-regulated RANKL expression and the LPS-induced osteoclastogenesis effect can be reversed by icariin (Fig. 3). Icariin scavenged LPS-induced osteoclastogenesis via RANKL/OPG pathway.


Icariin inhibits LPS-induced pro-inflammatory cytokines synthesis

In this experiment, the expression of LPS-mediated IL-6 and TNF-[alpha] protein synthesis and genes expression were enhanced by LPS (Fig. 4). Co-treatment with LPS and icariin ([10.sup.-8] M) can reduce LPS-mediated IL-6 protein on the 3rd day down to 90% of the positive control (n = 4, Fig. 4A), and decrease LPS-mediated IL-6 mRNA down to 86% and 28% of the positive control on the 1st and 3rd day, respectively (n = 3. Fig. 4B). Similar results were observed for TNF-[alpha] synthesis, where co-treatment with icariin can diminish its protein production to 90% of the positive control on the 3rd day (n = 4, Fig. 4C), and down-regulate TNF-[alpha] mRNA expression down to 89% and 57% of the positive control on the 1 st and 3rd day, respectively (n = 3, Fig. 4D). When osteoclasts co-culture was treated with [10.sup.-8] M icariin, the synthesis of LPS-induced IL-6 and TNF-[alpha] protein and mRNA expression were inhibited.


In this study, LPS-mediated COX-2 gene up-regulation was significantly inhibited by icariin (n-4, Fig. 5A and B). In either osteoblasts or osteoclast co-cultures, icariin also significantly suppressed the LPS-induced [PGE.sub.2] production (n = 4, Fig. 5C and D). Taken together, these results suggest that icariin suppresses LPS-induced [PGE.sub.2] production by inhibiting COX-2 activity in both osteoblasts and osteoclast co-cultures. Icariin can suppress LPS-mediated activation of the MAPKs and NF-[kappa]B pathway of osteoclasts


In this study, we used two kinds of cells (osteoblasts and osteoclasts co-culture) to investigate the effects of icariin on LPS-induced activation of MAPKs and I[kappa]B[alpha]. To this end, we treated freshly isolated osteoblasts or osteoclasts co-culture with LPS for 0, 5, 15, 30, and 60 min in the absence or presence of icariin, and the activation of the NF-[kappa]B, ERK1/2, p38, and JNK pathways was determined (Fig. 6A and B). This set of assays was also repeated with osteoclasts co-culture treated with LPS and/or icariin for 60 min (Fig. 6C).


On osteoclasts co-culture, icariin inhibited the LPS-mediated activation of the I[kappa]B[alpha], ERK1/2. p38. and JNK pathways at 60 min (Fig. 6A and C). For osteoblasts, icariin significantly decreased the LPS-mediated ERK1/2 activation at 5 min and I[kappa]B[alpha] activation at 60 min, and stimulated p38 phosphorylation at 60 min; while no effect was observed on JNK activation (Fig. 6B). This fact reflected that icariin was able to transiently reduce LPS-dependent activation of the I[kappa]B and ERK1/2 in both osteoblasts and osteoclasts. This fact reflected that icariin inhibited LPS-induced osteoclastogenesis by blocking I[kappa]B[alpha] and ERK1/2 activation on both osteoblasts and osteoclasts. Also, we found that icariin suppressed LPS-mediated activation of the p38 and JNK pathway on osteoclasts co-culture, but not on osteoblasts. This reflected that the inhibitory effect of icariin on LPS-induced osteoclastogenesis could be an indirect effect by suppressing the osteoclastogenesis-supporting activity of osteoblasts or, alternatively, be a direct effect on LPS-stimulated osteoclasts differentiation programs. We also suggested that there were different mechanisms for osteoblasts and osteoclasts for the inhibitory effect of icariin on LPS-induced osteoclastogenesis.

Icariin restrain osteoclastogenesis through suppression of LPS-regulated HIF-1[alpha] expression under normoxia condition

In this study, we found that LPS-treated osteoclasts had larger cell size than control and the treatment of icariin can reduce the osteoclasts cell size (Fig. 2B). In osteoclasts, icariin can inhibit LPS-mediated HIF-1[alpha] up-regulation at 8h (Fig. 7A); however, LPS did not had any effect on the HIF-1[alpha] production on osteoblasts (Fig. 7B). These data indicated that icariin restrain osteoclastogenesis through suppression of LPS-regulated HIF-1[alpha] expression from osteoclasts (but not osteoblasts) under normoxia condition.



The major modalities currently used in osteoporosis treatment primarily include estrogen replacement therapy, bisphospho-nates (e.g., alendronate, risedronate), selective estrogen receptor modulators (SERM), and calcitonin. However, such therapies are associated with adverse effects, including breast cancer, endometriosis, thromboembolism, hypercalcemia. GI problems, and hypertension (Lloyd 1998; Rodan and Martin 2000; O'Regan and Gradishar 2001; Body 2002; Watts 2003). Epimedium-derived flavonoids such as icariin, genistein, and daidzein are proved to be able to inhibit bone resorption, stimulate bone formation, and prevent ovariectomy-induced osteoporosis (Huang et al. 2007; Zhang et al. 2007). Icariin is the major flavonoids found in Herba epimedii (77%), and it has been reported to have a potential anabolic effect on bone (Hsieh et al. 2009). The molecular mechanisms regarding icariin function on osteogenesis is via the enhanced expression of BMP-2 and core-binding factor 1 (cbfa1) (Hsieh et al. 2009; Zhao et al. 2008). On osteoclasts, it has been reported that icariin can suppress osteoclastic differentiation and inhibit TRAP and RANK expressions (Chen et al. 2007; Huang et al. 2007). In our institute, we also noted that icariin can up-regulate OPG mRNA expression and down-regulate RANKL mRNA expression of osteoblasts (Hsieh et al. 2009). In this study, we examined the effects of icariin on the activity and differentiation of adult female mice osteoclasts. We found that icariin acted on osteoclasts to suppress osteoclasts cell differentiation and ACP and TRAP activities (Fig. 1).

LPS is a primary cell wall component of Gram negative bacteria, and LPS stimulation leads to the intracellular induction of p38, JNK phosphorylation and NF-[kappa]B in macrophages and monocytes and also the secretion of various inflammatory cell stimulating agents (Kim et al. 2006). Production of inflammatory factors induces pre-osteoclast fusion, supports the survival of mature osteoclasts, and stimulates osteoclastic bone resorption (Suda et al. 2002). In the model of LPS-induced osteoclastogenesis, we observed that icariin reduced LPS-induced ACP and TRAP activities, inhibited bone resorption, while with no effect on osteoclasts viability (Fig. 2). This suggested that the osteoclastogenesis suppression by icariin was not due to toxic effect on osteoclasts. The level of RANKL and OPG are critical in the regulation of the osteoclasts formation and differentiation (Chakravarti et al. 2009). Our study showed that icariin can significantly down-regulate LPS-mediated RANKL expression and up-regulate LPS-suppressed OPG expression (Fig. 3).

Many cytokines are involved in LPS-mediated osteoclasts formation. LPS stimulates the target cells to produce proinflammatory cytokines such as IL-6 and TNF-[alpha], which have been shown to support the survival of osteoclasts (Itoh et al. 2003). LPS stimulates osteoclast-mediated bone resorption through COX-2 induction and [PGE.sub.2] production (Sakuma et al. 2000). Enhanced [PGE.sub.2] release after LPS treatment can promote osteoclast differentiation (Miyaura et al. 2003). Previous reports have indicated that the production of [PGE.sub.2] after LPS treatment is dependent on the elevation of COX-2 (Kiji et al. 2007). There are two types of COX enzymes encoded by separate genes and are differentially expressed. Although both constitutive COX-1 and inducible COX-2 are expressed on osteoblasts or osteoclasts, the COX-2 is responsible for [PGE.sub.2] synthesis on osteoblasts or osteoclasts after LPS stimulation (Coon et al. 2007; Kaneko et al. 2007). There are two kinds of cell on primary osteoclasts co-culture, one is mature osteoclasts, and the other is osteoblasts. In order to differentiate the specific response from each cell type, we used both kinds of cells, i.e., osteoblast cells and osteoclasts, to test the effects of icariin on LPS-mediated COX-2 induction and [PGE.sub.2] production. Our results showed that icariin diminished LPS-induced IL-6 and TNF-[alpha] mRNA and protein expression on osteoclasts (Fig. 4); we also found that icariin decreased LPS-mediated [PGE.sub.2] production by inhibiting COX-2 on both cell types (Fig. 5).

In previous study, LPS has been shown to promote osteoclas-togenesis by activating various intracellular signaling pathways including NF-[kappa]B, JNK, ERK1/2, and p38. Binding of LPS to toll-like receptor 4 activates NF-[kappa]B and MAPKs, which induces production of pro-inflammatory cytokines such as IL-6 and TNF-[alpha] (Liu et al. 2009; Merck et al. 2005). Subsequently, the pro-inflammatory cytokines and [PGE.sub.2] abundant in sites of inflammation promote osteoclasts differentiation and activation (Caetano-Lopes et al. 2009; Lorenzo et al. 2008; Nakashima and Takayanagi 2009). The MAPK group of enzymes selectively phosphorylates serine and threonine residues in response to extracellular stimuli and transmits the stimuli from the cell surface to the nucleus. MAPK is primarily composed of JNK, ERK, and p38 in mammalian cells. RANKL and RANK receptor binding expressed in osteoclast precursors provides a link between distinct signaling molecules such as JNK, ERK, and p38 MAPK. The activation of signaling molecules induces transcription factors such as NF-[kappa]B, NFATc1, and AP-1 that are essential for osteoclasts differentiation (Takayanagi 2007), and p38 MAPK signaling is particularly crucial in the early stages of osteoclasts differentiation as it promotes the activity of micropthalmia-associated transcription factor (MITF) and TRAP expression. Inhibition of p38 MAPK with SB203580 has a negative effect on osteoclasts formation (Matsumoto et al. 2000).

NF-[kappa]B is an important signal mediator for inflammatory and immune reactions and is a major transcription factor for RANKL-activated osteoclastogenesis Qimi et al. 2004). Activation of NF-[kappa]B requires degradation of I[kappa]B, an inhibitory protein that forms a complex with NF-[kappa]B dimmers (Liu et al. 2009). I-[kappa]B is attached to NF-kB preventing it from migrating into the nucleus, and phosphorylation with I-[kappa]B kinase (IKK) separates the two proteins. Subsequent ubiq-uitination and proteosome degradation of I-[kappa]B allows the transfer of NF-[kappa]B into the nucleus and transcription of the target gene (Hayden and Ghosh 2004). Based on these observations, we reasoned that icariin exerted a potent inhibitory effect on LPS-induced osteoclastogenesis by targeting the intracellular signaling pathways. The LPS promote osteoclastogenesis via activating NF-[kappa]B, ERK1/2, JNK, and p38 signaling pathways (Carlson et al. 2009). In this study, we found that icariin suppresses LPS-mediated activation of the p38 and JNK on osteoclasts, but not on osteoblasts, supporting the idea that icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway (Fig. 6).

Hypoxia regulates the expression of many genes via the [alpha][beta] heterodimeric transcription factor HIF, and it includes pathways encompassing angiogenesis, apoptosis, glycolysis and pH regulation to process central to cell survival and expansion in an oxygen-deficient environment (Arany et al. 2008; Rius et al. 2008). Hypoxia inducible factor-1[alpha] (HIF-1[alpha]) can be induced by LPS in differentiated macrophages under condition of normoxia (Nishi et al. 2008). HIF-la plays an important role in the regulation of osteoclasts cell size, and has recently emerged as central regulators of bone biology (Bozec et al. 2008). Activation of HIF-1[alpha] enhances osteogenesis on osteoblasts, and also increases osteoclasts differentiation and bone resorption (Knowles and Athanasou 2009). LPS can induces HIF-1[alpha] activation on myeloid cell, monocyte, and the differentiated macrophage under normoxic condition (Nishi et al. 2008; Tacchini et al. 2008); however the effects of HIF-la induced by LPS on osetoclasts or osteoblasts are still not reported. In this study, we found that LPS can induce HIF-la expression on osteoclasts, but not osteoblasts (Fig. 7). This data suggested that icariin reduced bone resorption through suppression of LPS-regulated HIF-1[alpha] expression of osteoclasts under normoxia condition.


In our study, the effects of icariin on adult female mice osteoclasts were investigated. We demonstrate that low dose icariin can inhibits LPS-induced osteoclastogenesis without sacrifice cell viability. Icariin can inhibit LPS-induced pro-inflammatory cytokines synthesis and can scavenge LPS-induced RANKL up-regulation and OPG down-regulation. Icariin decreased LPS-mediated [PGE.sub.2] production by inhibiting COX-2 synthesis of osteoblasts and osteoclasts. In osteoclasts, icariin suppressed LPS-mediated activation of the I[kappa]B, JNK. ERK1/2, p38, and HIF-la pathways; while in osteoblasts, only IkB and ERK1/2 pathways were involved (Fig. 8). Our results indicate that icariin has an in vitro inhibitory effects on osteoclasts differentiation that can prevent inflammatory bone loss. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway. However, the in vivo molecular mechanisms about the icariin function on the LPS-induced osteoclastogenesis need to be further investigated.



The authors thank the National Science Council, Taiwan, ROC for its financial support. The authors also thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital, for technical support during this study.


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Abbreviations: ACP. acid phosphatease; BMD. bone mineral density; BM-MSCs. bone marrow-derived mesenchymal stem cells; BMP-2. bone morphogenetic protein-2; Cbfal/Runx2, core binding factor Al/runt-related transcription factor 2; COX-2, cyclo-oxygenase type-2; DMSO, dimethyl sulfoxide; ELISA. enzyme-linked immunosorbent assay; ERK1/2, extracellular signal-regulated kinasel/2; FBS. fetal bovine serum; HEF. flavonoids of Herba Epimedii; HIF-1[alpha], hypoxia inducible factor-1[alpha]; ICR mice. Imprinting Control Region mice; IL-6, interleukin-6; l[kappa]B[alpha], inhibitor of the nuclear transcription factor NF-[kappa]B; JNK. c-Jun N-terminal kinases; LPS, lipopolysaccharide; MAPKs, mitogen-activated protein kinases: M-CSF. macrophage/monocyte colony-stimulating factor; MTT assay, 3-[4,5-dimethylthiazol]-2,5-diphenylterazolium bromide assay; NF-[kappa]B. nuclear factor of kappa light polypeptide gene enhancer in B-cells: NO, nitric oxide; OPG, osteoprotegerin; p38, p38 MAPK; PCR, polymerase chain reaction; [PGE.sub.2], prostaglandin E2; RANKL, receptor activator of NF-[kappa]B ligand; TNF-[alpha], tumor necrosis factor-[alpha]; TRAP, tatrate resistances acid phosphatease: [alpha]-MEM. [alpha]-minimum essential medium.

* Corresponding author at: No. 25. Lane 442, Sec. 1. Jingguo Rd., Hsinchu City 30059. Taiwan, ROC. Tel.: +886 2 27361661 x3229/3 5326151: fax: +886 2 27390500.

** Corresponding author at: School of Pharmacy, College of Pharmacy. Taipei Medical University, No. 250. Wu-Shin Street, Taipei City 11031. Taiwan. ROC. Tel.: +886 2 27361661x6129: fax: +886 2 27361661x6129.

E-mail addresses: (S.-Y. Sheu)., (J.-S. Sun).

Tsai-Pei Hsieh (a), Shiow-Yunn Sheu (a), **, Jui-Sheng Sun (b), (c), *. Ming-Hong Chen (d)

(a) School of Pharmacy. College of Pharmacy. Taipei Medical University, Taiwan. ROC

(b) Graduate Institute of Clinical Medicine, Taipei Medical University. No. 250. WusingSt., Sinyi District, Taipei City 11031. Taiwan, ROC

(c) Department of Orthopedic Surgery, Hsin Chu General Hospital. Department of Health. Executive Yuan. Taiwan, ROC

(d) Institute of Biomedical Engineering, National Yang-Ming University, Taipei. Taiwan, ROC

doi: 10.1016/j.phymed.2010.04.003
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Author:Hsieh, Tsai-Pei; Sheu, Shiow-Yunn; Sun, Jui-Sheng; Chen, Ming-Hong
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Jan 15, 2011
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