Article pubs.acs.org/jnp
Lupeol Isolated from Sorbus commixta Suppresses 1α,25(OH)2D3‑Mediated Osteoclast Differentiation and Bone Loss in Vitro and in Vivo Nam Kyung Im,†,⊥ Dong-Sung Lee,‡,⊥ Seong-Ryong Lee,*,§ and Gil Saeng Jeong*,† †
College of Pharmacy, Keimyung University, Daegu 704-701, Republic of Korea College of Pharmacy, Chosun University, Dong-gu, Gwangju, 61452, Republic of Korea § Department of Pharmacology, School of Medicine and Brain Research Institute, Keimyung University, Daegu 704-701, Republic of Korea ‡
ABSTRACT: Lupeol is a lupane-type triterpene isolated from Sorbus commixta, an oriental medicine used to treat arthritis and inflammatory diseases. However, the antiosteoporotic effects of S. commixta or any of its constituents have not been studied yet. In the present study, we have examined the effect of lupeol (a major active triterpenoid isolated from S. commixta) on osteoclastogenesis and sought to elucidate its underlying molecular mechanisms. We evaluated whether lupeol antagonized osteoclast differentiation and bone resorption. Lupeol markedly inhibited osteoclast differentiation and bone resorption activity through its effects on MAP kinases and transcription factors (NF-κB, NFATc1, and c-Fos) downstream of the osteoclast differentiation factor receptor RANK. Furthermore, in vivo efficacy of lupeol was confirmed by using an animal model of hypercalcemic mediated bone loss. Taken together, lupeol showed strong inhibitory effects on osteoclastogenesis. Supplementation with S. commixta and lupeol could be beneficial for bone health or osteoclast-related diseases such as osteoporosis, Paget’s disease, osteolysis associated with periodontal disease, and multiple myeloma.
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c-Fos, nuclear factor-kappaB (NF-κB), and activator protein-1 (AP-1). These factors play an important role in osteoclast differentiation and osteoclast function.5 Calcium, vitamin D, hormone therapy, and bisphosphonates have been used for the treatment of osteoporosis. Bisphosphonates, the most common therapy for osteoporosis, are poorly absorbed from the gastrointestinal (GI) tract and have been associated with adverse GI events.6 Parathyroid hormone, a therapeutic peptide, cannot be given orally and is restricted for use for a maximum of 1 year due to the risk of developing osteosarcoma.7 In the context of the global burden of osteoporosis, alternative therapies should be developed. Interestingly, many natural products have long been used traditionally to prevent and cure bone loss.5,8−13 Thus, attention has recently been focused on searching for antiosteoporotic agents from natural sources, particularly from various edible and medicinal plants. In an attempt to develop new antiosteoporotic agents from natural sources, we have screened Sorbus commixta Hedlund for osteoclast differentiation activities. Lupeol is a major active triterpenoid isolated from S. commixta, a plant that has long been used as an traditional medicine in Korea, China, and Japan. S. commixta
one is a dynamic organ that is continuously and precisely remodeled by the combined roles of osteoblasts and osteoclasts. Bone homeostasis is maintained by the balance between the bone-forming activity of osteoblasts and the boneresorbing activity of osteoclasts. These two types of cells must simultaneously balance bone growth to achieve strength, resilience, and repair without overgrowth.1 While the balance between osteoblast and osteoclast function is maintained by bone remodeling, an excessive activity of osteoclasts and subsequent bone loss is evident in osteoporosis, Paget’s disease, osteolysis associated with periodontal disease, and multiple myeloma.2 Osteoclasts are multinucleated cells that differentiate from hematopoietic stem cells of the monocyte/ macrophage lineages. Differentiation and maturation of osteoclasts are controlled by many factors, including receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF).3 Osteoblasts express RANKL and M-CSF as the key regulators of osteoclastogenesis. These cells also express osteoprotegerin (OPG), a soluble decoy receptor for RANKL that blocks RANKL/RANK interactions. RANKL is a member of the tumor necrosis factor (TNF)-α family of cytokines that interacts with the osteoclast cell surface receptor RANK, which in turn results in the recruitment of TNF receptor-associated factors (TRAF6).4 TRAF6 induces the activation of mitogen-activated protein kinases (MAPKs) and transcription factors, including NFATc1, © XXXX American Chemical Society and American Society of Pharmacognosy
Received: December 10, 2015
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DOI: 10.1021/acs.jnatprod.5b01088 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. Structure of lupeol (A) and its effects on 1α,25(OH)2D3-induced osteoclastogenesis in mouse cocultures. Primary osteoblasts and BMM were cocultured in 24-well plates for 6 days in the presence of 1α,25(OH)2D3 (0.1 μM). After a 6-day coculture, the cells were fixed and stained for TRAP (B) and TRAP activity (D). TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts (E). Cell viability was determined by MTT assay (C). *P < 0.05 vs untreated control cells.
Figure 2. Effects of lupeol on cell viability in mouse calvarial osteoblasts. Mouse calvarial osteoblasts were cultured with lupeol at 1, 5, 10, and 20 μM for 2 days. The cell viability was determined by MTT assay (A). The ALP activity was measured on day 4, 7, and 14 after incubation of cells with or without lupeol. Mouse osteoblasts were cultured for 24 h in the presence of 1α,25(OH)2D3 (0.1 μM) and lupeol (B). Total RNA was then extracted from osteoblasts, and the expression levels of RANKL, OPG, and M-CSF mRNAs were analyzed by RT-PCR (C). Values represent the mean ± SD of data obtained from three independent experiments. #P < 0.05 vs untreated control cells. *P < 0.05 vs RANKL-treated cells.
has been reported to exhibit anti-inflammatory,14,15 antiatherosclerotic,16 antialcoholic,17 and vascular-relaxant effects,16 as well as antioxidant activity.18 Phytochemical analysis of S. commixta has yielded lupane-type triterpenes (lupeol and βsitosterol), ursane-type triterpenes (3β-acetoxy ursolic acid and ursolic acid), a lignin, namely, (−)-lyoniresinol 3-O-β-D-
xylopyranoside, and catechin-7-O-β-D-apiofuranoside.19 Lupeol also has disparate biological activities including anti-inflammatory and hepatoprotective activities.20,21 In addition, lupeol isolated from Loranthus micranthus has been shown to exhibit stimulatory effects on osteoblast differentiation as assessed by ALP assay, determination of osteogenic gene expression, and B
DOI: 10.1021/acs.jnatprod.5b01088 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 3. Effects of lupeol on actin ring formation and bone resorption. Mature osteoclasts were cultured on six-well plates with increasing concentrations of lupeol. After 48 h, the cells were fixed with paraformaldehyde and incubated with Alex Fluor 488-conjugated phalloidin. F-Actin was detected by fluoresence microscopy (100×) (A). Mature osteoclasts were cultured on dentin slices with increasing concentrations of lupeol. After 48 h, cells were removed and dentin slices were stained with Mayer’s hematoxylin to identify resorption pits by light microscopy (100×) (B). The numbers of resorption pits were counted (C). Values represent the mean ± SD of data obtained from three independent experiments. #P < 0.05 vs untreated control cells.
primary osteoblasts and BMM in 24-well plates for 6 days in the presence of 1α,25(OH)2D3. After the 6-day coculture, the cells were fixed and stained for TRAP and TRAP activity. As shown in Figure 1, lupeol was found to inhibit TRAP activity in osteoblasts stimulated with 1,25α-(OH)2D3. The inhibitory effects of lupeol on 1,25α-(OH)2D3-induced osteoclastogenesis were confirmed by the findings that the total number of TRAPpositive osteoclasts was significantly reduced by lupeol. Effects of Lupeol on the Differentiation of Osteoblast Cells. Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for assessing osteoblast activity. To investigate the effects of lupeol on bone metabolism, an osteoblast cell culture system was employed. Calvaria osteoblast cells were cultured for at least 4 days in media containing various concentrations of lupeol. The medium was exchanged every 2−3 days. The effect of lupeol on the viability of osteoblast cells was examined by the MTT assay. Incubation with lupeol for 4 days did not cause osteoblast cell death (Figure 2A). The ALP enzyme activity was measured after 4, 7, and 14 days treatment of MC3T3-E1 cells with lupeol. Treatment with lupeol did not alter the ALP activity when assessed after culturing cells for 7 and 14 days. However, incubation of cells with lupeol for 4 days decreased ALP activity at 10 or 20 μM (Figure 2B). These results indicate that lupeol does not induce osteoblast differentiation in osteoclasts. In addition, we examined the effects of various concentrations of lupeol on mouse calvarial osteoblasts cultured in the presence of 1α,25(OH)2D3 . Bone resorption requires numerous cytokines released from supporting cells such as osteoblasts and stromal cells to stimulate differentiation and maturation of osteoclasts, the only cell type capable of resorbing mineralized bone. Among the cytokines, RANKL is a key osteoclastogenic
BMP2 up-regulation. However, the antiosteoporotic effects and mechanisms of S. commixta and lupeol have not previously been studied especially in both in vitro and in vivo models.22 In the present study, we have examined the effect of lupeol on osteoclastogenesis and sought to elucidate its underlying molecular mechanisms.
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RESULTS AND DISCUSSION Effects of Lupeol in 1α,25-(OH)2D3-Induced Osteoclast Differentiation in Mouse Cocultured Primary Osteoblasts and BMM. Plant polyphenols, such as flavonoids, terpenoids, anthocyanins, and steroids, have been reported to inhibit osteoclastogenesis and prevent osteoporosis.23 A wide range of epidemiological studies have found a positive correlation between phytochemical consumption and the bone marrow density.24−26 From a preliminary screening of 70 natural products to suppress RANKL-induced tartrateresistant acid phosphatase (TRAP) activity in bone-marrowderived macrophages (BMM), S. commixta was found to be very potent (data not shown), and we have subsequently identified the triterpenoid lupeol as a major active constituent. As shown in Figure 1A, the chemical structure of lupeol was identified by 1H and 13C NMR. Next, we tested the effects of lupeol on 1α,25(OH)2D3-induced osteoclastogenesis in mouse cocultures. During osteoporosis, osteoblast cells migrate into the resorbing area of bone where osteoclasts cells are differentiated. This osteoblast migration is catalyzed by TRAP enzyme, which is induced by cytokines.4 We employed a coculture model comprising osteoblast cells with mouse bone marrow macrophages, a precursor of mature osteoclasts, followed by treatment with 1,25α-(OH)2D3. We cocultured C
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Figure 4. Effects of lupeol on RANKL-induced MAPKs signaling pathways. Mouse BMM cells were prepared from bone marrow cultures treated with M-CSF. BMM cells were treated with lupeol and RANKL for 15 min. The levels of phosphorylated and nonphosphorylated ERK, JNK, and p38 MAPK were determined by Western blotting. *P < 0.05 vs RANKL-treated cells.
Figure 5. Effects of lupeol on RANKL-induced activation of NF-κB, c-Fos, and NFATc1. Mouse BMM cells were prepared from bone marrow cultures treated with M-CSF. BMM cells were treated with lupeol and RANKL for 30 min. The levels of phosphorylated IκBα and NFκB were determined by Western blotting (A). BMM cells were treated with lupeol and RANKL for 24 h. The levels of c-fos and NFATc1 were determined by Western blotting (B). *P < 0.05 vs RANKL-treated cells.
resorption of the bone matrix. Mature osteoclasts undergo morphologic and functional polarization and begin to resorb the mineralized bone surface. Osteoclasts form a sealing zone by generating a ring structure made of F-actin.27−29 Staining Factin with Alexa Fluor 488-conjugated phalloidin enabled visualization of the actin ring in mature osteoclasts. When mature osteoclasts obtained from coculturing mouse calvaria
protein that directly binds to its cognate receptor, RANK, located on osteoclast precursor cells.3−5 As shown in Figure 2C, lupeol attenuated the RANKL mRNA expression in 1α,25(OH)2D3-induced mouse calvarial osteoblasts, but did not affect OPG or M-CSF mRNA expression. Effects of Lupeol on Actin Ring Formation and Bone Resorption. Osteoclast maturation plays a key role in the D
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Figure 6. Effects of lupeol evaluated by histological analysis and the body constitution of 1α,25(OH)2D3-treated mice. Mice were injected daily with PBS or lupeol at 5, 25, 50, and 100 μg/kg/day for 5 days and were sacrificed the following day (A). Histological sections of femur from these mice were stained for TRAP (B). Histomorphometric analysis of osteoclast numbers (C). Data were expressed as mean ± SD (n = 10/group). *P < 0.05 vs 1α,25(OH)2D3-treated groups.
concentration-dependent manner (Figure 4) without JNK. It has been known that the NF-κB transcription factor plays an important role in osteoclast differentiation. The phosphorylation-dependent degradation of IκBα, a cytosol JNK repressor of NF-κB, and subsequent nuclear translocation and phosphorylation of p65 are the key events in RANKL-induced activation of NF-κB.9,31,32 As shown in Figure 5A, treatment of BMMs with lupeol attenuated RANKL-induced phosphorylation of IκBα and p65 as well as the nuclear localization of p65. In addition, lupeol diminished RANKL-induced expression of cFos, a component of AP-1, and NFAT-c1 in BMM cells (Figure 5B). Thus, our findings that lupeol dampened the RANKLinduced phosphorylation of IκBα and p65 as well as the nuclear localization of p65 suggest that lupeol inhibited osteoclastogenesis partly by blocking NF-κB signaling. Recent studies have reported that MAP kinases are important for the induction of cFos and NFATc1 during osteoclast differentiation.1,4,33,34 Therefore, lupeol could inhibit the expression of c-Fos and NFATc1 through its suppression of RANKL-induced phosphorylation of MAP kinases in BMM cells. Effect of Lupeol on 1α,25-(OH)2D3-Induced Bone Loss Model. Vitamin D maintains serum calcium concentrations at a physiologically acceptable level to maximize a wide variety of metabolic functions, signal transduction, and neuromuscular activity.31,32 Excessive administration of vitamin D results in hypercalcemia, i.e., elevated calcium levels in the blood, and hypercalciuria, which may cause nephrolithiasis or renal calcinosis in the long term. These disorders are related to high bone-turnover rates. Both vitamin D2 and D3 undergo two
osteoblast and BMM cells in the presence of PGE2 and 1α,25(OH)2D3 on a collagen matrix were incubated with lupeol, there was remarkable disruption of the actin rings, as revealed by staining cells with Alexa Fluor 488-conjugated phalloidin and F-actin (Figure 3A). The actin rings in lupeol-treated mature osteoclasts appeared to have a loose and fuzzy structure. Furthermore, mature osteoclasts obtained as described above were replated on dentin slices and cultured in the presence or absence of lupeol to examine its effect on resorption pit formation. At a concentration of 20 μM, lupeol almost completely inhibited pit formation (Figure 3B and C), suggesting the protective effect of lupeol on bone resorption activity. Effects of Lupeol on RANKL-Induced Activation of MAPKs and Transcription Factors. To explore the mechanisms by which lupeol inhibits osteoclast differentiation, we observed that lupeol reduced protein expression levels of transcription factors c-Fos, NFATc1, and NF-κB in BMM osteoclast precursor cells stimulated with the osteoclast differentiation factor RANKL. RANKL-induced differentiation of osteoclasts involves inappropriate amplification of cell signaling mediated through MAPKs and the activation of downstream transcription factors, such as NF-κB, AP-1, and NFAT-c1.19,29,30 To gain insight into the molecular mechanisms by which lupeol inhibits osteoclast differentiation, we investigated its effect on intracellular signaling pathways in RANKL-stimulated BMMs. RANKL stimulation of BMMs for 15 min increased the phosphorylation of ERK, JNK, and p38 MAPK, which were blunted by treatment with lupeol in a E
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Figure 7. Effects of lupeol on serum levels of Ca (A), phosphate (B), ALP (C), and RANKL (D) in a murine model of 1α,25(OH)2D3-induced bone loss. Mice were injected daily with PBS or lupeol at 5, 25, 50, and 100 μg/kg/day for 5 days and were sacrificed the following day. *P < 0.05 vs 1α,25(OH)2D3-treated groups.
together, lupeol isolated from S. commixta has strong inhibitory effects on osteoclastogenesis.
hydroxylations to form the active metabolite 1,25-dihydroxyvitamin D (1α,25(OH) 2 D), termed calcitriol (1α,25(OH)2D3) when specifically referring to the synthesis from vitamin D3. Although 1α,25-(OH)2D3 is essential for calcium metabolism and bone development, hyperactivation of 1α,25(OH)2D3 receptor-mediated signaling promotes bone loss by inducing differentiation of osteoblast cells into osteoclasts.35 Therefore, we investigated the effects of lupeol on 1α,25(OH)2D3-induced bone loss in vivo. Mice were subcutaneously injected with 1α,25-(OH)2D3 and treated with or without lupeol (50 or 100 μg/kg). Control mice were left untreated or treated with lupeol (50 or 100 μg/kg) alone. Isolation and subsequent staining of femur bones with hematoxylin and TRAP revealed that treatment with lupeol attenuated osteoclast differentiation as assessed by counting TRAP-positive osteoclasts (Figure 6). While 1α,25-(OH)2D3 challenge increased the serum levels of calcium (Figure 7A), phosphate (Figure 7B), RANKL (Figure 7C), and OPG (Figure 7D), treatment with lupeol only reduced 1α,25-(OH)2D3-induced RANKL production, but did not affect the levels of calcium, phosphate, or OPG. The lowering of serum RANKL levels by lupeol in hypercalcemic mice suggests that lupeol prevents bone loss by interfering with RANKL-induced signal transduction pathways. Although vitamin D is essential for bone mineralization, excessive vitamin D3 causes resorption. Our findings that treatment with lupeol diminished TRAP activity, actin ring formation, and the number of resorption pits in mouse bone marrow cells as well as in mouse femur bone in vivo indicate the strong inhibitory effect of lupeol on osteoclastogenesis. In summary, the present study demonstrates that lupeol isolated a major active triterpenoid from S. commixta blocks RANKL-induced osteoclast differentiation and bone resorption activity through its effects on MAP kinases and transcription factors (NF-κB, NFATc1, and c-Fos) downstream of the osteoclast differentiation factor receptor RANK. Furthermore, in vivo efficacy of lupeol was confirmed by using an animal model of hypercalcemic mediated bone loss (Figure 8). Taken
Figure 8. Proposed scheme of lupeol activities in osteoclastogenesis. Model showing the mechanisms of the antiosteoporotic effect of lupeol.
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EXPERIMENTAL SECTION
General Experimental Procedures. NMR spectra were recorded using a JEOL JNM ECP-400 spectrometer (500 MHz for 1H and 100 MHz for 13C). TLC was performed on Kieselgel 60 F254 (1.05715; Merck Millipore, Darmstadt, Germany) or RP-18 F254s (Merck Millipore, Darmstadt, Germany) plates. Column chromatography was performed on silica gel (Kieselgel 60, 70−230 mesh and 230−400 mesh, Merck Millipore, Darmstadt, Germany) and YMC octadecylfunctionalized silica gel (C18). HPLC separations were performed on a Synergi semiprep-C18 column (21.2 × 150 mm; 4 μm particle size; 80 Å pore size, 5 mL/min). Compounds were detected by UV absorption at 210 and 254 nm. Plant Materials and Isolation of Lupeol. The stem bark of Sorbus commixta Hedlund (Rosaceae) was purchased from Yangnyeong herbal medicine market in Korea in November 2013. A F
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extracted from the cells using TRIzol solution (Life Technologies Inc., Grand Island, NY, USA). First-strand cDNA was synthesized from the total RNA extracts with random primers and subjected to PCR amplification with Ex Taq polymerase (Takara Biochemicals, Shiga, Japan) using specific PCR primers: mouse RANKL, 5′-CGC TCT GTT CCT GTA CTT TCG AGC G-3′ (forward) and 5′-TCG TGC TCC CTC CTT TCA TCA GGT T-3′ (reverse); mouse OPG, 5′TGG AGA TCG AAT TCT GCT TG-3′ (forward) and 5′-TCA AGT GCT TGA GGG CAT AC-3′ (reverse); mouse M-CSF, 5′-GAG AAG ACT GAT GGT ACA TCC-3′ (forward) and 5′-CTA TAC TGG CAG TTC CAC C-3′ (reverse); mouse GAPDH, 5′-ACC ACA GTC CAT GCC ATC AC-3′ (forward) and 5′-TCC ACC ACC CTG TTG CTG TA-3′ (reverse). The PCR products were separated by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining and UV light illumination. The sizes of the PCR products for mouse RANKL, OPG, M-CSF, and GAPDH were 587, 720, 516, and 452 bp, respectively. Osteoclast Differentiation. The protocol employed was a coculture system of mouse calvaria osteoblasts and BMM cells stimulated with various osteotropic factors, such as active vitamin D (1α,25-(OH)2D3) and PGE2. For primary cell cultures, mouse bone marrow cells (BMCs) were isolated from the tibiae of 4- to 6-week-old ICR male mice. Isolated BMCs were cultured in α-minimal essential medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL streptomycin with the addition of 50 ng/ mL macrophage stimulating factor. After 16 to 20 h of culture, adherent cells were used as BMM cells after washing out the nonadherents ones. Bone marrow cells (1 × 107 cells) were cocultured with isolated mouse calvariae osteoblasts (1 × 106 cells) in α-MEM containing 10% FBS with 1α,25-(OH)2D3 (0.1 μM) or PGE2 (10 μM) for 6 days. Differentiation of osteoclasts was monitored using TRAP staining. TRAP-positive multinucleated cells showing more than three nuclei were counted as osteoclast cells. TRAP Staining and the Measurement of TRAP Activity. TRAP activity is regarded as an important cytochemical and biochemical marker of osteoclasts. At differentiation on day 3−4, cells were fixed with 10% formaldehyde for 10 min and ethanol− acetone (1:1) for 1 min and then stained with TRAP staining solution (Sigma-Aldrich). The images of TRAP-positive cells were captured under a microscope. In order to measure TRAP activity, cells were fixed with 10% formaldehyde for 10 min and 95% ethanol for 1 min, and then 10 mM citrate buffer (pH 4.6) containing 10 mM sodium tartrate and 5 mM p-nitrophenylphosphate (Sigma-Aldrich) was added to the dried cell-containing wells of 96-well plates. After incubation for 1 h, the reaction mixtures were transferred into new plates containing an equal volume of 0.1 N NaOH. Absorbance was measured at 405 nm, and TRAP activity was calculated as percent of control. The experiment was performed in triplicate. Actin Ring Formation and Resorption Pit Assay. In the collagen gel coculture system, mouse bone marrow cells were cocultured with the calvaria osteoblast cells on collagen-coated culture dishs and incubated for 6−7 days in the presence of 1α,25-(OH)2D3 (0.1 μM) or PGE2 (10 μM). To obtain mature osteoclasts, cells were detached with 0.2% collagenase at 37 °C for 30 min. Upon removal of osteoblasts, crude mature osteoclasts were obtained. TRAP-positive multinucleated osteoclasts showed three or more nuclei. Actin rings of osteoclasts were stained with Alexa Fluor 488-conjugated phalloidin in the dark and then washed with cold PBS. The distribution of the actin rings was visualized and detected under a fluorescence microscope. For the resorption pit assay, osteoclasts in dentin slice were placed in 96well plates containing lupeol. After incubation, attached cells were completely removed from dentin slices and resorption pits were visualized by staining with hematoxylin solution. The number of resorption pits in each slice was counted. Western Blot Analysis. BMM cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM Nacl, 1 mM EDTA, 50 mM β-glycerophosphate, 1% NP-40, 1 mM Na3VO4 and 1× protease inhibitor cocktail. The lysates were centrifuged at 15000g for 15 min, and supernatants were collected. The concentrations of protein samples were measured using the Bradford method (Bio-Rad
voucher specimen (KMU-2013-11-03) was deposited at the Herbarium of the College of Pharmacy, Keimyung University, Daegu, Korea. The stem bark of S. commixta (2 kg) was extracted twice with MeOH (1 L) for 2 h at 85 °C. The MeOH extract (103.2 g) was dissolved in water and partitioned with n-hexane, EtOAc, and nbutanol. The EtOAc-soluble fraction (18.33 g) was partitioned on a silica gel column (6.5 × 60 cm) using n-hexane−CH2Cl2 (1:3) as the eluent to obtain six fractions (Fr. A−F). Fr. C (5.2 g) was subjected to Sephadex LH-20 (n-hexane−CHCl3−MeOH, 4:3:0.5) chromatography and purified by a silica gel column eluting with n-hexane−EtOAc (2:1) to yield compound 1 (2.3 g, 0.115 w/w%, purity >98.8%). Compound 1 was identified as lupeol by comparing its spectroscopic NMR data. The spectral data were identical with those reported in the literature.19 Compound 1 was determined to be >98.8% pure by HPLC analysis. Chemicals. All chemicals were obtained from Sigma-Aldrich Chemical (St. Louis, MO, USA) unless otherwise indicated. Fetal bovine serum (FBS), antibiotics, and cell culture medium were purchased from Gibco BRL (Rockville, MD, USA). Recombinant murine M-CSF and recombinant human RANKL were obtain from Peprotech Ltd. (London, UK). 1α,25-(OH)2D3 and prostaglandin E2 (PGE2) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Fast-red LB salt and naphthol AS-MX phosphate were purchased from Sigma-Aldrich. Primary antibodies for phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, p38, phospho I-κBα, polyclonal NF-κB (p65), and rabbit polyclonal antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). An anti-c-Fos mouse antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An anti-NFATc1 mouse antibody was purchased from BD Biosciences (San Jose, CA, USA). The enhanced chemiluminescence (ECL) Western blotting detection system and the Megaprime DNA labeling system were from Amersham (Arlington Heights, IL, USA). Actin antibody was purchased from Sigma-Aldrich. Alexa Fluor 488-conjugated phalloidin was purchased from Molecular Probes Inc. (Eugene, OR, USA). Isolation of Mouse Calvaria Osteoblastic Cells. Primary calvaria osteoblast cells were obtained from the 1−2-day-old newborn mice calvaria. Ten to twenty calvariae were subjected to digestions using 50 mL of enzyme solution containing 0.1% collagenase (Wako, Japan) and 0.2% dispase (Sigma-Aldrich) for 10 min at 37 °C in a shaking water bath (120 cycles/min). The collagenase solution was recovered, and fresh collagenase solution (10 mL) was added. Calvariae were further incubated for another 10 min at 37 °C in a shaking water bath (120 cycles/min). This digestion of calvaria with collagenase-dispase was repeated three or four times. The primary osteoblats isolated in fractions 2−5 were collected by centrifugation and cultured for 3 days in α-MEM containing 10% FBS in 10 cm culture dishes. Cells were then detached from culture dishes by trypsin-EDTA, suspended in α-MEM with 10% FBS, and used for coculture as osteoblastic cells. Alkaline Phosphatase Activity Assay. Calvaria osteoblast cells were grown in 96-well plates and treated with lupeol for 4, 7, and 14 days. After incubation, cells were washed twice with ice-cold phosphate-buffered saline (PBS, pH 7.4). Cells were fixed in 10% formaldehyde for 10 min and were lysed with Tris/glycine/Triton buffer (pH 10.5, 50 mM Tris, 100 mM glycine, 0.1% Triton X-100). The supernatant was collected by centrifugation at 5000g for 15 min at 4 °C. A 10 μL amount of p-nitrophenyl phosphate (p-NPP) substrate (100 mM) and 20 μL of 0.1 N glycine NaOH (pH 10.4) were added to 20 μL of the supernatant mixed with Tris/glycine buffer and incubated at 37 °C for 30 min. The enzymatic reaction was stopped by adding 200 μL of 0.1 N NaOH. The optical density (OD) of pnitrophenol (p-NP) was measured at 405 nm within 1 h. ALP activity was standardized as relative percent of control. The ALP activity was normalized to total protein, which was determined using a BCA protein assay kit. PCR Amplification of Reverse-Transcribed mRNA. For RTPCR analysis, mouse osteoblasts were cultured in α-MEM containing 10% FBS and 0.1 μM 1α,25-(OH)2D3 with or without lupeol on 60 mm culture dishes. After culture for 24 h, total cellular RNA was G
DOI: 10.1021/acs.jnatprod.5b01088 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Laboratories, Hercules, CA, USA). The protein was separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking with 5% nonfat milk, the membranes were hybridized with a 1:1000 dilution of primary antibodies for NFATc1, c-Fos, phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, p38, phospho I-κBα, I-κBα, and β-actin for 12 h at 4 °C. Membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) three times with 5 min intervals and hybridized with horseradish peroxidase-conjugated mouse or rabbit secondary antibodies for 1 h at room temperature. After washing three times with TBST, antigen−antibody complexes were visualized with an ECL Western blot detection kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. Animals and Experimental Design. C57BL/6 mice (6 weeks old, male) were purchased from Orient Bio (Gyeonggi-do, South Korea). Animals were housed in a climate-controlled environment (22 ± 2 °C, 50 ± 10% relative humidity) with a 12 h light/dark cycle and had free access to food and water. Mice were then randomly divided into six groups each consisting of five animals. Three groups of mice were given 1α,25-(OH)2D3 subcutaneously for three consecutive days followed by treatment with either PBS or lupeol at 25 or 100 μg/kg/ day for 3 days. The three other groups of mice were left unchallenged with 1α,25-(OH)2D3, but treated only with PBS or lupeol at 5, 25, 50, and 100 μg/kg/day for 5 days. Animals were sacrificed on day 8, and the serum and femur bones were collected. The animal study was approved by the Animal Experiment Committee of Keimyung University. The mice were maintained in accordance with the guidelines of the Animal Experiment Committee of Keimyung University (KM 2014-04). Serum Analysis and Histopathology. Collected blood was centrifuged to obtain serum. The serum concentrations of calcium and phosphate were measured using Quantichrom calcium and phosphate assat kits (Bioassay Systems, Hayward, CA, USA). The serum level of RANKL was measured using a sensitive ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). Femur bones harvested from mice treated with or without vitamin D3 in the presence of PBS or lupeol (25 or 100 μg/kg/day) were fixed with 10% formalin for 24 h, decalcified in 14% EDTA (pH 7.2) for 3 weeks, and embedded in paraffin. Paraffin sections were subjected to histochemical analysis of the TRAP activity. Statistical Analysis. All experiments were replicated at least three times. The means and standard deviations (SD) were calculated by SigmaPlot software. Student’s t test was used to assess the statistical significance of differences. Differences were considered significant at P < 0.05.
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AUTHOR INFORMATION
Corresponding Authors
*Tel (S.-R. Lee): +82-53-580-3862. E-mail:
[email protected]. *Tel (G.-S. Jeong): +82-53-580-6649. E-mail: gsjeong@kmu. ac.kr. Author Contributions ⊥
N. K. Im and D.-S. Lee contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No. 2014R1A5A2010008 and No. NRF2015M3A9A5031091).
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