Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway

Cyperenoic acid is a terpenoid isolated from the root of a medicinal plant Croton crassifolius with a wide range of biological activities. In this study, the effects of cyperenoic acid on osteoclast differentiation were investigated both in vitro and in vivo using receptor activator of nuclear factor-κB ligand (RANKL)-induced bone marrow-derived osteoclasts and senescence-accelerated mouse prone 6 (SAMP6). Cyperenoic acid significantly suppressed RANKL-induced osteoclast differentiation at the concentrations with no apparent cytotoxicity. The half maximum inhibitory concentration (IC50) for osteoclast differentiation was 36.69 μM ± 1.02. Cyperenoic acid treatment evidently reduced the expression of two key transcription factors in osteoclast differentiation, NFATc1 and c-Fos. Detailed signaling analysis revealed that cyperenoic acid did not affect MAPK pathways and canonical NF-κB pathway but impaired activation of p100/p52 in the non-canonical NF-κB pathway upon RANKL stimulation. Moreover, the expression of osteoclast-related genes, nfatc1, ctsk, irf8, acp5 and cfos were disrupted by cyperenoic acid treatment. The bone resorption activity by cyperenoic acid-treated osteoclasts were impaired. In a senile osteoporosis mouse model SAMP6, mice fed on diet supplemented with cyperenoic acid showed delay in bone loss, compared to the control. Taken together, plant-derived cyperenoic acid shows great potential as therapeutic agent for osteoporosis.

SCIEntIFIC REPORTS | (2018) 8:5625 | DOI: 10.1038/s41598-018-23912-3 relative cell viability (%) was expressed as percentages relative to the untreated control cells. The IC 50 was calculated using a GraphPad Prism 5.03 (GraphPad Software, USA). For anti-inflammation activity, RAW264.7 cells were treated with cyperenoic acid and stimulated with or without lipopolysaccharide (LPS) (100 ng/ml, Sigma Aldrich, MO, USA) and recombinant murine interferon-gamma (IFN-γ) (10 ng/ml, BioLegend, CA, USA) for 24 h. The amount of nitric oxide produced were measured by Griess reaction. The relative nitric oxide productivity (%) was expressed as a percentage relative to the untreated control and compared with nitrite standard.

Osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining assay.
Osteoclast differentiation from murine bone marrow cells were performed as described elsewhere with some modification 25 . Briefly, BMs-derived osteoclast precursors (3 × 10 6 cells/well in 60 mm culture tissue culture dish) were seeded and incubated with M-CSF (25 ng/ml) for 48 h to induce differentiation of osteoclast precursors. The precursors of osteoclasts were harvested by cold PBS and seeded at 2.5 × 10 5 cells/well in 96-well plates. Cells were pre-treated with cyperenoic acid or vehicle control DMSO for 30 min and stimulated with RANKL (100 ng/ ml) and M-CSF (25 ng/ml) for 5 days. After the incubation, cells were washed and fixed with 10% formaldehyde. Cells were stained with a TRAP-staining solution (50 mM acetate buffer, contained with 50 mM sodium tartrate, 0.1 mg/ml naphthol AS-MX phosphate and 0.6 mg/ml Fast red violet LB salt). TRAP+ multinucleated cells (MNCs) with three or more nuclei per cell were counted as mature osteoclast under a light microscope (Olympus, Tokyo, Japan).
Expression of osteoclast related genes by qPCR. Total RNA was isolated by using Trizol reagent (Thermo Fisher Scientific, MA, USA) or RNAiso reagent (Takara-Bio, Japan) according to the manufacturer's instruction. Isolated RNA was measured using Quan iT assays (Thermo Fisher Scientific, MA, USA). Primers for osteoclastogenic genes used in this study is listed in the Supplementary Table 1. A SYBR ® Green (Bio-Rad, USA) or Kapa SYBR (Kapa Biosystems, USA) was used for qPCR. Relative expression of osteoclast markers was determined and normalized, using the expression levels of β-actin or gapdh and calculated by 2 −ΔΔCt method 26 . Immunoblot. Cellular proteins were extracted using RIPA buffer and the amounts were measured by using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Proteins were separated with 8% or 12% SDS-PAGE and transferred onto PVDF membranes (Merck Millipore, MA, USA). The membranes were blocked with 3% non-fat-milk in PBS-T or 5% BSA in TBS-T and then probed with indicated antibodies. The signals were visualized by chemiluminescence with high performance chemiluminescence X-ray film.
Bone resorption assay. Osteoclast differentiation from murine bone marrow cells were performed as described elsewhere with some modification 25 . Briefly, osteoclast precursors were seeded in 96-well plates containing dentine or bone slices (IDS ® Immunodiagnostic Systems, UK) (2.5 × 10 5 cells/well), and stimulated with RANKL (100 ng/ml) and M-CSF (25 ng/ml) for 14 days. Cyperenoic acid (100 μM) or vehicle control DMSO was added to the culture at indicated times. At the end of the culture period, all cells were removed by sonication for 15 seconds in concentrated ammonium hydroxide and washed with double distilled water. The dentine or bone slices were stained for 10 min by Toluidine Blue (Sigma Aldrich, MO, USA), followed by extensive washing. The resorption areas were examined by a light microscope and photographed. The percentage of the resorbed areas were determined using Image-J software, area measurement, at 100-fold magnification.
Osteoblast differentiation by alkaline phosphatase activity (ALP) assay. Osteoblast differentiation using MC3T3-E1 cell line were performed as described elsewhere 27 . Briefly, MC3T3-E1 cell line (2.5 × 10 4 cells/well in 12-well plates) was induced to differentiate to osteoblasts by ascorbic acid (200 μM, Sigma Aldrich, MO, USA) or ascorbic acid in combination with β-glycerophosphate (8 mM, Sigma Aldrich, MO, USA) for 10 days. During this period, media were changed every 3 days. For ALP assay, cells were washed and harvested with 1xPBS and 0.1% triton x-100 for 10 min, respectively. The protein content was measured by using the BCA assay. Normalized protein lysates were incubated with 0.1 M sodium bicarbonate-carbonate buffer (pH 10) containing with 2 mM MgSO 4 and 6 mM p-nitrophenol phosphate for 30 min at 37 °C. The reaction was stopped by using 1.5 M NaOH (1:1 v/v) and absorbance was measured at 405 nm by microplate reader (Thermo Fisher Scientific, MA, USA).
A senile osteoporosis mouse model SAMP6. Female Senescence Accelerated Mouse Prone 6 (SAMP6) (7 weeks old) weight between 35-40 g were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were allowed to acclimatize for 1 week before the start of the experiment. All mice were randomly separated into two groups of control and cyperenoic acid treated group (6 mice per group). All experiments involved SAMP6 mice were conducted in accordance with the institutional guideline established by Shinshu University for laboratory animals.
The control group was fed with a standard rodent diet AIN-93M mixed with absolute ethanol (Oriental Yeast, Tokyo, Japan) and the cyperenoic acid treated group was fed with a standard diet mixed with 0.01% (w/w) cyperenoic acid powder. Mice were allowed free access to the diet and water. Food consumption and body weight were measured once a week. After 19 weeks, SAMP6 mice long bones (humerus, femur and tibia) were collected for BMs to be used to detect gene expression and for bone histomorphometry. Kidneys, livers and spleens were collected for pathohistological examination.
Bone histomorphometry. Bone histomorphometry was performed according to the modified methods of Orris and Arnett 28 and Vidal et al. 29 . Mice femur bones were fixed with 4.5% formaldehyde (pH 7.2) for 2 days, decalcified with 14% EDTA (pH 7.2) for 14 days and dehydrated with 70%, 80%, 96% and 100% ethanol, respectively. The processed bones were embedded in paraffin and the embedded bones were sectioned by microtome to a 5-µm thickness. The bone tissue sections were stained with hematoxylin, fast green CFC and safranin O and examined by a light microscope (model BX51TRF; Olympus, Tokyo, Japan). The analyzed areas covered the trabecular region (i.e., secondary spongiosa) of distal femur at 500 µm distal to the growth plate (orange color). The static histomorphometric parameters obtained from each section were bone volume/tissue volume (also known as bone volume fraction; BV/TV; %), trabecular thickness (Tb.Th; µm), trabecular separation (Tb.Sp; µm) and trabecular number (Tb.N; mm −1 ). These histomorphometric parameters were analyzed by the computer-assisted OsteoMeasure system with software version 7 (OsteoMetrics, Atlanta, USA), according to the standard of the ASBMR Histomorphometry Nomenclature Committee.

Statistical analyses.
All experiments were repeated independently three times unless otherwise specified. The results were expressed as the mean ± standard deviation (SD). The statistical significance of difference between an experimental group and its corresponding control were evaluated by one-way ANOVA, two-way ANOVA or Student's T-test (GraphPad Prism 5.03). All comparisons were made as specified, and p < 0.05 was considered statistical significance.

Results
Cytotoxicity and anti-inflammatory activity of cyperenoic acid. To determine the toxicity of cyperenoic acid on macrophage-like cell line RAW264.7 or BMs-derived osteoclast precursors, cells were treated with cyperenoic acid at various concentrations (0-100 μM) and the cell viability was measured. As shown in Fig. 1B,C, cyperenoic acid had no toxicity at the concentration as high as 100 μM in RAW264.7 or osteoclast precursors. Therefore, this concentration was used in further experiments. To examine the effect of cyperenoic acid on inflammatory response, the effect on nitric oxide production in RAW264.7 cell line stimulated with LPS/IFN-γ for 24 h was examined. As shown in Fig. 1D, this compound did not show any effect on nitric oxide production in LPS/IFN-γ-stimulated macrophages.
Anti-osteoclast differentiation activity of cyperenoic acid. A procedure to test the activity of cyperenoic acid against osteoclast differentiation was shown in Fig. 2A. To determine the dose dependent activity, various concentrations of cyperenoic acid were used. As shown in Fig. 2B, the percentages of differentiated osteoclasts as judged by TRAP+ MNCs, decreased upon cyperenoic acid treatment in a dose dependent manner. The IC 50 for osteoclastogenesis was 36.69 µM ± 1.02. The highest concentration (100 μM) used to determine an IC 50 completely suppressed RANKL-induced osteoclast differentiation (Fig. 2C). To test whether cyperenoic acid treatment affects cell-cell fusion, a hall mark step of osteoclast differentiation, BMs were treated with cyperenoic acid as described above and the TRAP+ MNCs were counted and divided into cells with 3-5 nuclei, 6-10 nuclei or more than 10 nuclei. As shown in Fig. 2C, 2D, cyperenoic acid treatment significantly reduced the percentages SCIEntIFIC REPORTS | (2018) 8:5625 | DOI:10.1038/s41598-018-23912-3 of cells with 3 or more nuclei per cell in a dose dependent manner. Therefore, cyperenoic acid showed strong inhibitory activity against RANKL-induced osteoclast differentiation in murine BMs.

Effect of cyperenoic acid on expression of osteoclast-related genes in osteoclast precursors.
As the first step to investigate how cyperenoic acid interferes with RANKL-induced osteoclast differentiation, its effect on expression of osteoclast-related genes (nfatc1, ctsk, irf8, acp5 and cfos) were determined. BMs were pre-treated with cyperenoic acid (100 μM) or vehicle control DMSO for 30 min and then stimulated with RANKL for various durations as indicated in Fig. 3. As shown in Fig. 3A, treatment of cyperenoic acid completely abrogated the expression of nfatc1 both at 24 and 48 hr. Because a gene encoding cathepsin K, ctsk, is under the regulation of NFATc1, the mRNA level of ctsk was examined. As shown in Fig. 3B and Supplementary Figure 1, treatment with cyperenoic acid clearly reduced the level of ctsk mRNA at 48 h to almost the base line level. IRF8 is an inhibitory factor functioning to inhibit osteoclast differentiation and its level must be reduced to allow osteoclast differentiation 30 . As expected, cyperenoic acid treatment delayed the downregulation of irf8 at 12 h (Fig. 3C). The levels of additional two genes, i.e. acp5 and cfos, that are involved in osteoclast function and differentiation were examined. The mRNA levels for both genes were significantly reduced in the presence of cyperenoic acid (Fig. 3D, E). Taken together, the expression of genes involved in an early stage of osteoclast differentiation was compromised by cyperenoic acid treatment.
Effect of cyperenoic acid on signaling pathways downstream of RANK/RANKL. To examine the effect of cyperenoic acid on the signaling cascades downstream of RANK upon RANKL engagement, the phosphorylation of signaling molecules in the MAPK and canonical NF-κB pathways were detected. BMMs were pre-treated with cyperenoic acid (100 μM) or vehicle control DMSO for 30 min, and stimulated with RANKL at indicated time points. As shown in Fig. 4A, phosphorylation of signaling molecules in the MAPK and canonical NF-κB pathways appeared unaltered in the presence of cyperenoic acid after RANKL stimulation. In contrast, when the activation of non-canonical NF-κB pathway was investigated, treatment with cyperenoic acid clearly suppressed the phosphorylation of p100 and the appearance of p52 at 5 and 60 min after RANKL stimulation (Fig. 4B, Supplementary Figure 2C). Quantitative analysis of band densities of phosphorylated p100 or p52 at 60 min confirmed that cyperenoic acid treatment reduced phosphorylation of p100 and the level of p52 (Fig. 4C,D). In addition, the level of TRAF3 was transiently reduced at 15 min in the presence of cyperenoic acid. In contrast, the effect of cyperenoic acid on RelB was only minimum. Taken together, cyperenoic acid affects mainly the activation of non-canonical NF-κB pathway but does not interfere with MAPK or canonical NF-κB pathway.   Effect of cyperenoic acid treatment on bone resorption by osteoclasts. To investigate whether cyperenoic acid treatment interrupts osteoclast function, dentine or bone slices were subjected to a bone resorption assay by osteoclasts differentiated in the presence or absence of cyperenoic acid for 14 days. First, cyperenoic acid was added from the start of osteoclast differentiation as schematically shown in Fig. 5A. As shown in Fig. 5B-E, cyperenoic acid treatment clearly reduced the area of osteoclast-mediated bone resorption, consistent with its negative effect on osteoclast differentiation. The areas of bone resorption were partially, but significantly, reduced when osteoclasts differentiated in the presence of cyperenoic acid were used for this assay.
To evaluate whether cyperenoic acid treatment after osteoclast maturation interferes with the bone resorption functions of osteoclasts, cyperenoic acid was added to the culture at day 7 after initiation of osteoclast differentiation (Fig. 6A). The areas of bone resorption were measured as described above. As shown in Fig. 6B-E, cyperenoic acid treatment even when added after the completion of osteoclast differentiation, also impinged upon bone resorption activity of osteoclasts. Taken together, this evidence strongly indicated anti-bone resorption activities of cyperenoic acid.

Effect of cyperenoic acid on osteoblast differentiation. Some compounds with anti-osteoclastogenesis
also possess pro-osteoblastogenic activity. To evaluate this possibility, we determined the cytotoxicity and the effect of cyperenoic acid on osteoblast differentiation induced by ascorbic acid or ascorbic acid in combination with β-glycerophosphate in the pre-osteoblast cell line MC3T3-E1. The cytotoxicity of cyperenoic acid on the MC3T3-E1 cell was first evaluated with cyperenoic acid of various concentrations (0-100 μM). The result revealed no apparent toxicity of cyperenoic acid in this cell line within the range of tested concentrations (Supplementary Figure 3A). Furthermore, cyperenoic acid had no effect on ALP activity induced by ascorbic acid or ascorbic acid in combination with β-glycerophosphate in osteoblasts as shown in Supplementary Figure 3B and C. Thus, cyperenoic acid appears to affect only the differentiation and function of osteoclasts, but not differentiation of osteoblasts.

Effect of cyperenoic acid on bone loss in SAMP6 mice of senescence-induced osteoporosis. SAMP6
mice were continually fed with normal diet or cyperenoic acid-supplemented diet for 19 weeks. The body weight and the diet consumption were measured once a week. As shown in Supplementary Figure 4A and B, there were no differences in the body weight and the diet consumption between both groups of mice. Furthermore, livers and kidneys were subjected to histopathological examination at the end of the experiment and no obvious toxicity to these tissues were found (Supplementary Figure 4C-F).
To determine the effect of cyperenoic acid on bone loss in SAMP6 mice, femur bones were subjected to bone histomorphometric measurement after 19 weeks of feeding with cyperenoic acid-supplemented diet. As shown in Fig. 7A and B, cyperenoic acid treatment clearly increased the area of trabecular bone, compared to the control group. To quantitatively evaluate the effect of cyperenoic acid on bone loss in SAMP6 mice, bone histomorphometry was performed to obtain four essential trabecular bone parameters. As shown in Fig. 7C-D, the bone volume fraction and trabecular thickness showed an increasing trend in cyperenoic acid-treated group, but the difference did not reach statistical significance. On the other hand, the trabecular separation was significantly less while the trabecular number was significantly increased in the cyperenoic acid-treated group as compared to the control   (Fig. 7E and F), indicating that cyperenoic acid was able to improve bone microstructure. A decrease in trabecular separation was also consistent with the anti-osteoclastic effect of this compound since an increased trabecular separation is often due to the enhanced osteoclast activity. Next, the mRNA expression of genes related to bone formation (col1a1, bglap, ibsp), bone resorption (acp5, mmp9) and pro-inflammatory cytokines (tnf-α, il-6) were detected in bone marrow cells isolated from SAMP6 mice (Fig. 8). Cyperenoic acid treatment enhanced the expression of ibsp, a gene encoding bone sialoprotein (BSP) which is an osteoblast-related gene and plays an important role in bone formation and bone mineralization (Fig. 8C). On the other hand, this compound did not have any detectable effect on the levels of mRNA of other genes detected here. Taken together, cyperenoic acid supplement in diet before the onset of bone loss in SAMP6 mice significantly delayed bone loss in vivo.

Discussion
C. crassifolius is a medicinal plant distributed mainly in the southeast Asia, especially in Thailand, Laos, Vietnam and China. The ethanol extract from this plant was demonstrated to have anti-nociceptive and anti-inflammatory effects 31 . Cyperenoic acid belongs to a group of terpenoids found in the extract from C. crassifolius and has been identified by Boonyarathanakornkit et al. (1987) 22 to possess anti-angiogenesis activity 23,24 . In this study, we reported the novel biological activity of this natural compound against RANKL-induced osteoclast differentiation, its possible mode of action and the effect in vivo on senile bone loss.
Cyperenoic acid suppressed RANKL-mediated osteoclast differentiation in BMs with the IC 50 of 36.69 µM ± 1.02. This compound showed no toxicity against osteoclast precursors, macrophage like cell line and osteoblast precursor in both short term and long term culture (up to 14 days). In addition, upon adding to a mouse diet and continuous feeding to mice for 19 weeks, no apparent organ toxicity was observed. Therefore, this compound shows low toxicity both in vitro and in vivo at least in mice.
The anti-inflammatory activity of cyperenoic acid was tested in LPS/IFNγ-activated macrophage-like cell line RAW264.7. The signaling downstream of LPS and its receptor shares some pathways with those utilized by RANK/RANKL for osteoclast differentiation 8,32 . Therefore, by preliminarily examining the anti-nitric oxide production in macrophages may help identifying the step(s) affected by cyperenoic acid treatment. In fact, the results obtained from signaling pathway analysis corroborated that cyperenoic acid did not interfere with MAPK and canonical NF-κB pathways. The molecular mechanism underlying the anti-osteoclastogenic activity of cyperenoic acid appeared to be partly at the non-canonical NF-κB signaling, while other signaling pathways, i.e. MAPK and canonical NF-κB pathways were largely unaffected by cyperenoic acid treatment. The non-canonical NF-κB signaling pathway is utilized by various cytokines, including RANK/RANKL, LTβ/LTβR and CD40/CD0L 10 . The immediate outcome of the non-canonical NF-κB signaling is the dimerization of partially degraded p52 and RelB and the translocation of this dimer to nuclei which, in turn, regulates target gene expression. TRAF2 and 3 are adaptor proteins that function to regulate the stability of NIK kinase via recruitment of cIAP1/2 33 . Cyperenoic acid treatment decreased the phosphorylation of p100, the appearance of p52 and also transiently affected the level of TRAF3 (Fig. 4). We proposed that cyperenoic acid may affect the activity of enzyme(s) that regulate phosphorylation of p100. The processing of p100 is critical for governing the development of lymphoid organs and bone metabolism 11 . Iotsova et al. 12 found that double knockout mice lacking both p50 and p52 exhibited an osteopetrotic phenotype while single knockout of p50 or p52 did not show bone formation abnormalities. Nevertheless, BMMs cells from p50 −/− p52 +/− mice differentiate and remain as osteoclast progenitors but the mature osteoclasts could not be detected. Interestingly, p50/p52 double KO mice show severe osteopetrosis and the overexpression of NFATc1 and c-Fos rescues this phenotype, indicating a crucial role of non-canonical NF-κB signaling in RANKL-induced osteoclastogenesis 34,35 . Cyperenoic acid treatment indeed substantially reduced the expression of both proteins in the presence of RANKL. These results strongly indicated that this compound affects the activation of the non-canonical NF-κB signaling and its downstream target genes involved in osteoclast differentiation.
NFATc1 is a master regulator of osteoclast differentiation which control the expression of osteoclast-specific genes encoding key enzymes required for osteoclast functions such as TRAP and cathepsin K via c-Fos 36 . NFATc1-deficient mice exhibit osteopetrotic phenotype because of the defect in osteoclastogenesis process 37 . Some studies reported that in an early stage of osteoclast differentiation, c-Fos is a necessary factor for the early activation of NFATc1 in osteoclastogenesis 38 . Cyperenoic acid treatment completely suppressed the activation of c-Fos at 48 h and this effect was translated to decreased NFATc1 level at 72 and 96 h. Expression of genes under the regulation of NFATc1 and c-Fos were all reduced by cyperenoic acid treatment, including acp5 and ctsk. Interestingly, cyperenoic acid treatment affected not only the positive regulators of osteoclast differentiation but also the negative regulator irf8. irf8 encodes a key modulatory protein for osteoclast differentiation. IRF8 suppresses the function and expression of NFATc1 and its level is reduced during osteoclast differentiation. In mice lacking of ifr8, a severe osteoporosis was manifested because of increased in osteoclast numbers 30 . Therefore, cyperenoic acid treatment suppresses the non-canonical NF-κB signaling and affects the expression of the target genes involved in both positive and negative regulation of osteoclast differentiation.
Cell-cell fusion is a critical step to generate MNCs during osteoclast differentiation. Various molecules were identified to play roles during this process such as Dendritic cell-specific transmembrane protein (DC-STAMP). DC-STAMP is under the regulation of NFATc1 and c-Fos 39 . In this study, cyperenoic acid reduced the numbers of TRAP+ cells with 3 or more nuclei during RANKL-induced osteoclast differentiation (Fig. 2D). Because the expressions of NFATc1 and c-Fos were compromised by cyperenoic acid treatment, it is highly likely that the expression of DC-STAMP and the step of cell-cell fusion leading to MNCs are also suppressed.
The effect of cyperenoic acid on functions of osteoclasts was evaluated by bone resorption assay. Addition of this compound at the same time as RANKL stimulation partially reduced the bone resorption areas (Fig. 5). This is an unexpected observation because the differentiation of osteoclasts was almost completely suppressed in vitro by TRAP staining with the same concentration of cyperenoic acid. The assay used in this study measured only the areas of resorption but not the depth of the resorbed pits 40 . It is possible that cyperenoic acid treatment may also affect the depth of mineralization. When cyperenoic acid was added after osteoclast differentiation, it also reduced the resorption areas, indicating that this compound may possess bone resorption activity independently of the osteoclast differentiation. How cyperenoic acid influences bone resorption remains unanswered.
Our studies provided compelling evidence that cyperenoic acid may be beneficial for senile osteoporosis in SAMP6 mice. SAMP6 is proposed to be a useful senile osteoporosis mouse model which shows sign of bone loss in relatively young age [41][42][43] . In our experiment, we supplemented the compound in the regular animal diet and allowed the animals free access to the diet. The delay in bone loss was clearly shown after long term consumption of modified diets with no apparent organ toxicity. Among the four bone microstructural parameters as analyzed by static bone histomorphometry, trabecular separation and trabecular number were significantly improved in cyperenoic acid-treated group. The bone volume and the trabecular thickness increased slightly but did not reach statistical significance between the two groups. These two latter parameters may need longer time of treatment to exhibit the improvement effect; however, an increased trabecular number and a decreased separation clearly confirmed that cyperenoic acid had beneficial effects on bone microstructure. Whether cyperenoic acid treatment can delay bone loss in other strains of mice or in other mouse model of osteoporosis induced by other manipulation needs further investigation.
Interestingly, cyperenoic acid enhanced expression of ibsp which encodes the bone sialoprotein (BSP). BSP is synthesized mainly by chondrocytes, a subset of osteoblasts, and osteoclasts. Mice with targeted deletion of ibsp show diminished bone growth and mineralization lead to reduce bone formation 44 , indicating that BSP is important for bone mineralization and bone formation. Further, BSP is a useful diagnostic marker of bone resorption reflecting osteoclast activity in human or animal serum 45 . In our study, cyperenoic acid slightly decreased BSP level in SAMP6 mice serum but did not reach the level of statistical significance (data not shown). In a study by Li et al. 20 , berberine, an isoquinoline alkaloid, diminished concentration of dexypyridinoline (Dpd) which is a bone resorption marker in urine, but it shows no effect on procollagen type I carboxyterminal extension peptide (PICP) in serum of SAMP6 mice 20 . The effect of cyperenoic acid treatment at the molecular level was examined by detecting gene expression in bone marrow cells in this study. Because our in vitro results indicated that cyperenoic acid affects early stage of osteoclast differentiation from BMs, we hypothesized that the cyperenoic acid treatment in vivo may affect gene expression profiles of osteoclast precursors reflecting bone formation and resorption.
Unexpectedly, we did not find any significant difference in the mRNA level of genes involved in bone resorption. Thus, more careful analysis of gene expression in a well defined cell subsets such as bone marrow stromal cells or osteoblasts and osteocytes may yield more informative results on the effect of this compound on bone formation.
In this study, cyperenoic acid has no impact on ALP activity of pre-osteoblast cell (MC3T3-E1) treated with ascorbic acid. Some natural compounds are shown to affect both osteoclast differentiation and osteoblast maturation/function 46 . Therefore, cyperenoic acid may indirectly affect the expression of ibsp. Cyperenoic acid treatment did not have any effect on the expression of il-6 and tnf-α which are related to inflammatory bone loss 47 . Furthermore, histomorphometric parameters are used to distinguish the bone condition. Typically, osteoporosis shows high and low level in bone turnover and bone volume, respectively 29 .
Taken together, we presented evidence that cyperenoic acid from C. crassifolius interferes with RANKL/RANK signaling at least at the non-canonical NF-kB pathway and the osteoclast differentiation process and might be a potential lead compound for osteoporosis therapeutic and prevention in senile osteoporosis.