Osteoporos Int DOI 10.1007/s00198-016-3496-8

ORIGINAL ARTICLE

Xanthotoxin prevents bone loss in ovariectomized mice through the inhibition of RANKL-induced osteoclastogenesis C. Dou 1,2 & Y. Chen 1 & N. Ding 1 & N. Li 1 & H. Jiang 1 & C. Zhao 1 & F. Kang 1 & Z. Cao 1 & H. Quan 1 & F. Luo 2 & J. Xu 2 & S. Dong 1

Received: 9 November 2015 / Accepted: 15 January 2016 # International Osteoporosis Foundation and National Osteoporosis Foundation 2016

Abstract Summary Xanthotoxin (XAT) is extracted from the seeds of Ammi majus. Here, we reported that XAT has an inhibitory effect on osteoclastogenesis in vitro through the suppression of both receptor activator of nuclear factor-κB ligand (RANKL)-induced ROS generation and Ca2+ oscillations. In vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, and restores bone strength in ovariectomized mice. Introduction Excessive osteoclast formation and the resultant increase in bone resorption activity are key pathogenic factors of osteoporosis. In the present study, we have investigated the effects of XAT, a natural furanocoumarin, on the RANKLmediated osteoclastogenesis in vitro and on ovariectomymediated bone loss in vivo. Methods Cytotoxicity of XAT was evaluated using bone marrow macrophages (BMMs). Osteoclast differentiation, formation, and fusion were assessed using the tartrate-resistant acid phosphatase (TRAP) stain, the actin cytoskeleton and focal adhesion (FAK) stain, and the fusion assay, respectively. Osteoclastic bone resorption was evaluated using the pit formation assay. Reactive oxygen species (ROS) generation and Electronic supplementary material The online version of this article (doi:10.1007/s00198-016-3496-8) contains supplementary material, which is available to authorized users. * S. Dong [email protected]

1

Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing 400038, China

2

Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing 400038, China

removal were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). Ca2+ oscillations and their downstream signaling targets were then detected. The ovariectomized (OVX) mouse model was adopted for our in vivo studies. Results In vitro assays revealed that XAT inhibited the differentiation, formation, fusion, and bone resorption activity of osteoclasts. The inhibitory effect of XAT on osteoclastogenesis was associated with decreased intracellular ROS generation. XAT treatment also suppressed RANKL-induced Ca2+ oscillations and the activation of the resultant downstream calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT downregulated the key osteoclastogenic factors nuclear factor of activated T cells c1 (NFATc1) and c-FOS. Our in vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice. Conclusion Our findings indicate that XAT is protective against ovariectomy-mediated bone loss through the inhibition of RANKL-mediated osteoclastogenesis. Therefore, XAT may be considered to be a new therapeutic candidate for treating osteoporosis. Keywords Bone loss . Calcium oscillation . Osteoclasts . ROS . Xanthotoxin

Introduction Osteoporosis is a serious public health concern. According to the International Osteoporosis Foundation, more than 200 million people suffer from this disease worldwide [1, 2]. Postmenopausal osteoporosis is the most common type of osteoporosis. Approximately 30 % of all postmenopausal women in the USA and Europe have osteoporosis. At least

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40 % of these women are expected to sustain one or more fractures in their lifetime [3]. Bone homeostasis is maintained and orchestrated by osteoclast (OC)-mediated bone resorption and osteoblast (OB)-mediated bone formation. Osteoporosis may be resulted from reduced bone stock and greater bone loss than normal level. For postmenopausal osteoporosis, bone loss increases due to lower levels of estrogen. Mature osteoclasts are bone-specific polykaryons derived from hematopoietic stem cells (HSCs). Two of the most important regulating factors during osteoclast differentiation are receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) [4]. During RANKL-induced osteoclast formation, reactive oxygen species (ROS) have been shown to play important roles in differentiation, survival, activation, and bone resorption [5–8]. Excessive ROS generation has been proved to be associated with estrogen-deficient osteoporosis [9, 10]. Another important regulating factor in osteoclasts is calcium. During RANKL-mediated osteoclastogenesis, Ca2+ oscillations activate Ca 2+ /calmodulin-dependent protein kinases IV (CaMKIV) and cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) [11]. Additionally, mitochondrial ROS is also involved in the activation of CREB [12]. Ca2+ and ROS upregulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), the master regulator of osteoclastogenesis through the CaMKIV/ CREB pathway [13, 14]. Xanthotoxin (XAT), also known as 8-methoxypsoralen, is extracted from the seeds of Ammi majus, a plant of the family Apiaceae [15]. XAT belongs to the family of naturally occurring photoactive compounds that can be used in the treatment of a number of epidermal proliferative disorders, including psoriasis, vitiligo, and leprosy [16, 17]. XAT is also used in psoralen plus ultraviolet A radiation (PUVA) therapy for the treatment of cutaneous T cell lymphomas and autoimmune disorders, such as rheumatoid arthritis and scleroderma [18, 19]. Recently, XAT has also been used to induce antitumor activity through the induction of apoptosis and cell cycle arrest [20, 21]. Interestingly, XAT exhibits powerful anti-lipid peroxidation activity, suggesting that it has antioxidant activity [22, 23]. Another study showed that XAT could affect the intracellular Ca 2+ concentration in melanocytes, resulting in cytoskeletal actin reorganization [24]. Based on these results, we hypothesize that XAT has strong regulatory effects on osteoclastogenesis and bone homeostasis, which have not been previously reported. In this study, we report that XAT inhibits osteoclastogenesis through ROS scavenging activity and the regulation of RANKL-mediated Ca2+ signaling pathways. XAT administration in ovariectomized (OVX) mice decreases the OC number in vivo and prevents bone loss.

Materials and methods Reagents Recombinant mouse RANKL and recombinant mouse MCSF were purchased from R&D Systems (Minneapolis, MN). Antibodies against CaMKKβ, Pyk2, c-FOS, NFATc1, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Osteo assay surface plates for the assessment of bone resorption were purchased from Corning (NY, USA). Bovine cortical bone slices were obtained from Boneslices.com (Jelling, Denmark). Cell counting kit-8 was obtained from Dojindo Molecular Technologies (Dojindo, Japan). Tartrate-resistant acid phosphatase (TRAP) stain kits were obtained from Sigma-Aldrich (NY, USA). Actin cytoskeleton and focal adhesion staining kits were purchased from Millipore (Darmstadt, Germany). Membrane dye DiI, CellTracker Green, Fluo-4 AM, Pluronic F-127, and Hank’s balanced salt solution (HBSS) were obtained from Life Technologies (Carlsbad, CA). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kits were obtained from Abcam (Cambridge, UK). Alpha minimal essential medium (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, USA). Penicillinstreptomycin solution was obtained from Hyclone (Thermo Scientific, USA). Xanthotoxin was purchased from SigmaAldrich (NY, USA). Mice The 8-week-old female C57BL/6 mice used in this study were provided by the animal center of the Third Military Medical University. All experimental procedures were approved by the Third Military Medical University and performed according to the guidelines of laboratory animal care and use. All efforts were made to reduce both the number of animals that were tested and their suffering. Mice were divided into four groups: sham-operated mice (sham, n = 5), OVX mice (control, n = 5), low-dose XAT-treated OVX mice (0.5 mg/kg, n = 5), and high-dose XAT-treated OVX mice (5 mg/kg, n = 5). TWEEN 80 (Sigma-P4780) was used as a solvent for XAT. Vehicle or XAT solution was injected intraperitoneally three times a week for 4 weeks. Mice were weighed daily, and the required concentration of XAT was calculated. The control groups received only TWEEN 80 solution. All treated mice were sacrificed by cervical dislocation a day after the last administration. Cell viability analysis Bone marrow macrophages (BMMs) were seeded (2 × 103 per well) into 96-well plates and were cultured overnight. The cells were treated with M-CSF (50 ng/ml) and RANKL

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(100 ng/ml) for 24 h or 72 h along with varying doses of XAT. The cell proliferation and viability were evaluated at 24 and 72 h by adding the cell counting kit-8 (CCK-8) reagent and measuring the absorbance at 450 nm using a 96-well plate reader according to the manufacturer’s instructions. The wells containing only the CCK-8 reagent were used as blank controls. Annexin-V/PI staining Cell apoptosis was assessed using annexin V/propidium iodide (PI) staining. BMMs were treated with RANKL (100 ng/ ml) and M-CSF (50 ng/ml) for 72 h along with XAT at different dosages (0, 0.1, 1, 5 μM). The cells were washed twice with cold PBS and then resuspended in 500 μl of binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2] at a concentration of 1 × 106 cells/ml. The cells were then stained with 5 μl of annexin V-FITC (Life Technologies) and 10 μl of 20 μg/ml PI. Apoptosis was analyzed using a FACStar flow cytometer (BD Biosciences). In vitro assays for osteoclasts differentiation, fusion, and function Bone marrow cells were isolated and cultured with M-CSF (50 ng/ml) for 24 h to obtain BMMs. BMMs were cultured in α-MEM containing 10 % FBS and 1 % penicillinstreptomycin solution. For TRAP staining, the cells were cultured in 96-well plates at a density of 5 × 103 cells/well and treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 3 days. The cells were then fixed in 4 % paraformaldehyde for 2 min and stained with TRAP staining solution according to the manufacturers’ instructions. The relative TRAP activity was measured using a colorimetric method. For the actin cytoskeleton and focal adhesion staining, cells were seeded at a density of 4 × 104 cells/well on glass coverslips placed in 12-well plates and treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 3 days. The procedures for FAK staining have been described in a previous study [25]. Briefly, on day 4, the cells were washed, fixed for permeabilization, and blocked. After blocking, the cells were incubated in the primary antibody solution for 1 h at room temperature followed by washing. The cells were then incubated with secondary antibody and TRITC-conjugated phalloidin for 1 h at room temperature. The nuclei were counterstained with DAPI for 5 minutes, and the results were viewed using a fluorescent microscope. For the fusion assay, cells were cultured in six-well plates at a density of 8 × 104 cells/well and treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 3 days. The cells were then incubated with either membrane dye DiI or cell content marker CellTracker Green for 30 min at room temperature.

The cells labeled with DiI were scraped and transferred to the wells containing the cells labeled with CellTracker Green. The co-plated cells were then incubated together for 2 h followed by the removal of the culture medium. The cells were then viewed using a fluorescence microscope. The membrane merge data were analyzed using the ImageJ software. For the pit formation assay, cells were seeded on 96-well plates (Corning Osteo Assay Surface) at a density of 2 × 103 cells/well and on bovine bone slices placed in 48-well plates at a density of 1 × 104 cells/well. The cells were treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 5 days. Methylene blue staining was performed to evaluate the resorption area on the bone slices. The cells were removed using a bleach solution. Detailed analysis of pit formation area was performed as described previously [26]. Intracellular ROS detection Intracellular ROS levels were detected using a DCFDA cellular ROS detection assay kit (Abcam). BMMs (5 × 103 cells/ well in 96-well plates) were treated with RANKL (100 ng/ml), M-CSF (50 ng/ml), and XAT doses according to the assigned groups for 72 h. Intracellular ROS levels were measured using 2′,7′-dichlorofluorescein diacetate (DCFH), which oxidizes into fluorescent DCF in the presence of ROS. Cells were washed in PBS and incubated in the dark for 30 min with 10 μM DCFH-DA. Images were obtained using a fluorescence microscope (Olympus). Intracellular Ca2+ oscillation measurement BMMs (5 × 103) were seeded on 96-well plates, treated with supplements based on the assigned groups, and cultured for 72 h. Cells in the treatment groups were treated with RANKL (100 ng/ml), M-CSF (50 ng/ml), and XAT (1 μM); cells in the positive control group were treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml); and cells in the control group were treated with M-CSF (50 ng/ml) only. At the end of 72 hours, the cells were incubated in 5 μM Fluo-4 AM and 0.05 % Pluronic F-127 (Invitrogen) in HBSS supplemented with 1 % FCS/1 mM probenecid (assay buffer) for 30 min at 37 °C. Cells were washed twice with the assay buffer and incubated at room temperature for 20 minutes. Cells were excited at 488 nm using an inverted fluorescent microscope (Olympus). The relative intracellular calcium levels in individual cells were monitored for 5 min at 5-s intervals using the fluorescence intensity of Fluo-4 at ×200 magnification. Cells with at least two oscillations were counted as oscillating cells. A minimum of 40 cells were monitored in triplicate wells. The average amplitude of calcium oscillation in each cell was calculated using the TuneR and SeeWave packages for the R programming language [27].

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μCT analysis and histological analysis For μCT analysis, a Bruker MicroCT Skyscan 1272 system (Kontich, Belgium) with an isotropic voxel size of 10.0 μm was used to image the whole femur. The scans were obtained in 4 % paraformaldehyde using an X-ray tube potential of 60 kV, an X-ray intensity of 166 μA, and an exposure time of 1700 ms. The threshold for the trabecular bones was set at 86–255 (8-bit gray scale bitmap). μCT scans of whole bodies of mice (except the skulls) were obtained using an isotropic voxel size of 148 μm. Reconstruction was accomplished using NRecon (Ver. 1.6.10). 3D images were obtained from contoured 2D images using methods based on the distance transformation of the original gray scale images (CTvox, Ver. 3.0.0). 3D and 2D analyses were performed using CT Analyzer software (Ver. 1.15.4.0). All images presented are representative of their respective groups. For the histological analysis of the bones, the femurs were dissected and fixed in 4 % paraformaldehyde-PBS for 48 h. The femurs were then decalcified by daily changes of a 15 % tetrasodium EDTA soaking solution for 2 weeks. The decalcified femurs were dehydrated by passing them through a series of increasing concentrations of ethanol, cleared in xylene twice, embedded in paraffin, and sectioned into 8-μmthick sections along the coronal plate from anterior to posterior. The decalcified femoral sections were stained with TRAP. RT-qPCR Total RNA was isolated using the Trizol reagent (Life Technologies). Single-stranded cDNA was prepared from 1 μg of total RNA using reverse transcriptase and oligo-dT primers according to the manufacturer’s instructions (Promega, USA). Two microliters of cDNA from each sample was subjected to PCR amplification using specific primers as shown in STable 1.

expressed as the mean ± SD. One-way ANOVA followed by the Student-Newman-Keuls post hoc tests were used to determine the significance of difference between results, with *p < 0.05 and **p < 0.01 considered statistically significant.

Results XAT toxicity evaluation and its inhibitory effects on TRAP-positive cell formation For toxicity evaluation, a CCK-8 assay was conducted to assess the effects of XAT on cell viability. BMMs were treated with varying doses of XAT with or without RANKL for 24 and 72 h, respectively. Annexin-V/PI Staining was analyzed using FCM techniques for BMMs treated with RANKL and XAT (0, 0.1, 1, 5 μM) (Fig. 1b). The CCK-8 results showed that XAT concentrations greater than 5 μM strongly inhibited cell proliferation regardless of RANKL treatment (p < 0.01) (Fig. 1c). Consistently, the XAT dose at 5 μM significantly increased the rates of both early and late apoptosis (p < 0.01) (Fig. 1d). Based on the results of the toxicity evaluation, XAT concentrations of 0.1 and 1 μM were chosen for further tests. TRAP staining was then conducted to evaluate the effects of XAT on osteoclastogenesis (Fig. 1e). TRAP-positive cells with more than three nuclei were counted as osteoclasts. Quantitative analysis revealed that XAT significantly decreased the osteoclast number and TRAP activity at 0.1 μM (p < 0.01), and when the XAT concentration reached 1 μM, almost no osteoclasts were detected (Fig. 1f). This was confirmed by the qPCR results, which also showed that the two OC-specific markers, Ctsk and DC-STMAP, were significantly suppressed by XAT treatment (p < 0.01) (Fig. 1g). XAT inhibits the formation of multinucleated, mature osteoclasts

The cells were lysed in a lysis buffer containing 10 mM Tris, pH 7.2, 150 mM NaCl, 5 mM EDTA, 0.1 % SDS, 1 % Triton X100, and 1 % deoxycholic acid. For western blots, 30 μg of protein samples were subjected to SDS-PAGE, followed by their transfer onto PVDF membranes. After blocking in 5 % skim milk, the membranes were incubated with rabbit primary antibodies against CaMKKβ, Pyk2, c-FOS, NFATc1, and β-actin at 4 °C overnight followed by 1 h incubation with the secondary antibody (1:2000). β-Actin was served as the loading control.

FAK staining was conducted to better visualize the OC cytoskeleton and to study the effects of XAT on RANKL-induced osteoclastogenesis. The FAK staining results showed that actin ring formation was inhibited by the XAT treatments (Fig. 2a). Apart from the decrease inthe OC number (p < 0.01) (Fig. 2c), the average number of nuclei also significantly decreased due to XAT treatments (p < 0.01), suggesting that OC fusion was inhibited (Fig. 2d). The fusion assay confirmed this result (Fig. 2b) and showed that the membrane merge rate was decreased by the XAT treatments (p < 0.01) (Fig. 2e).

Statistical analysis

XAT inhibited osteoclast bone resorption activity

All data are representative of at least three experiments performed in triplicate, unless otherwise indicated. Data are

To test the effects of XAT on OC function, a pit formation assay was conducted on bone slices and on osteo assay surface

Immunoblotting

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Fig. 1 XAT toxicity evaluation and its inhibitory effects on TRAPpositive cell formation. a Chemical formula of xanthotoxin. b FCM analysis of the apoptosis rate of BMMs treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 72 h with varying doses of XAT. c CCK-8 was performed in triplicate to analyze the cell viability of BMMs treated with varying doses of XAT for 24 and 72 h with or without RANKL (100 ng/ ml) and M-CSF (50 ng/ml). d. Quantitative analysis of the early and late stage apoptosis rates. e Representative images of BMMs stained for

TRAP (red) treated with XAT at different doses (0, 0.1, 1 μM). Experiments were done in triplicate; scale bar represents 200 μm. f Quantification of TRAP (+) cells with more than three nuclei in each well (96-well plate). Relative TRAP activity was measured by colorimetric analysis. g Relative mRNA expression levels of Ctsk and DC-STAMP (n = 5). The data in the figures represent the averages ± SD. Statistically significant differences between the treatment and control groups are indicated as *(p < 0.05) or **(p < 0.01).

plates (Fig. 3a, c). BMMs were seeded on bone slices and osteo assay surfaces and treated according to the assigned groups. After 5 days of incubation, the cells were removed and the resorption pits were quantified. Consistent with previous results, XAT showed strong inhibitory effects on OC resorption activity on both the bone slices and the osteo assay surfaces (p < 0.01) (Fig. 3b, d).

DA. BMMs treated with RANKL alone exhibited high intracellular ROS levels, which were inhibited by XAT treatments (Fig. 4A). Quantitative analysis showed that both the number of ROS-positive cells and the levels of ROS were significantly decreased by XAT (p < 0.01) (Fig. 4b). We further tested the effects of XAT on Ca2+ oscillation (Fig. 4d). Our results indicate that XAT inhibits both the average amplitude and the frequency of Ca2+ oscillations induced by RANKL (Fig. 4e). Additionally, we tested the expressions of CaMKKβ and Pyk2 during calcium signaling using qPCR and western blots. The results suggest that the expression of CaMKKβ and Pyk2 is suppressed by XAT treatments both at the mRNA level

XAT scavenges ROS production and attenuates Ca2+ oscillation To explain the inhibitory effects of XAT on osteoclastogenesis, intracellular ROS was measured using DCFH-

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Fig. 2 XAT inhibited the formation of multinucleated, mature osteoclasts. a Representative images of focal and adhesion staining of BMMs treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 72 h with varying doses of XAT. Experiments were done in triplicate; scale bar represents 100 μm. b Representative fusion assay images of BMMs from different groups. Experiments were done in triplicate; scale

bar represents 100 μm. c Quantification of osteoclasts (with three or more nuclei) in each well (96-well plate). d Quantification of the average number of nuclei in each group. e Quantification of the membrane merge rate of osteoclasts in each well (six-well plate). The data in the figures indicate the averages ± SD. Statistically significant differences between the treatment and control groups are indicated as *(p < 0.05) or **(p < 0.01).

and at the protein level (Fig. 4c, f). The western blot results showed that XAT also inhibits the expressions of NFATc1 and c-FOS (Fig. 1f), which are downstream targets of CaMKKβ and Pyk2 [28].

central epiphysis and was contoured as shown in the dashed red box (Fig. 5a). Quantitative analysis showed that XAT administration (0.5 and 5 mg/kg) in OVX mice significantly increased the bone mineral density (BMD), trabecular bone volume fraction (BV/TV), and trabecular number (Tb. N) and decreased trabecular separation (Tb. Sp) (p < 0.01) (Fig. 5b). TRAP staining of the distal femurs show that the OC surface/bone surface ratio was significantly higher in OVX mice compared to the control group (Fig. 5c). XAT treatments considerably decreased the OC surface area (Fig. 5d). In addition, Masson stain results showed that XAT significantly increased the new bone formation rate (p < 0.01) and rescued the serum biochemical markers change in OVX mice (p < 0.01) (SFig. 1). The in vivo results indicated that XAT administration prevented bone loss and increased new bone formation in OVX mice through the inhibition of osteoclastogenesis.

XAT prevents bone loss in OVX mice We used OVX mice to test if the anti-osteoclastogenesis effects of XAT could be observed in an in vivo model. Mice were divided into four groups: control group (sham operated), OVX group, low XAT dose (0.5 mg/kg), and high XAT dose (5 mg/kg). XAT was dissolved in Tween 80 and injected intraperitoneally three times a week for 4 weeks. The control group received only Tween 80. After killing, μCT analysis was conducted on the dissected femurs. Trabecular bone analysis of the distal femur was conducted on the upper 3mm region of the femur beginning at 0.8 mm proximal to the

Osteoporos Int Fig. 3 XAT inhibited osteoclast bone resorption activity. a Representative images of BMMs cultured on bovine slices treated with RANKL (100 ng/ml) and MCSF (50 ng/ml) for 5 days with varying concentrations of XAT. Experiments were done in triplicate; scale bar represents 400 μm. b Quantification of bone resorption area on the bone slices. c Representative images of osteo assay surface 96-well plates after the removal of osteoclasts. Experiments were done in triplicate; scale bar represents 400 μm. d Quantification of bone resorption area on the osteo surface. The data in the figures represent the averages ± SD. Statistically significant differences between the treatment and control groups are indicated as *(p < 0.05) or **(p < 0.01).

Discussion In the present study, we showed that XAT could prevent ovariectomy-induced bone loss through the inhibition of RANKL-induced osteoclastogenesis. XAT inhibited osteoclastogenesis through two mechanisms: (1) XAT exhibited antioxidant activity during osteoclastogenesis, which downregulated the intracellular ROS levels that were elevated by RANKL activation. (2) The presence of XAT during osteoclastogenesis suppressed the RANKLinduced Ca 2+ oscillations and the activation of downstream targets of calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT suppressed the induction of two key osteoclastogenic factors, NFATc1 and c-FOS. Postmenopausal osteoporosis is a disease marked with excessive bone resorption that is caused by estrogen deficiency. Estrogen is crucial in maintaining bone health due to its antioxidant activity in OCs and stimulating effects on OBs. Although the details of the mechanisms are unclear, excess production of ROS due to estrogen deficiency is one of the main pathogenic factors in postmenopausal osteoporosis [9, 29]. ROS plays an essential role in RANKL-induced osteoclastogenesis. Mitogen-activated protein (MAP) kinases, including c-Jun N-terminal kinase

(JNK) and p38 MAP kinase (p38), are crucial for OC differentiation, and both of these two genes can be activated by ROS [7, 30]. Recently, it has also been proven that ROS production and intracellular hydrogen peroxide accumulation in OCs is critical for osteoclastogenesis and skeletal homeostasis [31]. RANKL-mediated Ca2+ oscillations provide Ca2+ signaling that induces OCs to up regulate and autoamplify NFATc1 [13]. Ca2+ mobilization and influx are essential for OC differentiation, cytoskeleton organization and bone resorption activity [32]. Current OC-targeted anti-resorptive therapies for osteoporosis are mainly based on the RANKL/RANK/OPG regulatory axis [33]. A recent study has drawn attention to ROS as a potential target in treating osteoporosis [34]. This study showed that a SNP of NADPH oxidase 4 (NOX4) was associated with elevated circulating markers of bone turnover and reduced bone density in women and showed that Nox4−/− mice displayed higher bone density with reduced number of OCs. However, only a few studies have considered Ca2+ signaling pathways as a target for treating osteoporosis. XAT can be extracted from Sichuan pepper, also known as Chinese coriander. Chongqing district is distinct from other regions in China, specifically for its extensive use of Sichuan pepper in everyday dishes, such as hotpot, and even in

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Fig. 4 XAT scavenges ROS production and attenuates Ca2+ oscillation. a Representative images of ROS-positive BMMs during RANKLinduced osteoclastogenesis treated with varying concentrations of XAT. Experiments were done in triplicate; scale bar represents 100 μm. b Quantification of the ROS-positive cell numbers and green fluorescence intensity in each well (96-well plate). c Relative mRNA expression levels of CaMKKβ and Pyk2 (n = 5). d Representative fluo-4 fluorescent images of BMMs in different groups (day 3). Pseudo-color-labeled (purple) area represents cells that are actively undergoing fluorescence ratio changes.

Experiments were done in triplicate; scale bar represents 200 μm. e Representative traces of three randomly chosen BMMs in different groups. The fluorescence ratio change was recorded every 5 s for 300 s. f Representative western blot images of CaMKKβ, Pyk2, c-FOS, NFATc1, and β-actin from BMMs in different groups (day 3). Quantification of normalized intensity of the western blot bands. The data in the figures represent the averages ± SD. Statistically significant differences between the treatment and control groups are indicated as *(p < 0.05) or **(p < 0.01).

desserts. According to a recent study regarding osteoporosis, the prevalence of osteoporotic fracture is lower in Chongqing district, at 14 %, compared to that of mainland China, at 26.6 % [35, 36]. These data motivated us to investigate whether XAT could be protective against osteoporotic bone loss. Compared to other osteoclastogenesis inhibitory antioxidants that are focused on ROS scavenging [37],

XAT showed additional suppression of RANKL-mediated Ca2+ oscillations, which makes it a more efficient inhibitor of osteoporosis. The results of the present study demonstrate that XAT treatment prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice. These effects resulted from the inhibition of RANKL-mediated osteoclastogenesis.

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Fig. 5 XAT prevents bone loss in OVX mice. a Representative microCT images of the longitudinal section of the femurs, cross-sectional view of the distal femurs, and reconstructed trabecular structure of the ROI (red dashed box). Color scale bar represents the bone mineral density level. (n = 5) b Quantitative microCT analysis of distal femoral volumetric bone mineral density (BMD), trabecular bone volume fraction (BV/TV), trabecular number (Tb. N), and trabecular separation (Tb. Sp) in each

group. c Representative images of TRAP-stained histological slides focusing on the metaphyseal region of the distal femur from mice of different groups. Scale bar represents 800 μm. (n = 5) d Quantitative analysis of OC surface/bone surface ratio. The data in the figures represent the averages ± SD. Statistically significant differences between the treatment and control groups are indicated as *(p < 0.05) or **(p < 0.01).

The results revealed that the underlying mechanisms of XAT that induced inhibition of osteoclastogenesis include ROS scavenging activity and suppression of Ca2+ oscillation signaling. This is the first report to describe the use of XAT in treating OVX-induced osteoporosis. XAT is a potential therapeutic agent for the prevention of postmenopausal osteoporosis.

References

Acknowledgments This work was funded by grants from the Nature Science Foundation of China (81572164), the National Key Technology Research and Development Program of China (2012BAI42G01), the National High-tech R&D Program of China (863 Program, 2015AA020315), Basic and Cutting-edge Research Program of Chongqing (cstc2014jcyjA10095) and Key project of Logistics Research Plan of PLA (BWS13C014).

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Compliance with ethical standards Conflicts of interest None 6. Ethics approval All experimental procedures were approved by the Third Military Medical University and performed according to the guidelines of laboratory animal care and use.

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