Article Cite This: J. Agric. Food Chem. 2017, 65, 9647-9654
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Sophoridine from Sophora Flower Attenuates Ovariectomy Induced Osteoporosis through the RANKL-ERK-NFAT Pathway Xiaoying Zhao,†,‡ Lijuan Mei,§ Jinjin Pei,§,∥ Zenggen Liu,§ Yun Shao,§ Yanduo Tao,§ Xiaoling Zhang,*,†,‡ and Lei Jiang*,§ §
Key Laboratory of Tibetan Medicine Research, Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining 810001, P. R. China † The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai 200025, China ‡ Department of Orthopedic Surgery, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200092, China ∥ Shaanxi Key Laboratory of Bioresources and Biology, Shaanxi University of Technology, Hanzhong 723001, P. R. China S Supporting Information *
ABSTRACT: An imbalance in osteogenesis and osteoclastogenesis is a crucial pathological factor in the development of osteoporosis. Osteoclasts (OCs) play a pivotal role in osteoporosis, whose new therapy exploration has been focused on the suppression of OC formation. Sophoridine is found from the Chinese traditional food sophora flower to exhibit anti-osteoporosis capacity by screening. This study is focused on its anti-osteoporosis mechanism evaluation. The anti-osteoporosis effect of sophoridine, (15 mg kg−1 body), was evaluated in ovariectomized (OVX) mice by monitoring changes in bone histomorphometry index, formation of osteoclasts from blood-derived mononuclear cells, and changes in the synthesis of pro-osteoclastogenic cytokines. Signal pathways were investigated by QPCR, Western blot, and immunofluorescence. Sophoridine has a significant anti-osteoporosis effect in vivo, which can inhibit RANKL-induced OC formation, the appearance of OC-specific marker genes, and OC marker protein in vitro. Mechanistically, sophoridine dose- and time-dependently blocks the RANKL-induced OC formation and the activation of ERK and c-Fos as well as the induction and nucleus translocation of NFATc1. Sophora flower might be a useful alternative functional food in preventing or treating osteoporosis. KEYWORDS: osteoporosis, sophora flower, sophoridine, osteoclast differentiation, MAPK, NFATc1
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ingredient in many cuisines such as sophora flower cranberry rice cakes (Figure 1C), sophora flower-egg pancakes (Figure 1D), sophora flower-pork dumplings (Figure 1E), and steamed sophora flower (Figure 1F). Sophora flower is also a traditional Chinese medicine listed in the pharmacopoeia of the People’s Republic of China. In some literature, it is reported that extracts of sophora flower have anti-osteoporosis capacity.9 However, the mechanism of the therapeutic effects on osteoporosis still remains unknown. It is very necessary to study the active compound responsible for the anti-osteoporosis effect. Sophoridine (SOP), the structure of which is shown in Figure 1G, is a natural quinolizidine alkaloid. SOP has been reported to have analgesia,10 antitumor,11 anti-inflammatory,12 and anti-hepatitis B virus13 effects in previous literature. Moreover, it has many of druggable advantages such as special chemical scaffold, flexible structure, high solubility, and good safety profiles, suggesting that it is an ideal lead compound for further exploring.11 In our previous study, sophoridine was separated from sophora flower and it was proved that SOP exhibited anti-
INTRODUCTION Osteoporosis is a progressive, age-related skeletal disorder characterized by increased bone resorption by osteoclasts, reduced bone mineral density, and increased fracture risk.1 Osteoclasts (OCs) are the only cells in the human body that are capable of bone resorption, and their formation and function are critical for both bone homeostasis and pathological bone loss disorders, including osteoporosis,2 rheumatoid arthritis,3 aseptic loosening of prostheses,4 multiple myeloma,5 and metastatic bone tumors.6 Excessive bone resorption resulting from enhanced osteoclast formation and/or function is the most important cellular cause responsible for osteolytic diseases. Therefore, osteoclasts are important targets for the treatment of the diseases above.2−7 Osteoclasts, formed by the differentiation and fusion of hematopoietic mononuclear precursor cells after stimulation of M-CSF and RANKL, are multinucleated giant cells responsible for bone-resorbing. After binding to receptor, Rankl can stimulate TRAF6 to activate many downstream signal pathways including AKT-PLCγ and MAPKs. This signaling will then promote the activation of NFATc1 and c-Fos, which are two of the most important transcription factors essential for osteoclast differentiation.8 Sophora flower (flower of Sophora japonica Linn.) is a very common food in China (Figures 1A and 1B). It is an essential © 2017 American Chemical Society
Received: Revised: Accepted: Published: 9647
August 7, 2017 October 23, 2017 October 23, 2017 October 23, 2017 DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
Article
Journal of Agricultural and Food Chemistry
For differentiation, cells were plated at 2 × 103 cells per well of 96well plates in DMEM supplemented with 75 ng/mL recombinant RANKL and 15 ng/mL M-CSF. For drug assays, SOP was added at different concentrations to the cell medium. Then cells were incubated in a humidified incubator at 37 °C with 5% CO2 and fed daily with RANKL and M-CSF medium for 7 days. MTT Assay. BMM cells were plated at 1 × 104 cells per well of 96well plates. BMM were plated at 1 × 104 cells per well of 96-well plates. After 12 h incubation, different concentrations of SOP were added to the plates. After further incubation for 3 days, cell amount was calculated with a microplate reader (Tecan, Männedorf, Switzerland). The proliferation percentage was calculated according to the following formula:
proliferation rate (%) =
Drug Induced Apoptosis Evaluation. Different concentrations of SOP (0, 5, 10, or 15 μg/mL) were added to plates of BMM cells for 48 h and then harvested. The SOP pretreated BMM cells were then stained with Annexin V-PE and 7-amino-actinomycin (7-AAD) for 15 min at room temperature respectively without light. Cell fluorescent labeling was activated at 488 nm, and signals were collected from 10,000 cells at 702/64 (670−735 nm) and 585/42 (564−606 nm) in a FACS Canto II (BD, NJ, USA). Results were analyzed with FlowJo software (BD, NJ, USA). TRAP Staining. The mature osteoclasts were first fixed with 4% paraformaldehyde solution for 60 s. Then the fixed cells were treated with 30 μL of TRAP staining solution prepared for 20 min according to the manufacturer’s instruction (Sigma, St. Louis, MO, USA). After staining, all the stained wells were washed with PBS solution 3 times. At last photomicrographs of all the wells were obtained by an Olympus microscope at 10.0 magnification. Western Blot. Proteins from SOP treated BMM cells or mature osteoclasts were extracted using RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with 50 μg/mL phenylmethylsulfonyl fluoride (Thermo Scientific, Waltham, MA, USA). 30 μg of total cell proteins was mixed with loading buffer and separated on 10% SDS−PAGE gels, and the proteins in the gels were electrotransferred onto nitrocellulose membranes (GE, Marlborough, MA, USA). Membranes were blocked for 1 h with TBS-Tween (TBST: 0.2% Tween-20, 0.05 M Tris, pH 7.5 and 0.15 M NaCl) containing 5% (w/v) BSA and then incubated with primary antibodies diluted in TBST containing 5% BSA for 12 h at 4 °C. Membranes were then incubated for 2 h at room temperature with the appropriate secondary antibodies after three washes with TBST. The blotted protein spots were determined in a Tanon LAS-4000 Gel Documentation System (Pujiang, Shanghai, China) after exposure to an ECL substrate (Millipore, Darmstadt, German). The relative expression of proteins was measured using ImageJ (NIH, Bethesda, MD, USA) and normalized to that of GAPDH. Q-PCR. Total RNA of SOP treated BMM cells or mature osteoclasts was isolated using the PureLin RNA Mini Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. 2 μg of total RNA was used to synthesize cDNA with OligodT primer and High Capacity RNA-to-cDNA Kit (Thermo Scientific, Waltham, MA, USA). PCR reactions were started with 1 μL of cDNA, appropriate primers (Supplementary Table 1), and 30 cycles of 94 °C for 40 s, the appropriate annealing temperature (Supplementary Table 1), and 72 °C for 40 s. QPCR was performed with SYBR Green Master Mix (Takara, Dalian, China) in combination with a 7500 RealTime PCR Detection System (Applied Biosystems, Foster City, CA, USA) using GAPDH for normalization. Immunofluorescence Confocal Microscopy. SOP treated BMM cells or mature osteoclasts were fixed for 4 min at room temperature with 4% paraformaldehyde. Then each well was permeabilized for 5 min with 0.1% Triton X-100 in PBS and nonspecific binding was blocked by incubating with 5% BSA in PBS for 30 min. After incubation with NFATc1 antibody dissolved in 5% BSA-PBS for 12 h at 4 °C, cells were washed with 5% BSA in PBS 3
Figure 1. Sophora flower and SOP. (A) Sophora flower is the flower of Sophora japonica Linn. (B) Sophora flower is collected in every April and May as an important crop. (C) Sophora flower is used to make a lot of food, such as sophora flower cranberry rice cakes, (D) sophora flower-egg pancakes, (E) sophora flower-pork dumplings, and (F) steamed sophora flower. Sophoridine (G) is an active compound in sophora flower.
osteoclastogenesis activities in vitro by screening. However, little is known about the effect of SOP against OVX-induced osteoporosis in vivo and the mechanisms associated with its activity. Here, we assessed the anti-osteoporosis evaluation assay with SOP in vivo and RANKL-induced osteoclast differentiation assay in vitro, with a focus on determining SOP’s mechanism of action and providing some basis for the further development of sophora flower.
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A492 (sample) × 100% A492 (control)
MATERIALS AND METHODS
Activity Evaluation, Work Associated Media, Reagents, and Antibodies. Media: Dulbecco’s modified Eagle’s medium (DMEM), Alpha modification of Eagle’s medium (α-MEM), FBS, L-glutamine, and penicillin/streptomycin were purchased from Gibco-BRL (Gaithersburg, MD, USA). Reagents and kits: Sophoridine, with molecular formula C15H24N2O, molecular weight 248.37, and CAS No. 6882-68-4, was purified from sophora flower. The purity of SOP is 99.2%. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Diagnostic Acid Phosphatase kit (TRAP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rhodamineconjugated phalloidin was from Thermo Scientific (Waltham, MA, USA). GST-RANKL (rRANKL), and macrophage colony-stimulating factor (M-CSF) was purchased from R&D Systems, Inc. (Minneapolis, MO, USA). Antibodies: Primary antibodies to p-ERK, p-P38, c-Fos, pAKT, NFATc1, AKT, p-P38, t-ERK, t-JNK, t-P38, GAPDH, and pJNK were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies against mice IgG and rabbit IgG were purchased from Signaling Technology (Danvers, MA, USA). All antibodies were used at the recommended dilutions. Isolation and Culture of Mice Bone Marrow Macrophage (BMM). BMM cells were isolated from FVB mice bone marrow and cultured in α-MEM medium containing 10% fetal bovine serum (FBS) and 5 ng/mL M-CSF overnight. Floating cells were collected and further cultured with α-MEM medium with 10% FBS and 5 ng/mL MCSF for 3 days. Then all the floating cells were washed away with PBS solution, and adherent cells were used for osteoclast formation. In the current study, all cells were cultured at 37 °C in 5% CO2. 9648
DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
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Journal of Agricultural and Food Chemistry
Figure 2. Anti-osteoporosis activity of SOP. SOP suppressed bone loss in OVX mice. (A) Representative micro-CT images in Sham mice, OVX mice, Sham mice with SOP treatment, and OVX mice with SOP treatment. (B) Quantitative statistics of bone histomorphometry factors BV/TV, Conn-Dens., Tb.N, Tb.Th, Tb.Sp, SMI, and BMD. (C) Quantitative statistics of CTX-I and OCN levels in serum. (D) Tibia section H&E staining of Sham mice, OVX mice, Sham mice with SOP, and OVX mice with SOP treatment. (E) Tibia section TRAP staining of Sham mice, OVX mice, Sham mice with SOP, and OVX mice with SOP treatment. (F) Quantitative statistics of number of TRAP+ve OC per bone surface (OC/BS) and area of TRAP+ve OC per bone surface (OC/BS). All data are shown as the mean ± SD. *p < 0.05, **p < 0.01. induce osteoporosis under chloral hydrate anesthesia, and the rest of the mice received a sham procedure in which the ovaries were exteriorized but not resected. All the mice were housed 5 per cage at 25 °C with a 12 h light/12 h dark cycle. All the in vivo study procedures were agreed upon by the Animal Ethics Committee of Chinese Academy of Sciences and were carried out according to Guidelines for the Care and Use of Laboratory Animals. Forty mice were allocated into four groups randomly: sham operation plus corn oil group (Sham Cn), OVX plus corn oil (OVX Cn), sham plus SOP solution (OVX SOP 15 mg/kg/d), and OVX
times and incubated with an Alexa Fluor-488 conjugated secondary antibody (Thermo Scientific, Waltham, MA, USA). After the NFATc1 was stained, F-actin was labeled with Rhodamine-conjugated phalloidin staining solution and then nuclei with Hoechst 33258 dye (Thermo Scientific, Waltham, MA, USA). Images were obtained with a NIKON A1Si confocal microscope with 40× (oil immersion) lenses and analyzed using Image-Pro software (GE healthcare, PA, USA). In Vivo Experiments. Animals. Forty SPF grade female FVB mice, 8 weeks of age, were obtained from the SLAC Company Limited (Pujiang, Shanghai, China). Bilateral oophorectomy was carried out to 9649
DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
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Journal of Agricultural and Food Chemistry
Figure 3. SOP would not affect BMM proliferation or induce apoptosis. (A) BMMs treated with a range of SOP concentrations for 48 h were stained with Annexin V-PE and 7-AAD and FACS used to determine the percentage of dead and apoptotic cells (quadrants 2 and 3) within each population. (B) Proliferation and survival curve for BMM exposed to SOP for 72 h.
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plus SOP solution (OVX SOP 15 mg/kg/d). The injection volume was 50 μL. SOP sample was dispersed in corn oil (vehicle) and was administered by intraperitoneal injection 8 h after the lights were turned on every day. Mouse body weights were recorded every week to adjust dosage. SOP administration began 4 weeks after surgery, and daily administration continued for 28 days until euthanasia. Blood Sample Analysis. Blood samples were collected after the mice were fasted (>12 h) and stored at −80 °C. Serum CTX-I and OCN levels were measured using ELISA kit (HCB, Ottawa, Canada). Histological Evaluation of Tibias. After careful separated from the muscle and connective tissue dissection, the entire tibias were fixed with 4% formaldehyde solution for 24 h. The tibias were then decalcified with 10% EDTA solution for 12 weeks. The decalcified tibias were embedded in paraffin. Coronal sections (5 μm thickness) were cut, and each section was stained by TRAP dye for 2 h. After the photograph of TRAP straining, each section was then strained by H&E dye (Thermo Scientific, Waltham, MA, USA). The number of TRAP+ve osteoclasts, number of TRAP+ve osteoclasts per bone surface (OC/BS), the area of TRAP+ve osteoclasts, and area of TRAP+ve osteoclasts per bone surface (OC/BS) were determined using Image Pro Plus software (GE Healthcare, PA, USA). Micro-CT Analysis. The proximal femurs were carefully separated from the muscle and connective tissue. The clean femurs were then fixed with 4% paraformaldehyde for 24 h. The fixed samples were subsequently washed with 10% sucrose solution 12 h later. Micro-CT data were collected with tube voltage of 60 kV, at a resolution of 9.088 mm, tube current of 220 mA, and exposure time of 800 ms in each of the 360 rotational steps. A typical examination consisted of selection of the examination volume, measurement, a scout view, off line reconstruction, automatic positioning, and evaluation. Three dimensional reconstructions were produced with two dimensional images using multimodal 3D visualization software (Inveon Research Workplace; Siemens, Munich, Germany). Parameters were calculated using an Inveon Research Workplace (Siemens) as follows: trabecular thickness (Tb.Th), structure model index (SMI), bone volume/total volume (BV/TV), trabecular separation (Tb.Sp), bone mineral density (BMD), trabecular connectivity density (Conn-Dens.) and trabecular number (Tb.N) in the trabecular region (0.5 to 1 mm distal to the proximal epiphysis) according to guidelines of the American Society for Bone and Mineral Research (ASBMR) Statistical Analysis. All statistical analyses were performed using ANOVA test. Results were represented as the mean ± SEM. Values were considered to be significant at *p < 0.05 and **p < 0.01.
RESULTS SOP Protects against OVX-Induced Osteoporosis. To show the clinical significance of our findings, we explored whether the SOP could treat osteoporosis effectively. Forty 4 week old female mice were divided into four groups of 10 mice: Sham Cn, OVX Cn, Sham SOP, and OVX SOP. As shown in Figures 2A and 2D, micro-CT and H&E staining showed that there was significant difference between Sham Cn mice and OVX Cn mice, whereas bone loss was blocked in OVX SOP mice treated with SOP for 28 days. Compared with the Sham mice, BV/TV, Conn-Dens., Tb.N, Tb.Th, and BMD of OVX Cn mice were reduced significantly, and Tb.Sp and SMI of OVX Cn mice increased significantly at the distal femur. This is the obvious symptom of osteoporosis, which means that the in vivo model is built successfully. However, SOP treatment for the OVX mice totally reversed this bone loss situation (Figures 2A and 2B). Compared with the OVX mice, BV/TV, ConnDens., Tb.N, Tb.Th, and BMD of OVX SOP mice increased significantly, and Tb.Sp and SMI of OVX mice were reduced significantly. This means that osteoporosis of OVX mice is greatly cured by SOP successfully. Animal serum ELISA test reveals that SOP can reduce the concentration of osteoclastogenesis marker CTX-1 instead of increasing osteogenesis marker OCN, which indicated that SOP treats osteoporosis only by inhibiting osteoclastogenesis. In addition, TRAP staining assay indicated that the number of TRAP+ve osteoclasts and area of TRAP+ve osteoclasts were reduced (Figures 2E and 2F), and the number of TRAP+ve OC per bone surface (OC/ BS) and area of TRAP+ve OC per bone surface (OC/BS) increased in the OVX group and reduced with SOP treatment (Figures 2E and 2F). TRAP staining results prove that SOP treats osteoporosis by inhibiting osteoclast formation in vivo, which is consistent with serum test results. Compared with the Sham Cn group, the Sham SOP group exhibited no differences in micro-CT or H&E assay, which indicated that the active compound SOP has no effect on healthy animals. SOP Cannot Induce BMM Cell Apoptosis. To study the anti-osteoclastogenesis activity of SOP, we had to confirm that the reduction of the formation of osteoclasts was not caused by cell apoptosis. The cellular apoptosis could be detected by 9650
DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
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Figure 4. SOP dose- and time-dependently inhibited osteoclast differentiation. (A) A representative image showing SOP’s dose-dependent inhibition of OC formation. BMMs were cultured for 5 days with rRANKL (75 ng/mL) in the presence of various concentrations of SOP and then stained for TRAP. (B) The number and average area (percentage of area) of TRAP+ve OC with ≥3 nuclei (mean ± SD of 3 experiments). (C) The effect of different times of addition of SOP (15 μg/mL) during the 5-day process of OC formation from rRANKL treated BMM. (D) The number and average area (percentage of area) of TRAP+ve OC with ≥3 nuclei (mean ± SD of 3 experiments). (E) Relative expression of OC marker gene mRNAs (CTSK, NFATc1, CTR, OSCAR, and DC-STAMP) from 5 day rRANKL-induced BMM incubated in the presence of various concentrations of SOP. The levels of mRNA were assessed by QPCR, normalized to β-actin expression, and converted to a fold of control. *P < 0.05, **P < 0.01 compared to control.
(CTR), and osteoclast associated, immunoglobulin-like receptor (OSCAR) dose-dependently (Figure 4E). To determine at which stage of OC formation SOP exerted its inhibitory effects, the administration of 15 μg/mL SOP started at day 0 (the day RANKL was added), day 2 (2 days after RANKL was added), day 4 (4 days after RANKL was added), and day 5 (5 days after RANKL was added) in different groups. Neither in the day 4 nor day 5 treatment groups was significant altered number or size of TRAP+ve OCs observed compared to the CN group (Figures 4C and 4D). However, when SOP was added on the day of RANKL stimulation (day 0), the number or size of TRAP+ve osteoclasts was greatly reduced (Figure 4C). If SOP was added 2 days after RANKL stimulation, the decrease could also be observed, but the inhibition effect was weakened (Figure 4C). Collectively, these results indicate that, in order to suppress osteoclast formation effectively, SOP has to be added during the early stages and throughout the course of RANKL-induced differentiation. SOP Inhibits RANKL-Induced MAPK Activation in Vitro. To study the mechanism of SOP responsible for its inhibitory capacity on the differentiation of BMM into osteoclasts, we next examined the effects of SOP on the MAPK and AKT signal pathways. Within 15 min of RANKL stimulation of BMM, three MAPK family members, ERK, JNK, P38, and AKT show increased phosphorylation (Figure 5D). By comparison, cotreatment of BMM with RANKL and
measuring the expression of Annexin-V on the cell surface, which is routinely used as a marker of cellular apoptosis. Therefore, after treatment with SOP of different concentrations, we used flow cytometry to assess the number of Annexin-V positive cells. It is clearly demonstrated from the results that 10 or 15 μg/mL SOP did not increase the number of Annexin-V+ve BMMs compared to control cells (Figure 3A). On the contrary, at a higher dose (20 μg/mL), an increased Annexin-V+ve cells group was observed, indicating that SOP induces apoptosis at 20 μg/mL. In the next work, 20 μg/mL was set as the highest concentration. We set 12 h, 24 h, 36 h, and 48 h as four time points to evaluate the inhibitory effect of SOP on BMM cell proliferation (Figure 3B). In the concentration range 10 μg/mL to 90 μg/ mL, the reduction of BMM cell proliferation cannot be observed compared to untreated control group. The results indicate that SOP is nontoxic for BMM cell in the safe concentration range. The inhibitory effects of SOP on osteoclast differentiation were not due to the cytotoxic effect in the concentration range 10 μg/mL to 90 μg/mL. SOP Inhibits Osteoclast Differentiation. SOP suppressed the area occupied by and the formation of TRAP+ve osteoclasts dose-dependently (Figures 4A and 4B). Consistent with this, SOP also suppressed the expression of OC marker genes including cathepsin K (CTSK), calcitonin receptor 9651
DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
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Journal of Agricultural and Food Chemistry
Figure 5. OC differentiation marker proteins and pathway detection. Extracts from BMMs treated with different concentration of SOP (0, 5, 10, or 15 μg/mL) with RANKL (75 ng/mL) for the indicated times were subject to Western blot analysis and membranes immunoblotted with antibodies to various components of the MAPK and AKT (D) pathway. Extracts from BMMs treated with different concentration of SOP (0, 5, 10, 15, or 20 μg/mL) with RANKL (75 ng/mL) for 4 days were subject to Western blot analysis and membranes immunoblotted with antibodies to various components of the NFATc1 and c-FOS protein (A). The levels of protein expressions were normalized to GAPDH expression and converted to a fold of control (B, C, E, F, G, H).
μg/mL and SOP 15 μg/mL, number or size of OCs was greatly
different concentration of SOP (15 μg/mL) could significantly inhibit RANKL-induced phosphorylation of ERK at 15 min (Figure 5D). SOP Attenuates c-Fos and NFATc1 Expression. NFATc1 is the most important transcriptional regulator of osteoclast formation, which is dependent on c-Fos signaling and the MAPK pathways.14 RANKL stimulation of BMM could increase c-Fos protein expression after 4 days (Figure 5A). The downstream transcriptional targets of NFATc1 and CTSK were greatly induced 4 days after RANKL stimulation (Figures 4E and 5A). Coculturing with SOP (15 μg/mL) significantly inhibited the expression of c-Fos and NFATc1 in RANKL treated BMM (Figure 5A). These results indicate that SOP can inhibit RANKL-induced activation of c-Fos and NFATc1 signal pathways. SOP Suppresses RANKL-Induced NFATc1 Expression but Not Translocation. The key transcriptional regulator NFATc1 needs to translocate from the cytoplasm to nucleus in order for transactivation of target genes to occur. We employed immunofluorescence microscopy to study the nuclear translocation of NFATc1. Treatment with SOP significantly reduced the expression of NFATc1, which led to the reduction of osteoclasts. Although SOP could dose-dependently inhibit number or size of OCs, it could not reduce nuclear localization of NFATc1. It is easily observed that in groups of CN, SOP 10
reduced but green NFATc1 molecules still concentrate upon nuclei (Figure 6). These results indicate that SOP suppressed osteoclastogenesis by reduced activation of the ERK signaling
Figure 6. SOP could not inhibit NFATc1 nuclear localization. BMMs cultured on glass coverslips treated with rRANKL and different concentration of SOP (0, 5, 10, or 15 μg/mL) for 5 days were fixed and stained with DAPI (nuclei), NFATc1, and rhodamine-phalloidin (F-actin) and examined under immunofluorescence confocal microscopy. 9652
DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
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Journal of Agricultural and Food Chemistry to inhibit the expressions of NFATc1, but cannot influence the nucleus translocation of NFATc1.
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DISCUSSION Osteoporosis is associated with severe complications, such as skeletal deformity and increased bone fractures; therefore, efficient treatments for osteoporosis are required urgently.15 Nonequilibrium of bone metabolism caused by overactivation of osteoclastogenesis are reasons for a lot of bone lytic disorders.16 Osteoclasts are considered as one of the most important targets for the exploration of new therapies.17 In this study, we have found for the first time that SOP, a chemical compound in ordinary food sophora flower, exerts antiosteoporosis activity in vivo and anti-osteoclastogenic effects in vitro via suppression of MAPK pathway and c-Fos leading to attenuation of NFATc1 expression and transcriptional activity. We confirmed the significant therapeutic effect of SOP on OVX-induced osteoporotic mice in vivo. To confirm the therapeutic target of SOP, we found that number of TRAP+ve OC per bone surface (OC/BS) and area of TRAP+ve OC per bone surface (OC/BS) of SOP group significantly decreased compared with the OVX Cn group (Figures 2E and 2F); this was the first evidence that SOP targets osteoclasts. Then, we tested the CTX-I (osteoclast marker protein) and OCN (osteoblast marker protein) in the serum. It was found that CTX-I decreased much in the SOP treated group compared with the OVX group but OCN did not change (Figure 2C), which indicated that the activities of osteoclasts were inhibited but the osteogenesis remained the same. This was the second evidence that SOP targeted osteoclasts. So the next in vitro experiments focused on the effect of SOP on osteoclasts. Consistent with the in vivo results, RANKL induced osteoclast formation was suppressed by SOP. At the same time, expression of OC marker genes such as CTR, OSCAR, and CTSK was greatly attenuated following SOP treatment (Figure 4), which supports this perspective that SOP has antiosteoclastogenesis activities. The anti-osteoclastogenesis effects of SOP are also time dependent (Figure 7). It is found that with the initial administration of SOP at day 0 and day 2 after RANKL stimulation, OC differentiation was inhibited well, but at day 4 or day 5, SOP had no effect on osteoclast formation. The decreased suppressing effect indicates that early signals have already induced the expression of NFATc1 and that OC precursors have become fully committed to the OC differentiation pathway. All in all, current results suggest that downregulation of NFATc1 by SOP is likely to be the result of inhibition of signals from the ERK and c-Fos pathways. SOP only has the ability to inhibit the expression of NFATc1 indirectly by suppressing the upstream signaling pathway but cannot directly inhibit NFATc1 expression. Once the proper inhibition time (day 0, day 1, and day 2) is missed or the upstream signaling pathway has already been activated, SOP has no effect on OC formation. Imbalances in bone homeostasis result in exacerbated osteoporosis. Finding more chemicals that reduce this excessive bone resorption is central for exploring new therapies. In the current research, we shed new light on the role of SOP in osteoclast differentiation and found that treatment with SOP is a promising and novel therapeutic approach for osteoporosis. Mechanically, we have demonstrated that SOP exhibited antiosteoclastogenic capacity by attenuating RANKL induced activation of ERK and c-Fos, and sequentially then leading to
Figure 7. Mechanism of SOP anti-osteoclast-formation activity. SOP dose- and time-dependently blocked the RANKL-induced activation of ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation.
the decreasing expression of NFATc1, the most important factor for regulating osteoclastogenesis. The results of this research reveal that SOP may exhibit its therapeutic effect of anti-osteoporosis by inhibiting OC formation, and SOP may potentially be applied for the treatment of osteoporosis.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03666. Table of primers (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*No. 23 Xinning Road, Xining, Qinghai, China. Tel: +86 15297217193. E-mail:
[email protected]. *No. 320 Yueyang Road, Shanghai, China. E-mial: xlzhang@ sibs.ac.cn. ORCID
Lei Jiang: 0000-0003-4701-8112 9653
DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
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This work was supported by grants from Natural Science Foundation of Qinghai (No. 2016-ZJ-942Q), West Light Foundation of the Chinese Academy of Sciences (No. Y629071211), National Natural Science Foundation of China (No. 81572123, No. 31701243), Science and Technology Commission of Shanghai Municipality (No. 14431900900, No. 115411951100, No. 116430723500), and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No. 20161314). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED BMMs, bone marrow-derived macrophages; BV/TV, bone volume:tissue volume ratio; CTR, calcitonin receptor; CTSK, cathepsin K; DC-STAMP, dendritic cell-specific transmembrane protein; H&E, hematoxylin and eosin; M-CSF, macrophage colony-stimulating factor; micro-CT, microcomputed tomography; RANKL, receptor activator of NF-κB ligand; Tb.N, trabecular number; Tb.Sp, trabecular space; Tb.Th, trabecular thickness; TRAF6, TNF receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase; Conn-Dens, trabecular connectivity density; Tb.Sp, trabecular separation; BMD, bone mineral density; SMI, structure model index; OSCAR, osteoclast associated and immunoglobulin-like receptor; MAPK, mitogen activated protein kinases; NFATc1, nuclear factor of activated T cells cytoplasmic 1; RANK, receptor activator of nuclear factor kappa B; SPF, specific pathogen free
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DOI: 10.1021/acs.jafc.7b03666 J. Agric. Food Chem. 2017, 65, 9647−9654
