Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL‐induced osteoclastogenesis

Abstract Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL‐induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F‐actin ring formation and tartrate‐resistant acid phosphatase (TRAP) staining in dose‐dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK‐related trigger RANKL by phosphorylation JNK/ERK/p38‐MAPK, IκBα/p65‐NF‐κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K‐AKT‐NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL‐induced RANK‐TRAF6 association and RANKL‐related gene and protein markers such as NFATc1, Cathepsin K, MMP‐9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.


| INTRODUC TI ON
Bone maintains in a homeostasis because of osteogenesis of osteoblast and osteoclastogenesis of osteoclast. 1 Once this condition upset and there will occur bone diseases, such as osteoporosis, osteopetrosis, rheumatoid arthritis and so on. Osteoporosis is a metabolic disease that could diminish bone mineral density, alter micro-structure and lead to much bone loss. Postmenopausal osteoporosis (PMOP) is the most familiar disease that occurred in older women owing to an imbalanced situation between osteoblast and osteoclast. PMOP usually results in osteoporotic fracture and body pain. 2 Oestrogen is very important for osteoprotectiving that can urge osteoblasts to secrete osteoprotegerin (OPG) and attenuate osteoclastogenesis, osteoclast precursor differentiation and osteoporosis formation. 3 Oestrogen deficiency has become an independent key factor of pathologic PMOP condition. 4 So, it is a wise therapeutic way to restrain osteoclast differentiation and formation by diminishing the progress of PMOP. 5 Osteoclasts are multinucleated huge cells that result from a monocyte-macrophage lineage by stimulation of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). 6 The cytokine M-CSF is a basis of osteoclast precursor to proliferation and survival; meanwhile, cytokine RANKL which comes from a tumour necrosis factor (TNF) family that regulates the interaction with its receptor RANK to exert the function of becoming mature osteoclast. 7 With the binding of M-CSF and extracellular signal-related kinase (ERK 1/2), osteoclast precursor gradual proliferation and survival. 8 With the binding of RANKL and its receptor RANK that recruit tumour necrosis factor receptor-associated factors (TRAFs), many intracellular signalling pathways were activated, including PI3K-AKT and ERK 1/2-MAPK signalling. 9 Nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) is the vital transcriptional factor for osteoclast and its related transcriptional genes, such as matrix metalloproteinase 9 (MMP-9), cathepsin K (CTSK), calcitonin receptor (CTR) and tartrate-resistant acid phosphatase (TRAP). 10 Subsequently, the NFATc1 and osteoclast are activated and promoted intracellular Ca 2+ releasing. 11 Thus, based on former works, suppressing RANKL-induced osteoclastogenesis maybe a good idea to cure PMOP.
Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae). 12 It has been used to as a partial agonist of high-affinity acetylcholine receptors to withdraw nicotine cessation. 13 From the former research, we found that Cytisine has several pharmacological functions, such as anti-depressant like property, 14 anti-diabetes, 15 antivirus, 16 against human influenza virus A (H1N1) 16 and against cerebral ischemia-reperfusion 17 properties and so on. Furthermore, there has been reported that Cytisine alleviated liver fibrosis via PI3K/AKT/Smad pathway. 18 In addition, Cytisine has been implied to exert anti-tumour effects on lung cancer cells by regulating reactive oxygen species-induced and MAPK, NF-κB signalling pathways 19 and inhibited human breast cancer cells through apoptosis-induced activity. 20 However, both the function of treatment for osteoporosis and the mechanism by Cytisine suppresses OCs have not yet been studied. So, we managed this study to detect whether Cytisine has the protecting function of ovariectomy-induced bone loss and the possible deeper molecular mechanism.
In this study, we illustrated the phenomenon of Cytisine suppressed RANKL-induced osteoclastogenesis and its possible mechanism in vitro. We implied that Cytisine inhibited osteoclast-precursor become mature osteoclast and decreased bone absorption via impacting NF-κB, MAPKs and PI3K/AKT signalling pathways. In addition, our work showed that Cytisine can take a positive role in decreasing bone loss at oestrogen deficiency-induced osteoporosis mouse model. Thereby, our research showed that Cytisine may have latent effects against osteolytic diseases, implying its value of curing for osteoporosis.

| Cell viability assay
We used a cell counting kit-8 (CCK-8) assay to detect cell viability at the guide of manufacturing protocol. The cells were cultured into 96-well plates (5 × 10 3 cells/well for RAW 264.7 and 1 × 10 4 cells/well for BMMs, respectively) and incubated with RANKL (50 ng/mL) and different concentration of Cytisine at 37℃ for 24, 48 and 96 hours. After every time point treatment that the plate was washed with PBS and added 100 μL/well CCK-8 reagent and incubated at 37℃ for 1 hour. The absorbance of the wells was detected at 490 nm by a micro-plate reader (Thermo Fisher, Waltham, MA, USA).

| In vitro osteogenesis assay
To detect the function of Cytisine on the differentiation and mineralization of osteoblast, BMSCs cells were isolated from femurs of six-week-old C57BL/6 mice. Bone marrow was washed by PBS till the bone marrow cavity bleached and sent to centrifugate (1000 g, 10 minutes, 4℃). The extracted cells were planted at 9 cm dish plates for 24 hours, then collected the unattached cells for osteoclastogenesis assay and the adherent cells passaged 2-3 generations for the present study. The BMSCs' differentiation to osteoblast was induced by complex culture medium (α-MEM media with 10% FBS, 1% antibiotic mixture of penicillin and streptomycin, 100 nmol/L dexamethasone, 50 µmol/L ascorbic acid and 10 mmol/L glycerophosphate). Cytisine (25 μmol/L and 12.5 μmol/L) was added into the above medium during the differentiation. Alkaline phosphatase (ALP) assay and alizarin staining assay were executed to mark the osteoblast differentiation and mineralization through ALP and alizarin staining kit (Beyotime Biotechnology, Shanghai, China) separately.     Table 1.

| Western blotting
To determine the effect of Cytisine on RANKL-induced osteoclast Silverwater, Australia). The membranes were blocked with 5% skim milk at room temperature for 2 hours and incubated at 4℃ overnight with one of the following two groups primary antibodies: NFATc1, CTR, MMP9, TRAP and CTSK for first group while p-JNK, p-ERK, p-p38-MAPK, p-IκBα, p-p65(NF-κB), p-PI3K, p-AKT and NFATc1 for second group. Subsequently, membranes were incubated with accordance of secondary antibodies for 1 hour at room temperature. Protein bands were tested through an enhanced chemiluminescence (ECL) liquid (Teye, Bioteke Cor. Beijing, China) and system (Amersham Pharmacia Biotech, Sydney, NSW, Australia).

| Immunoprecipitation
To make sure whether Cytisine can impact RANKL-induced relationship between RANK and TRAF6, the immunoprecipitation was performed. In a word, RAW264.7 cells (1 × 10 6 cells/ well) were planted into a 6 cm-dish and cultured with or without Cytisine 25 μmol/L for 3 hours. After stimulated by RANKL (100 ng/mL) for a half hour, then all the cells were lysed in EP-40 assay buffer and centrifuged at 6000 g for 10 minutes at 4℃. Take out of 50 μL supernatant liquid protein of per microtube for WB Input assay, then the RANK or TRAF6 antibody was added into the remaindered supernatant and incubated at 4℃ overnight with rotation. Protein A or G agarose beads were added and cultured at 4℃ for 3 hours with rotation. After centrifugation (1100 g, 4℃, 5 minutes) at 4 times, added loading buffer and boiled protein at 95℃ which the proteins were collected to Western blot.

| Animals and experiments
All experiments were executed in the Specific Pathogen Free

| Bone histological analysis
The left femurs were excised from all mice and fixed in 4% paraformaldehyde at room temperature for 4 days and decalcified in 10% tetrasodium-ethylenediaminetetraacetic acid (T-EDTA) for 2 weeks.
Then, femur bone samples were paraffin-embedded, sectioned 4 μm thickness and prepared with a microtome (Jung, Heidelberg, Germany). Haematoxylin and eosin (H&E) were executed to measure trabecular bone. TRAP staining was performed to detect osteoclast in the distal femur metaphysis bone tissue. All standard histologic bone slices measures were analysed by microscope which primary magnification was × 10 (BX53, Olympus), and all data were calculated by Image J software (NIH).

| Miro-computed tomography analysis
The right femurs were measured by micro-computed tomography (Skyscan1172, Bruker, Kontich, Belgium). The trabecular bone selected from distal femur metaphysis were scanned at follow-

| Statistical analysis
All experiments were performed at least three times. All results were expressed as means ± SD. Statistical analysis was measured by GraphPad Prism version 7.0 software (GraphPad software, San Diego, CA, USA). Intergroup and intragroup comparisons were executed using one-way ANOVA followed by Tukey test. Values of P < 0.05 were considered statistically significant.

| Cytisine inhibited BMMs and RAW264.7 cells into osteoclast in vitro
The chemical structure of Cytisine is shown in Figure 1A. To explore the effect of Cytisine to RANKL-induced osteoclastogenesis, we used two standard osteoclast differentiation in vitro.

| Cytisine suppressed RANKL-induced osteoclast differentiation only at early stage
Bone marrow monocytes cells or RAW264.7 cells transfer osteoclast is a multistep progress which contains proliferation, differentiation, cell fusion and multinucleation and so on. It is necessary to detect which stage that Cytisine could suppress osteoclast differentiation.
The Cytisine was added to culture at beginning on day 1 to day 7 for BMMs cells (Figure 2A,C) and day 1 to day 4 for RAW264.7 cells  Figure 2B,D). The results showed that Cytisine inhibited osteoclastogenesis totally at day 1 to day 3 for BMMs cells and day 1 to day 2 for RAW264.7 cells; meanwhile, there was no suppressible effect to exposure precursor osteoclast at later stage.

| Cytisine suppressed RANKL-induced F-actin ring and bone-pit formation
To further examine the effect of Cytisine on osteoclastogenesis, we determined whether Cytisine effected osteoclast fibrous actin To deep examine, whether Cytisine suppressed bone pit formation function by using biomimetic synthetic bone surface of osteoclast. BMMs cells were planted into 48-well plates which coated with bone slices and treated with or without Cytisine for 72 hours. The osteoclast activity was weakened obviously owning to Cytisine treatment, and the pit-forming of bone slices was inhibited severely at 12.5 μmol/L and 25 μmol/L (Figure 3B,F,G). The same situation of TRAP staining for OCs of bone slices ( Figure 3C).
Therefore, the bone pit formation activity and TRAP staining for hydroxyapatite-coated plates of the mature osteoclast were both attenuated by Cytisine.

| Cytisine had no effect on differentiation and mineralization of osteogenesis in vitro
As is known, bone sustains homeostasis and remodelling owning to a balance which between osteogenesis of osteoblast and osteoclastogenesis of osteoclast. To explore the effect of differentiation on osteoblast, we executed ALP and alizarin red staining assay for BMSCs cells. As results displayed, there was no significant effect differentiation and mineralization compared with control group of Cytisine on osteoblast ( Figure S1A,B) by using Cytisine at experiment dosage range of 0 to 25 μmol/L ( Figure S1C,D). Thus, all data demonstrated that Cytisine had no effect of osteogenesis on differentiation and mineralization in vitro.

F I G U R E 4
Cytisine down-regulated osteoclastogenesis gene and protein marker expression. A, Real-time PCR was performed onto RAW264.7 cells to detect Cytisine with or without RANKL that focused on osteoclast-related gene expression of Cathepsin K, MMP-9, TRAP and NFATc1. The expression levels of these genes were normalized with GAPDH. B, Western blotting was measured to discover Cytisine with or without RANKL onto RAW264.7 cells that focused on osteoclast-related protein marker expression of Cathepsin K, CTR, TRAP, MMP-9 and NFATc1 at different time point. C, Quantification of osteoclast-related protein expression at different time point according to Western blotting. Data are presented with mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001

| Cytisine suppressed RANKL-induced osteoclast-related gene expression
Osteoclast differentiation and function because of the expression of some related symbol gene, such as NFATc1, Cathepsin K, MMP-9 and TRAP, which base sequence was listed in Table. 1. We deter-

| Cytisine suppressed RANKL-induced osteoclast-related protein expression
Osteoclast differentiation and function cannot be separated from the expression of a lot of related symbol protein, such as Cathepsin K, CTR, MMP-9, TRAP and NFATc1. So, we determined the infect of Cytisine on RANKL-induced NFATc1 and other related protein by Western blot. As our expected, results indicated that RANKL increased above protein expression at day 1 or day3 and Cytisine prominently diminished CTR and TRAP at day 1 while suppressed Cathepsin K, MMP-9 at day 3. Interestingly, the Cytisine suppressed protein expression of NFATc1 from day 1 to day 5, especially at day 1. (Figure 4B,C).

| Cytisine inhibited RANKL-induced activation of MAPK signalling pathway
It is important that RANKL-induced MAPK activation for osteoclas-

| Cytisine suppressed RANKL-induced activation of NF-κB signalling pathway
Besides of the MAPK signalling pathway, NF-κB signalling pathway may take part in the modulation of osteoclast proliferation and function. To determine whether Cytisine suppresses NF-κB-mediated osteoclastogenesis, two different ways were performed for NF-κB activation. Firstly, we have been suggested that Cytisine could suppress RANKL-induced phosphorylation and degradation of NF-κB/ IκBα and NF-κB/p65. The results of Western blot assays were shown in Figure 5C,D that with the RANKL stimulation, phosphorylation of IκBα and p65 were activated at 15 or 30 minutes, respectively; then, both of them were inhibited almost at the same time point after added Cytisine. Secondly, the immunofluorescence staining was performed of location for p65 in the presence or absence of RANKL on RAW 264.7 cells. As shown in the Figure 5E, most p65 were inactive, unphosphorylated and located in the cytoplasm without of RANKL.
Whereas, with the presence of RANKL, almost all p65 was transferred into nucleus after 30 minutes of stimulation while this phenomenon was blocked when Cytisine and RANKL were incubated together.
However, we examined the ratio of fluorescence intensities which indicated the fluorescence intensity at nuclear site relative of whole-cell fluorescence intensity ( Figure 5F). Therefore, the results implied that Cytisine could suppressed RANKL-induced activation of NF-κB signalling pathway.  Figure 6A,B, the phosphorylated PI3K, phosphorylated AKT and total NFATc1 which belong to PI3K-AKT-NFATc1 pathway illustrated a significant increase on RANKL stimulation. Whereas, this phosphorylation was partly suppressed with the treatment of Cytisine. Therefore, these results implied that Cytisine can suppress RANKL-induced activation of PI3K-AKT-NFATc1 signalling pathway.

| Rescue assay of osteoclastogenesisrelated protein and PI3K-AKT-NFATc1 pathway by activator SC79
Through above work, we found that Cytisine can suppress RANKLinduced osteoclast-related protein and activation of PI3K-AKT-NFATc1 signalling pathway. Whether is there something could reverse this inhibited situation? So, we select the AKT activator SC79 to detect if it has the rescue effect for osteoclastogenesis-related protein and PI3K-AKT-NFATc1 pathway. As we expected, firstly, we found that osteoclast-related proteins were up-regulated after added the SC79 into Cytisine group reduced by RANKL through Western blot ( Figure 6C). Secondly, we also observed that the activation of p-PI3K, p-AKT and NFATc1 were all up-regulated after interfered with SC79 and RANKL ( Figure 6D). Lastly, the rescued phenomenon for BMMs cells (Figure 6E-a,b,c,d) and RAW264.7 cells ( Figure 6E-e,f,g,h) which induced by RANKL combination of Cytisine with or without SC79 ( Figure 6E) were similar to former results. Thus, above results illustrated that the activator SC79 combination with RANKL can rescue the negative situation of Cytisine for RANKL-induced osteoclast-related protein expression and PI3K-AKT-NFATc1 pathway.

| Cytisine suppressed RANKL-induced RANK-TRAF6 association
From the above results, the MAPK, NF-κB and PI3K-AKT pathways showed us the similar regulation of molecule mechanism. It was known that the TRAF6 which was recruited by RANK while activated by RANKL, we then wanted to detect whether Cytisine affected the relationship of RANK and TRAF6 through coimmunoprecipitation assay. RAW 264.7 cells were planted into the 6 cm dish at presence or absence of Cytisine and then added with or without RANKL. The cells lysates were harvested for immunoprecipitation with anti-RANK and then blotted with anti-TRAF6 ( Figure 6F).
The results illustrated that RANKL could promote the relation between RANK and TRAF6; however, Cytisine can inhibit this action.
Interestingly, Cytisine can also suppress the action of RANK and TRAF6 at absence of RANKL, implying that Cytisine maybe has an effect for RANKL-induced recruitment of TRAF6 by RANK at endogenous cells.

| Cytisine prevented bone loss by suppressing osteoclast activity in vivo
According to above study of Cytisine inhibited RANKL-induced osteoclast in vitro, it is necessary to explore the potency in vivo that using OVX mice model to mimic post-menopause osteoporo-  Figure 7B.
We further explored whether Cytisine suppressed OVX bone loss via inhibited osteoclast differentiation. Compared with OVX model mice, the H&E staining for trabecular density ( Figure 7C,D,F) and TRAP-positive multinucleate cells in distal femurs were significant ameliorated of OVX + Cytisine-treated mice ( Figure 7E). According to data, that Cytisine prevented bone loss in OVX mice by suppressing osteoclast activity more than by encouraging osteoblast in vivo.

| D ISCUSS I ON
In our present study, we illustrated that Cytisine could suppress bone loss by diminishing the activity of osteoclastogenesis in vivo and vitro. Furthermore, we introduced the effect of Cytisine on oestrogen deficiency osteoporosis and bone metabolism which via ovariectomy mice model to mimic the whole process. These downstream typical pathways like as MAPK, NF-κB and PI3K-AKT were activated by RANKL during osteoclast precursor cells differentiation and function which were inhibited by Cytisine completely (Figure 8).
Because of undoubted effect of Cytisine on protecting bone mineral, according to its structure, it is necessary to detect whether the Cytisine has better anti-osteoporotic activities.
Oestrogen deficiency is the general phenomenon in older women which could cause bone mineral loss, pathological fracture and increased formation and activation of osteoclast. 21 Continuous and gradually bone resorption of osteoclast could lead to metabolic bone disorder, such as PMOP. 22 Thus, suppressing osteoclast differentiation and activation may be a potential strategy for PMOP treatment. 23 Therefore, to inhabit oestrogen withdrawing and osteoclast activation may provide us a choice for PMOP treatment and restoration.
According to amount of previous reports, numerous biological plant active monomers root in nature products has been studied to display inhibitory effects on osteoclast activation and differentiation. [24][25][26][27] Cytisine is an alkaloid that was found in plants and seeds of the Leguminosae (Fabaceae) family and has been shown to have several multipotent pharmacological activities. [13][14][15][16][17][18][19][20]   NFATc1 is an important transcription factors in osteoclast differentiation. 10 NFATc1 is an NFAT family member that has been reported to modulate expression of several osteoclast-related genes including TRAP, cathepsin K and MMP-9. 33 In pro-osteoclast cells, NFATc1 can accelerate transcriptional processes. Therefore, the factor NFATc1 deficiency could cause severely osteopetrotic phenotypes. 34 These key genes were all up-regulated by RANKL-induced osteoclastogenesis and were down-regulated by Cytisine treatment, indicating that Cytisine affected NFATc1 and downstream gene expression.
Because of osteoblasts are vital for bone formation, structure and mineralization, [35][36][37][38] the effect on osteoblast of Cytisine was managed at our study. However, there were no significant statistics between Cytisine treatment group and control group below  Figure 7C, 250 μm in Figure 7D and 200 μm in Figure 7E. Data are presented with mean ± SD. *P < 0.05, **P < 0.01 Owning to without saving the serum of mice, so there are deficiency data of serum levels for OCN, OPG, Runx2 and osteogenesis related gene markers, so we have responsibility to explore the relationship of Cytisine and osteogenesis in the future. However, followed the whole study data, we speculated that the level of OPG may be down-regulated in the OVX model group, while be up-regulated in the OVX + Cytisine treatment group.
To sum up, our study illustrated that Cytisine could act as an effective and novel agent for PMOP by inhabiting partly cellular signalling pathway. This may supply a reference to exploit novel therapeutic effect drugs for osteoclast-associated disorders. University, Shanghai, China) for technical guidance and support.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data, models and code generated or used during the study appear in the submitted article.