Stachydrine prevents LPS‐induced bone loss by inhibiting osteoclastogenesis via NF‐κB and Akt signalling

Abstract Osteoclast overactivation‐induced imbalance in bone remodelling leads to pathological bone destruction, which is a characteristic of many osteolytic diseases such as rheumatoid arthritis, osteoporosis, periprosthetic osteolysis and periodontitis. Natural compounds that suppress osteoclast formation and function have therapeutic potential for treating these diseases. Stachydrine (STA) is a bioactive alkaloid isolated from Leonurus heterophyllus Sweet and possesses antioxidant, anti‐inflammatory, anticancer and cardioprotective properties. However, its effects on osteoclast formation and function have been rarely described. In the present study, we found that STA suppressed receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL)‐induced osteoclast formation and bone resorption, and reduced osteoclast‐related gene expression in vitro. Mechanistically, STA inhibited RANKL‐induced activation of NF‐κB and Akt signalling, thus suppressing nuclear factor of activated T cells c1 induction and nuclear translocation. In addition, STA alleviated bone loss and reduced osteoclast number in a murine model of LPS‐induced inflammatory bone loss. STA also inhibited the activities of NF‐κB and NFATc1 in vivo. Together, these results suggest that STA effectively inhibits osteoclastogenesis both in vitro and in vivo and therefore is a potential option for treating osteoclast‐related diseases.

Osteoclasts, which are derived from hematopoietic monocyte/ macrophage precursors, are multinucleated giant cells that are mainly involved in bone resorption. Macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factor-kappa B (NF-κB) ligand (RANKL) are the key cytokines involved in osteoclast differentiation. The binding of RANKL to its receptor RANK promotes the recruitment of adaptor molecules called tumour necrosis factor receptor-associated factors (TRAFs), which activate downstream signalling pathways, including NF-κB and mitogen-activated protein kinase (MAPK) pathways. [6][7][8] This leads to the activation and accumulation of two pivotal transcription factors involved in osteoclast differentiation, namely, c-Fos and nuclear factor of activated T cells c1 (NFATc1). 9,10 These factors induce the expression of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), cathepsin K (CTSK) and calcitonin receptor (CTR), leading to the formation of mature osteoclasts. 11,12 Subsequently, mature osteoclasts undergo polarization and structural changes to produce a tight sealing zone. Secretion of acids and proteolytic enzymes into this sealing zone results in the dissolution and degradation of the underlying bone. 13,14 Several recent studies have focused on natural compounds because of their pharmacological activity against human diseases. Over several years, our group has screened natural compounds for treating osteoclast-related diseases. [13][14][15][16] Stachydrine (STA), a bioactive constituent extracted from a medicinal herb Leonurus heterophyllus Sweet, has extensive pharmacological properties, including antioxidant, anti-inflammatory, anticancer and cardioprotective properties. [17][18][19][20] Recent studies have reported that STA inhibits NF-κB and MAPK pathways in different cell types. [20][21][22] As the NF-κB and MAPK pathways are crucial for osteoclastogenesis and because STA exerts inhibitory effects on these pathways, we hypothesized that STA may represent a novel candidate for treating osteoclast-related diseases by inducing the targeted suppression of osteoclastogenesis.
In the present study, we first investigated the effects of STA on RANKL-induced osteoclast formation and bone resorption. Second, we assessed the effects of STA on crucial signalling events in RANKLinduced osteoclastogenesis to determine its molecular target. Third, we explored the therapeutic potential of STA in a murine model of LPS-induced inflammatory bone loss. Thus, our study determined the effect of STA on osteoclastogenesis and inflammatory bone loss and elucidated mechanisms underlying its mechanism of action.
Cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technology. TRAP staining kit, DMSO, SC-514 and other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.

| In vitro bone marrow-derived macrophage isolation and osteoclast differentiation
Bone marrow cells were obtained from the long bones of 8-week-old male C57BL/6 mice, as described previously, 23 and were differentiated into bone marrow-derived macrophages (BMMs) in α-MEM supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 25 ng/mL M-CSF at 37°C for 5 days in a humidified incubator with 5% CO 2 . Next, BMMs were seeded into 48-well plates (density, 1 × 10 4 cells/well) in triplicate and were treated with various concentrations of STA (0, 12.5, 25, 50, 100 or 200 μmol/L) in the presence of 25 ng/mL M-CSF and 50 ng/mL RANKL. Culture medium was replaced every 2 days. After culturing for 5 days, the cells were fixed with 4% paraformaldehyde (PFA) and stained for TRAP according to the manufacturer's protocol. TRAP-positive multinucleated cells (nuclei number, ≥3) were counted using a light microscope (BX51; Olympus).

| Cell viability assay
The potential cytotoxic effects of STA on BMMs were determined by performing CCK-8 assay. BMMs were seeded in 96-well plates (density, 5 × 10 3 cells/well) in the presence of 25 ng/mL M-CSF for 24 hours. The cells were then treated with different concentrations of STA (0-800 µmol/L) for 48 or 96 hours. Next, 10 μL CCK-8 buffer was added to each well, and the plates were incubated at 37°C for 2 hours. Absorbance was measured using ELX800 absorbance microplate reader (BioTek) at a wavelength of 450 nm (reference wavelength, 650 nm).

| F-actin ring immunofluorescence assay and resorption pit assay
To observe F-actin ring formation, BMMs were cultured in the presence of 25 ng/mL M-CSF and 50 ng/mL RANKL for 4 days.
Next, an equal number of mature osteoclasts were seeded on an Osteo Assay Surface Multiple Well Plate (Corning) coated with hydroxyapatite. After culturing overnight to promote adhesion, the cells were treated with 0, 25, 50 and 100 µmol/L STA for 2 days.
Next, the cells were fixed with 4% PFA for 20 minutes and were permeabilized using 0.5% Triton X-100 for 20 minutes. The cells were then washed three times with PBS and were stained with rhodamine-conjugated phalloidin (dilution, 1:200; Invitrogen Life Technologies) diluted in 1% bovine serum albumin (BSA) for 30 minutes. Images of F-actin rings were captured using a fluorescence microscope (EU5888; Leica) and were analysed using ImageJ software (National Institutes of Health). To observe resorption pits, the cells adhering to the plates were removed by incubating with 5% NaClO for 10 minutes. Resorption pits were photographed using a light microscope (Olympus), and their areas were analysed using the ImageJ software.

| Western blotting analysis
To determine the main signalling pathways targeted by STA, BMMs were seeded in six-well plates (density, 8 × 10 5 cells/well) with complete α-MEM supplemented with 25 ng/mL M-CSF and allowed to adhere overnight. After treatment with DMSO or 100 μmol/L STA for 4 hours, the cells were stimulated with 50 ng/mL RANKL for 0, 5, 10, 20, 30 or 60 minutes. To examine the effects of STA on the expression of osteoclast-related markers, BMMs were seeded in six-well plates (density, 1 × 10 5 cells/well) and were stimulated with 25 ng/mL M-CSF and 50 ng/mL RANKL in the presence or absence of 100 μmol/L STA for 0, 1, 3 or 5 days. Next, the cells were washed twice with PBS, lysed using a radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) on ice for 30 minutes. Each protein lysate containing 30 μg protein was analysed on 8%-12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% skimmed milk for 1 hours, followed by overnight incubation at 4°C with the primary antibodies. After three washes, the membranes were incubated with appropriate horseradish peroxidaseconjugated secondary antibodies at 4°C for 2 hours. Signals were detected using an electrochemical luminescence reagent (Millipore) and were visualized using XRS chemiluminescence detection system (Bio-Rad).

| Analysis of NFATc1 nuclear translocation
Extracts for determining NFATc1 nuclear translocation were prepared from BMMs stimulated with 25 ng/mL M-CSF and 50 ng/mL RANKL in the presence or absence of 100 μmol/L STA for 0, 2 or 4 days.
Cytoplasmic and nuclear proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime), according to the manufacturer's protocol. Western blotting analysis was performed to detect NFATc1 abundance in the cytoplasmic and nuclear fractions, which was expressed as the ratio of NFATc1 level in control cells treated with RANKL alone. PCNA and β-tubulin were used as internal reference proteins for the cytoplasmic and nuclear fractions, respectively.

| Immunofluorescence analysis
To determine p65 nuclear translocation, BMMs were treated with 100 μmol/L STA for 4 hours, followed by stimulation with

| Histological and immunohistochemical analyses
The mouse tibias (n = 5 per group) were fixed in 4% PFA for 2 days, decalcified in 10% EDTA (pH = 7.4) for 4 weeks and embedded in paraffin. Next, the tibias were cut into 4-μm-thick histological sections for performing haematoxylin and eosin (H&E) and TRAP

| Statistical analysis
Data were expressed as mean ± SEM of at least three independent experiments. Statistical analysis was performed using Prism 6.01 (GraphPad Software). Differences between two groups were compared using a two-tailed unpaired Student's t test. One-way ANOVA with post hoc Newman-Keuls test was performed to analyse differences in multiple group comparisons. P < 0.05 was considered to be statistically significant.

| STA inhibits RANKL-induced osteoclast formation in vitro
The potential cytotoxic effect of STA on BMMs was determined by To clarify at which stage of osteoclastogenesis STA exerted its inhibitory effect, BMMs were treated with 100 μmol/L STA on days 1-3 (early stage), 3-5 (late stage) and 1-5 (early + late stage).
Treatment of BMMs with 100 µmol/L STA in the early stage markedly inhibited osteoclast formation ( Figure 1F)

| STA attenuates osteoclastic bone resorption and F-actin ring formation in vitro
Given that STA markedly suppressed osteoclast formation, we assessed its effect on the resorptive function of mature osteoclasts.
Mature osteoclasts were plated onto the Osteo Assay Plate and were treated with the indicated STA doses in an osteoclastogenic-inducing medium for 2 days. Optical images showed extensive resorption of the bone surface by osteoclasts in the control group (Figure 2A).
In contrast, bone resorption area was significantly reduced to 54% and 23% after treatment with 25 and 50 μmol/L STA, respectively; moreover, rare resorption pit was observed in the plate containing cells treated with 100 μmol/L STA (Figure 2A,B).
A tight F-actin ring is a prerequisite for osteoclastic bone resorption and is an observable marker of mature osteoclasts. 28,29 Immunofluorescence analysis indicated that STA disrupted the morphology and size of F-actin rings ( Figure 2C). Statistically, STA decreased the size of F-actin rings in a dose-dependent manner ( Figure 2D). Together, our results suggest that STA suppresses the bone resorptive activity and F-actin ring formation of mature osteoclasts in vitro.

| STA inhibits RANKL-induced NF-κB and Akt signalling
To determine the exact mechanisms through which STA inhibited osteoclastogenesis, we investigated the effects of STA on NF-κB, To determine the molecular target of STA, we explored upstream molecules involved in the RANKL-induced NF-κB pathway. We observed that STA suppressed IKKα/β phosphorylation ( Figure 4A,B) but did not affect TAK1 phosphorylation ( Figure 4D and Figure S1).
Next, we compared the efficiency of STA to SC-514, a well-established NF-κB inhibitor. Our data showed when compared to SC-514, STA exerted a similar inhibitory effect on RANKL-induced NF-κB signalling, and even slightly better inhibition on osteoclast differentiation ( Figures S2 and S3). To further determine the effect of STA on NF-κB activity in longer time-points, we performed NF-κB luciferase gene reporter assay. Treatment with 100 μmol/L STA was shown to inhibit NF-κB activity at 24 and 48 hours ( Figure 4E). The MAPK pathways (including ERK, JNK and p38 pathways) also play a crucial role in RANKL-induced osteoclastogenesis. 31 We observed that STA did not affect RANKL-induced activation of the ERK, JNK and p38 pathways ( Figure 4F and Figure S4), indicating that it did not affect the MAPK pathways.
As the Akt-GSK3β signalling pathway is important for RANKL-induced osteoclast differentiation, 32 we next investigated whether STA affected the activation of Akt and GSK3β. As shown in Figure 4G, STA inhibited RANKL-induced phosphorylation of Akt and GSK3β. Quantitative analysis confirmed these observations ( Figure 4H). To further confirm these results, we determined whether combining SC79 (an AKT agonist) can rescue osteoclast differentiation. As expected, osteoclast differentiation was suppressed in cells treated with STA alone. However, in cells co-treated with SC79, the impaired osteoclastogenesis was partly rescued, as indicated by an increased number and larger size of osteoclasts ( Figure 4I,J).

| STA alleviates bone loss by inhibiting osteoclast activity through NF-κB-NFATc1 signalling
Considering the importance of osteoclasts in inflammatory bone loss, we performed TRAP staining to investigate the effect of STA on osteoclast formation and activity in vivo. LPS induced the formation of TRAP-positive osteoclasts along the trabecular bone surface, whereas STA decreased the number of osteoclasts ( Figure 7A).  Previous studies have established that Akt-GSK3β-NFATc1 signalling cascade is also critical for osteoclast formation. 32 Furthermore, STA markedly inhibited NF-κB and NFATc1 activities in vivo, which was consistent with the in vitro results. These results obtained here provided evidence that STA exerts a therapeutic effect on inflammatory bone loss and this effect of STA is mainly mediated by suppressing osteoclast activity and NF-κB-NFATc1 signalling.

| D ISCUSS I ON
Despite these promising results, our study has some limitations.
First, LPS-induced inflammatory bone loss in vivo is a complex process involving many cell types. In the present study, we investigate the effects of STA on osteoclast formation and osteoclastic bone resorption. However, we cannot exclude the possibility that STA might affect osteoblastic bone formation. Therefore, further studies are needed to assess the effect of STA on osteoblastic bone formation.
Second, our study only focused on LPS-induced pathological bone loss, which is dominated by excessive osteoclast activity. Therefore, additional studies assessing the effects of STA on the normal bone or other pathological conditions are needed to completely elucidate the effects and mechanisms of action of STA in bone remodelling.
In summary, the present study is the first to show that STA inhibits both osteoclastogenesis and bone resorption in vitro. These inhibitory effects of STA are mediated by the inhibition of the RANKL-induced NF-κB and Akt signalling pathways, leading to suppression of the induction, accumulation and nuclear translocation of NFATc1. Furthermore, STA exerts protective effects against LPS-induced inflammatory bone loss in vivo, suggesting its potential for preventing or treating osteoclast-related diseases.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (No. 81772360 and No. 31771106).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y
All data used to support the findings of this study are available from the corresponding authors upon request.