Bisphosphonate‐enoxacin inhibit osteoclast formation and function by abrogating RANKL‐induced JNK signalling pathways during osteoporosis treatment

Abstract Osteoporosis is an age‐related disease characterized by low mineral density, compromised bone strength and increased risk of fragility fracture. Most agents for treating osteoporosis focus primarily on anti‐resorption by inhibiting osteoclast activity. Bisphosphonate (BP) is a potent anti‐resorptive agent that has been used clinically for decades and is proven to be effective. However, BP has a variety of side effects and is far from being an ideal anti‐osteoporosis agent. BP selectively binds to calcium crystals, which are subsequently taken up or released by osteoclasts. Based on the action of BP, we previously demonstrated the inhibitory effect of a novel bone‐targeting BP derivative, bisphosphonate‐enoxacin (BE). In the current study, we used bone marrow‐derived osteoclast cultures to further assess the inhibitory effect of BE on osteoclastogenesis and employed reverse transcription PCR and real‐time PCR to examine expression of osteoclast‐specific genes. Additionally, we used bone resorption and F‐actin immunofluorescence assays to evaluate the effect of BE on osteoclast function and investigated the potential mechanisms affecting osteoclast differentiation and function in vitro. Furthermore, an ovariectomized (OVX) rat model was established to evaluate the therapeutic effects of BE on preventing bone loss. Results showed that BE exerted potent inhibitory effects on osteoclast formation and bone resorption by specifically abrogating RANKL‐induced JNK signalling, and that it preserved OVX rat bone mass in vivo without any notable side effects. Collectively, these results indicated that the BP derivative BE may have significant potential as a treatment for osteoporosis and other osteolytic diseases.


| INTRODUC TI ON
Bone homeostasis is maintained through two coordinated actions, osteoblast bone formation and osteoclast bone resorption, which is a process called 'bone remodelling'. 1 If the balance is disrupted, there will be an accordingly increase or decrease in bone mass. Excess osteoclast formation or activation is responsible for most osteolytic diseases, including osteoporosis, rheumatoid arthritis and aseptic osteolysis following total joint replacement. [2][3][4][5] Thus, osteoclasts are considered key targets in treating these diseases. Identification of agents that can modulate aberrant osteoclast formation and resorptive function is a viable strategy in the development of boneprotective therapies.
As first reported by Fleisch, bisphosphonates (BP) have high affinity for calcium crystals, leading to the impairment of their formation and dissolution in vivo and thereby inhibiting bone resorption. [6][7][8] As a result of this activity, BP has been used clinically over the past two decades as a treatment for osteoporosis and has been proven effective in improving bone strength and decreasing the risk of fracture. [9][10][11] However, other research has indicated that BP may be incorporated into bone during prolonged treatment, resulting in bone metabolism being affected for extended periods, even after the cessation of BP intake. 12,13 Although the deposition of BP in bone tissue is considered relatively safe, events such as gastrointestinal discomfort, atypical femora fracture and osteonecrosis of the jaw do occur. [14][15][16][17] Given these adverse side effects, the use of BP is somewhat limited and there is a clinical need to identify novel antiresorptive agents that can be developed for treating osteoporosis and other osteolytic diseases.
Enoxacin, a fluoroquinolone antibiotic, was reported to have the properties of inhibiting osteoclast formation and function by interfering the interaction between V-ATPase B2-subunit and microfilaments. 18,19 Furthermore, we previously reported that enoxacin has an inhibitory effect on osteoclastogenesis and reduces titaniumparticle-induced osteolysis through suppression of the JNK signalling pathway. 20 It is intriguing that this widely used antibiotic has these unexpected properties, which may translate into its clinical use for preventing osteoporosis and other osteoclast-related bone diseases. However, it is of concern that patients undergoing longterm treatment with enoxacin may experience adverse effects, such as dysbacteriosis and gastrointestinal discomfort. To overcome these potential difficulties, we developed a BP derivative of enoxacin (BE), aiming to target bone with the high affinity of BP to calcium crystals and thereby preventing the side effects associated with other systems.
Previous research has shown that BE has a significant inhibitory effect on osteoclast formation in vitro and that it is beneficial for preventing orthodontic tooth movement and alveolar bone resorption in vivo. 21,22 However, the potential mechanisms regarding the inhibitory effect of BE on osteoclastogenesis remain to be investigated. Furthermore, the efficacy of BE on preventing ovariectomized (OVX)-induced bone loss and the safety of this novel agent are currently unclear. Therefore, in the current study, we investigated the direct effect of BE on osteoclast formation and resorption function in vitro and compared it with zoledronate, a widely used nitrogen-containing bisphosphonate. We also explored the molecular mechanisms of BE activity and established an OVX rat model to evaluate this novel agent in vivo with respect to its safety, therapeutic effects on preserving bone mass, and effect on bone turnover. We found that BE functioned differently than BP and provided equal or better therapeutic efficacy, both in vitro and in vivo. Taken together, we believe that the use of BP derivatives may be a viable approach towards reducing the occurrence of side effects associated with long-term BP treatment and may reduce the occurrence of unsatisfactory situations when treating osteoporosis and other osteoclast-related bone destructive diseases. Specific antibodies against ERK, JNK, p38, IκBα, phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho-IκBα (Ser32) and GAPDH were obtained from Cell Signaling Technology. The tartrate-resistant acid phosphatase (TRAP) staining kit (Diagnostic Acid Phosphatase Kit) and all other reagents were purchased from Sigma Aldrich, unless stated otherwise.

| Cell culture and osteoclast differentiation assays
We harvested bone marrow monocyte/macrophage cells (BMMs) from four to six-week-old C57BL/6 mice, as previously described. 23 Briefly, cells were isolated from the femur and tibiae bone marrow and cultured for 24 h in T75 flasks containing α-MEM supplemented with 10% FBS, 1% penicillin/streptomycin and 10 ng/mL M-CSF. Non-adherent cells were removed, and the adherent cells were cultured in a 37 °C, 5% CO 2 incubator for an additional 3-4 days. When the BMMs were 90% confluent, they were washed three times with phosphate-buffered saline (PBS) and trypsinised. We then seeded the cells into 96-well plates at a density of 8 × 10 3 cells/well 24 in complete α-MEM supplemented with 30 ng/ml M-CSF, 50 ng/ml RANKL, 2.5 μM zoledronate (ZOL) or various concentrations of BE (0, 5 or 10 μM). The cell culture media were replaced every 2 days until mature osteoclasts formed. The cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 20 min and stained for TRAP using the Diagnostic Acid Phosphatase Kit. The cells were evaluated by light microscopy, and TRAP-positive cells with more than three nuclei were counted.

| Cell viability assays
To assess the cytotoxicity of ZOL and BE on BMMs, we performed CCK-8 assays. According to the manufacturer's instructions, BMMs were plated into 96-well plates at a density of 8 x 10 3 cells/well and cultured in complete α-MEM supplemented with 30 ng/mL M-CSF.
After 24 h, the cells were treated with different concentrations of ZOL and BE for 2 days. Next, 10 μl of CCK-8 buffer was added to each well and incubated at 37°C, 5% CO2 for an additional 2 h. We then measured absorbance at a wavelength of 450 nm (650 nm reference) using an ELX800 Absorbance Microplate Reader (Bio-Tek).
The following formula was used to calculate cell viability compared to control cells: (experimental group OD -zeroing OD)/(control group OD -zeroing OD).

| Resorption pit and F-actin ring immunofluorescence
We seeded BMMs onto bovine bone slices in 96-well plates at a density of 8 x 10 3 cells/well. The cells were then induced every other day with complete α-MEM supplemented with 30 ng/ml M-CSF and 50 ng/ml RANKL. Once mature osteoclast were observed, cells were subsequently treated with ZOL (2.5 μM) and different concentrations of BE (0, 5 and 10 μM) for another 48 h.
Cells treated with PBS were used as negative controls. Adherent cells on the bone slices were removed, and resorption pits were visualized using an FEI Quanta 250 scanning electron microscope (SEM). The percentage of absorbance area was quantified for the bone slices using Image J software (National Institutes of Health). In addition, we observed F-actin ring (Ruffled membrane of osteoclast) formation using immunofluorescence assays as a more in depth investigation of osteoclast bone resorption function. For this, BMMs were cultured and treated with ZOL, varying concentrations of BE on bovine bone slices as the same way.

| Reverse transcription and quantitative PCR
We seeded BMMs into 6-well plates at a density of 8 x 10 4 cells/ well and cultured them in complete α-MEM supplemented with 30 ng/ml M-CSF and 50 ng/ml RANKL. The media were changed every other day. The cells were then treated with 5 or 10 μM BE until mature osteoclasts formed. We isolated total RNA mature os-

| Western blot analysis
To evaluate protein expression, we seeded RAW264.7 cells into 6-well plates at a density of 5 × 10 5 cells/well. Once confluent, The membranes were then incubated overnight at 4 ℃ with primary antibodies diluted in TBS-Tween buffer containing 1% (w/v) skimmed milk powder. We then washed the membranes and incubated them with the appropriate secondary antibodies conjugated with IRDye 800CW (molecular weight 1162 Da). Antibody reactivity was detected by exposure using an Odyssey Infrared Imaging System (LI-COR).

| OVX rat model
To analyse the protective effects of BE on osteoporotic bone, we established an OVX rat model as previously described. 25,26 Briefly, 60 6-month-old female Sprague-Dawley (SD) rats were randomly divided into five groups (n = 12/group), including a sham-surgery group (Sham), mock drug-treated OVX rat group

| Evaluation of bone turnover markers
We analysed tartrate-resistant acid phosphatase (TRAP5b) and procollagen type I amino-terminal propeptide (PINP) and as bone turnover markers. TRAP5b is secreted by osteoclasts, and its levels are proportional to the number of osteoclasts. Therefore, TRAP5b is considered a reliable and sensitive marker of bone resorption. 27 Meanwhile, PINP is produced by osteoblasts and correlates with bone formation and can be used as a specific bone formation marker in postmenopausal osteoporosis. 28 In the current study, TRAP5b and PINP were quantified using ELISA assays (Rat MidTM Osteocalcin ELISA Kit and Rat TRAPTM Assay Kit, respectively; IDS Inc., Fountain Hills, AZ, USA), according to the manufacturers' instructions.

| Histomorphometric analysis
After micro-CT scanning, the distal right femurs were paraformaldehyde fixed, decalcified in EDTA buffer and paraffin embedded.
Sections (5 mm organs, including the hearts, livers and kidneys, were also sectioned and subjected to H&E staining as described for the femur samples.

| Bone formation analysis
We also harvest the left femurs when the animals were euthanized and dehydrated them for a week in graded concentrations of ethanol (70%-100%). We then embedded the specimens in polymethyl methacrylate (PMMA). Sagittal sections of the distal femurs were prepared and ground to approximately 30 μM thickness and then examined using a confocal fluorescence microscope for the presence of Alizarin Red S and Calcein green, which marked bone formation surfaces. The mineral apposition rate (MAR) was analysed using Image J software.

| Statistical analysis
All the data were expressed as means ± standard deviations (SD).
Data analysis was conducted using one-way analysis of variance (ANOVA). Pairwise comparisons were conducted using Student Neuman-Keuls (SNK) post hoc test to determine differences.
Statistically significant differences were considered as p < 0.05.

| BE inhibited RANKL-induced osteoclastogenesis in vitro without cytotoxicity
First, we used CCK-8 cell viability/cytotoxicity assays to examine the effect of BE and ZOL on BMM viability. M-CSF-dependent BMMs were treated for 96 h with different concentrations of BE and ZOL.
As shown in Figure 1A,C, BE and ZOL had no effect on the viability of BMMs at concentrations of ≤20 μM and ≤2.5 μM, respectively. To determine the stage that BE exerted its inhibitory affect, BMMs stimulated with RANKL were treated with BE during the early, middle and late stages of osteoclastogenesis. We treated BMMs with 0 or 10 μM BE at 3, 5 and 7 d of development. BE clearly inhibited osteoclast formation at all stages ( Figure 1H-J). This suggested that the inhibition of osteoclast differentiation by BE may be both dose and time dependent.

| BE attenuated osteoclastic bone resorption and F-actin ring formation
To determine whether BE impaired the function of osteoclasts, we eval-

| BE down-regulated osteoclast-specific gene expression
It is known that RANKL can up-regulate the expression of osteoclastrelated genes during osteoclast differentiation. 29 Therefore, to confirm the suppressive effect of BE on osteoclast formation, we evaluated the effects of BE on the expression of osteoclast-related genes TRAP, c-fos, NFATc1, V-ATPased2, V-ATPasea3, CTR, DC-STAMP and Cathepsin K. Compared with the control group, treatment with 5 and 10 μM BE inhibited expression of these genes in a dose-dependent manner ( Figure 3A,B). These findings confirmed that BE was able to downregulate osteoclast formation and osteoclast-specific gene expression.  (Figure 4A,B). These results suggested that BE treatment inhibited JNK phosphorylation during osteoclast differentiation. We then used Western blotting to investigate other signal pathways closely related to osteoclast differentiation, including p38, ERK and NF-κB signalling pathways. Interestingly, we found that when RAW264.7 cells were stimulated with RANKL, BE had no significant effect on signal transduction of the NF-κB pathway as it was related to proteins IκBα, p38 or ERK, an observation further supported by the quantitative data ( Figure 4C-F). These data suggested that BE primarily suppressed osteoclast formation by inhibiting the JNK signal pathway, without affecting p38, ERK or NF-κB.

| BE prevented OVX-induced bone loss in vivo by inhibiting osteoclast activity
To determine whether BE could prevent in vivo bone loss caused by oestrogen deficiency, we used the OVX rat model, which mimics week group compared to that in the 4-week group.
Next, we measured the level of TRAP5b released by osteoclasts into the serum, which is a well-known marker of osteoclast activity and bone resorption. Figure 7A,C shows that TRAP5b activity in OVX rats was significantly decreased in the BE-treated groups and ZOL-treated group. Moreover, the effect of BE on TRAP5b levels showed a dose-dependent inhibitory trend. A greater extent in the reduction of TRAP5b was observed in the high concentration BEtreated group compared to that in the low concentration BE-treated group.
Since PINP is a marker of bone formation, 31 we also measured PINP levels in the serum. Figure 7B,D shows that both the highdose and low-dose BE-treated groups and the ZOL-treated group had significantly higher levels of serum PINP compared to that in the untreated OVX group. Furthermore, PINP levels seemed to increase significantly in the BE-treated groups compared with that in the ZOL-treated group.
Fluorescent dyes injected in rats can specifically bind with mineralized tissues in vivo and auto-fluoresce during the osteogenic repair process. Therefore, the growth rate of new bone tissue in the defect area can be analysed based on the distance between the two fluorescent bands. As shown in Figure 7E Band intensity corresponding to P-JNK/JNK was quantitated and plotted using Image J software. (C-F) Western blotting was used to analyse protein expression levels of IκBα, phospho-ERK, ERK, phospho-p38 and p38. No differences were observed in the phosphorylation of the cascade between the control and BE-treated groups. The findings were confirmed that no significant differences existed in IκBα/GAPDH, p-ERK/ERK and p-p38/p38 quantities. All the experiments were carried out at least three times. The data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 vs. the control group that in the sham control group, which indicated that BE and ZOL effectively promoted new bone formation and significantly increased MAR ( Figure 7E-G). It is intriguing that both the low-dose and highdose of BE appeared to be superior in restoring bone loss compared to that of ZOL.

| BE in vivo treatment appeared to be safe in major organs
To evaluate the safety of BE and ZOL in vivo, we carefully monitored the condition of the rats at 4 and 8 weeks after intraperitoneal injection of BE and ZOL. No abnormal behaviour was exhibited by animals in any of the groups of rats. This indicated that BE and ZOL were safe and did not induce any obvious side effects in the rats.
Tissue sections of major organs including the heart, liver and kidney were stained with H&E 4 and 8 weeks after BE treatment and histopathologically evaluated. All the treated groups showed similar histological morphology to that of the sham control group with clear cell structures, normal nuclei morphology and no abnormal histological manifestations such as shrinkage, oedema and necrosis being found ( Figure 8). Additionally, as shown in Table 1 . The BE appears to have superior efficacy compared to that of ZOL in terms of the significant restored MAR. All the experiments were carried out at least three times. The data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 vs. the vehicle group had no effect. The results of these in vivo experiments suggested that BE was even safer than ZOL on the occurrence of side effects associated with long-term treatment.

| DISCUSS ION
Osteoporosis is a challenging diagnosis in the initial stages of the disease and is rarely diagnosed before a fracture occurs. 32 Osteoporotic fractures severely affect public health and increase medical costs, thereby being a significant economic burden on soci- We also used an in vivo osteoporosis model induced by oestrogen deficiency in ovariectomized rats, which causes reduced bone density and bone microstructure changes similar to those observed in humans. 45 We found that the BE-treated group and Subsequently, stimulation of microRNAs, rescue of p53 48 or stimulation of the JNK pathway may produce a variety of cellular effects that are distinct from BP action on bone.
In our current work, we observed that BE had an anti-osteoclastic effect, which indicates that it may have enormous potential for wide application in humans as an anti-osteoporosis treatment and even possibly as an anti-osteoclast-related osteolytic disease therapy. Certainly, there are several reasons for this assumption.
The primary reason for the minimal or non-existent toxicity seen in the rats after treatment with BE during the time frame of the Moreover, our observed success in vivo in part was based on the fact that treatment with BE prevented bone loss in OVX rats, but also because the histopathological analysis and a series of biochemical indicators tests showed that BE did not induce any noticeable toxic effects on major organs, including the heart, liver and kidney. Thus, our study not only considered the efficacy of BE, but also systematically evaluated the safety of BE, both in vitro and in vivo.
Overall, our findings are novel and contribute towards the biological applications and clinical transformation of BE as a treatment for osteoporosis and other osteolytic diseases. However, there are some limitations to our studies. The pathological process of osteoporosis involves both osteoclastic bone resorption and osteoblastic bone formation.
While our present study confirmed that BE significantly inhibited osteoclast differentiation in vitro and in vivo and reduced OVX-induced bone loss, the effects of BE on bone formation and its exact mechanism still need to be fully addressed. Additionally, although we demonstrated that BE affected osteoclastogenesis by inhibiting the expression of NFATc1 and other osteoclast-related markers by targeting the JNK-MAPKs signalling pathway, the specific kinases involved need to be resolved.

| CON CLUS IONS
In summary, the results of our present study indicated that BE inhibited osteoclast differentiation and functioned both in vitro and in vivo.
We also found that BE inhibited the RANK-induced JNK signalling pathway and ultimately attenuated expression of the transcription factor NFATc1, resulting in the downregulation of osteoclast-specific genes and thereby inhibiting osteoclast formation. Moreover, to our knowledge, this is the first report of BE preventing OVX-induced bone loss under the premise that it highly safe both in vivo and in vitro and also provided the first real insight into the relevant mechanisms involved. We also demonstrated that BE was equal or superior at preventing and treating bone loss compared with zoledronate, which is widely used in anti-osteoporosis treatment. Furthermore, we showed that BE was even safer than zoledronate and may prove to be a feasible approach to reduce the occurrence of side effects associated with long-term treatment. Based on these findings, we believe that BE is a novel bone-targeting derivative with unique advantages and may provide a potentially new strategy in the treatment of osteoporosis.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation (No. 81601912, 81860404 and 81860405).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.