Curcumin has immunomodulatory effects on RANKL‐stimulated osteoclastogenesis in vitro and titanium nanoparticle‐induced bone loss in vivo

Abstract Wear particle‐stimulated inflammatory bone destruction and the consequent aseptic loosening remain the primary causes of artificial prosthesis failure and revision. Previous studies have demonstrated that curcumin has a protective effect on bone disorders and inflammatory diseases and can ameliorate polymethylmethacrylate‐induced osteolysis in vivo. However, the effect on immunomodulation and the definitive mechanism by which curcumin reduces the receptor activators of nuclear factor‐kappa B ligand (RANKL)‐stimulated osteoclast formation and prevents the activation of osteoclastic signalling pathways are unclear. In this work, the immunomodulation effect and anti‐osteoclastogenesis capacities exerted by curcumin on titanium nanoparticle‐stimulated macrophage polarization and on RANKL‐mediated osteoclast activation and differentiation in osteoclastic precursor cells in vitro were investigated. As expected, curcumin inhibited RANKL‐stimulated osteoclast maturation and formation and had an immunomodulatory effect on macrophage polarization in vitro. Furthermore, studies aimed to identify the potential molecular and cellular mechanisms revealed that this protective effect of curcumin on osteoclastogenesis occurred through the amelioration of the activation of Akt/NF‐κB/NFATc1 pathways. Additionally, an in vivo mouse calvarial bone destruction model further confirmed that curcumin ameliorated the severity of titanium nanoparticle‐stimulated bone loss and destruction. Our results conclusively indicated that curcumin, a major biologic component of Curcuma longa with anti‐inflammatory and immunomodulatory properties, may serve as a potential therapeutic agent for osteoclastic diseases.

induced osteolysis in vivo. However, the effect on immunomodulation and the definitive mechanism by which curcumin reduces the receptor activators of nuclear factor-kappa B ligand (RANKL)-stimulated osteoclast formation and prevents the activation of osteoclastic signalling pathways are unclear. In this work, the immunomodulation effect and anti-osteoclastogenesis capacities exerted by curcumin on titanium nanoparticle-stimulated macrophage polarization and on RANKL-mediated osteoclast activation and differentiation in osteoclastic precursor cells in vitro were investigated. As expected, curcumin inhibited RANKL-stimulated osteoclast maturation and formation and had an immunomodulatory effect on macrophage polarization in vitro. Furthermore, studies aimed to identify the potential molecular and cellular mechanisms revealed that this protective effect of curcumin on osteoclastogenesis occurred through the amelioration of the activation of Akt/NF-κB/NFATc1 pathways. Additionally, an in vivo mouse calvarial bone destruction model further confirmed that curcumin ameliorated the severity of titanium nanoparticle-stimulated bone loss and destruction. Our results conclusively indicated that curcumin, a major biologic component of Curcuma longa with anti-inflammatory and immunomodulatory properties, may serve as a potential therapeutic agent for osteoclastic diseases.

| INTRODUC TI ON
Total joint replacement (TJR) has achieved great success in the field of orthopedic surgery in the last few decades. TJR can reduce the pain and restore the joint function of patients with severe joint diseases. 1,2 However, wear debris-mediated bone loss and the consequent implant loosening remain major obstacles for TJR. 3 Studies have demonstrated that wear debris from the implant materials, including titanium particles (TiPs), ultra-high molecular weight polyethylene particles and CoCrMo particles, enhance the production of proinflammatory cytokines and the activation of osteoclastic signalling pathways. 4,5 Finally, the balance between osteoblasts and osteoclasts becomes disrupted, resulting in pathological osteolytic diseases.
As mentioned, wear debris-stimulated inflammatory reactions and a proinflammatory microenvironment are critical for the activation of osteoclast precursor cells and the subsequent osteoclast formation. 6,7 Therefore, inhibiting the inflammatory response and reducing the release of proinflammatory cytokines are considered an effective strategy for preventing and treating wear debris-mediated periprosthetic osteolysis. In recent years, the immunoregulation of macrophage polarization was identified as a critical mechanism in the process of implant loosening. 8 Wear debris-stimulated M0 macrophages turn into proinflammatory macrophages (M1 phenotype) and subsequently produce of proinflammatory chemokines. This proinflammatory microenvironment triggers the activation of osteoclastic signalling pathways and induces the differentiation of the hematopoietic monocyte/macrophage linage. However, M2 phenotype macrophages secrete anti-inflammatory cytokines and create an anti-inflammatory microenvironment, which subsequently inhibit the differentiation and formation of osteoclasts. [9][10][11] It has been reported that the regulation of macrophage polarization can mitigate wear debris-stimulated osteolysis. 8 Similar to this finding, in our previous study, we reported that curcumin ameliorated inflammatory reactions by inhibiting M1 polarization in a mouse air-pouch model. 12 Therefore, we have been suggested that the modulation of M1/M2 macrophages represents a potential therapeutic strategy to alleviate wear debris-mediated osteolysis.
Curcumin, a polyphenol and a major biologic component in the extract of the root of Curcuma longa, exhibits biologic properties because of its anti-inflammatory, antioxidant, anti-tumour and antimicrobial activities. [13][14][15] We have previously reported that curcumin attenuated polymethylmethacrylate-stimulated bone destruction in mice and alleviated Ti particle (TiP)-stimulated inflammation by modulating macrophage polarization. 12,16 In addition, previous studies have suggested that curcumin exerted a protective effect on bone disorders and inflammatory diseases, such as osteolysis, rheumatoid arthritis and osteoporosis. 17,18 Moreover, curcumin alleviates the up-regulation of nuclear factor-kappa B (NF-κB) phosphorylation in bone loss, which is associated with the functional state of osteoclasts. 15 Previous studies have shown that several osteoclastic signalling pathways, including the mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB) and phosphatidylinositol 3-kinase/AKT (PI3k/Akt) pathways, are regulated by the activation of receptor activators of nuclear factor-kappa B ligand (RANKL) and RANK and that cross-talk occurs in these pathways. However, the definitive mechanism by which curcumin ameliorates RANKLstimulated osteoclastogenesis and up-regulates osteoclastic signalling pathways is unclear. Based on previous findings, we have been suggested that curcumin prevents the formation of an inflammatory microenvironment by regulating the ratio of M1/M2 macrophages and then further attenuates RANKL-mediated osteoclast maturation and formation via suppressing the related osteoclastic signalling pathway.
The purpose of this study was to evaluate the immunomodulatory effects of curcumin on wear debris-stimulated inflammatory responses and identify the definitive mechanism by which it affects osteoclastogenesis, and classical mouse and cell models were used to provide a reliable basis for clinical applications in the future. Therefore, the direct anti-osteoclastogenesis and immunomodulatory effects of curcumin on RANKL-stimulated osteoclast differentiation and TiP-mediated changes in macrophage polarization in vitro were investigated. Furthermore, the responsible mechanisms at the cellular level were also explored by Western blotting. Then, micro-computed tomography (micro-CT) and histological staining were used to investigate the therapeutic effectiveness of curcumin in vivo in a mouse calvarial model, and immunofluorescence staining was used to evaluate macrophage polarization in vivo.

| Osteoclast precursor cell isolation and culture
Bone marrow-derived macrophages (BMMs) and RAW264.7 macrophages were used for in vitro experiments. BMMs were obtained from the bone marrow of 4-week-old male C57BL/6J mice and cultured in complete α-MEM containing 10 ng/mL M-CSF for 1 day.
Then, the suspension cells were resuspended and incubated with 30 ng/mL M-CSF for 3 days. The BMMs were used for further experiments at approximately 80% confluence. RAW cells were cultured in complete DMEM.

| Cell viability assay
The BMMs (1 × 10 4 ) were seeded on a 96-well plate and cultured in complete α-MEM containing 30 ng/mL M-CSF for 1 day. The medium was replaced with fresh complete medium containing various concentrations of curcumin (0, 0.5, 1. 25, 5, 10, 20, 30, 40, 50 or 100 μmol/L) in the next day. After culturing for 3 days, the medium was replaced with fresh complete α-MEM containing 10% CCK-8 solution and the cells were cultured for an additional 3 hours. A microplate reader was used to evaluate cell viability at a wavelength of 450 nm.

| Curcumin attenuated RANKL-mediated osteoclast maturation
Bone marrow-derived macrophages were used to investigate the direct anti-osteoclastogenic effect of curcumin on osteoclast formation. The cells were induced in complete medium supplemented with 30 ng/mL M-CSF, 100 ng/mL RANKL and 0, 1.25, 5 or 20 μmol/L curcumin. In addition, BMMs were plated and induced in osteoclastic induction medium and 20 μmol/L curcumin was added at day 0, 2, or 4, respectively. After 6 days, BMMs were rinsed three times and fixed for 15 minutes. A tartrate-resistant acid phosphatase (TRAP; Sigma) staining kit was applied to stain osteoclasts.

| F-actin ring formation and bone resorption area assays
To measure the functional state of osteoclasts, F-actin ring formation and osteoclastic resorption were assessed to evaluate the inhibitory effect of curcumin. BMMs were seeded and cultured as described above. After 6 days, the cells were fixed and permeabilized. Then, the cells were stained with phalloidin and DAPI for 15 minutes to visualize the cytoskeleton and nucleus, respectively. Fluorescence microscopy (Leica) was used to observe F-actin ring formation. In addition, BMMs were plated on an Osteo Assay Plate (OAP; Corning) and induced as described above. When mature osteoclasts were observed on day 4, the osteoclastic induction medium and 0, 1.25, 5 or 20 μmol/L curcumin were replaced and cultured for an additional 2 days. At day 6, osteoclasts were removed via sonication, and the resorption pits were observed with a light microscope (Leica). The percentage of the bone resorption areas was measured using Image-Pro Plus software.

| Osteoclastic-related gene expression
Bone marrow-derived macrophages were seeded and incubated in osteoclast induction medium containing 0, 1.25, 5 or 20 μmol/L curcumin for 5 days. Furthermore, the expression of osteoclasticrelated genes with or without curcumin pre-treatment at different stages was also investigated. Briefly, cells (1 × 10 5 ) were plated on a 6-well plate and cultured in osteoclast induction medium with or without curcumin (20 μmol/L) for 1, 3 and 5 days. TRIzol reagent (Invitrogen) was used to extract total RNA. Then, 1μg of total RNA was used to synthesize complementary DNA using M-MLV reverse transcriptase (Takara). SYBR Premix Ex Taq (Takara) was applied for quantitative gene analysis. Gene primers are shown in Table 1 with GAPDH as a housekeeping gene.

| Immunofluorescence staining of p65 in RAW264.7 cells
The cells were added to a 24-well plate and pretreated with or with-

| Analysis of macrophage polarization by ELISA and immunofluorescence staining
RAW264.7 cells were seeded and cultured as described above.

| Western blotting
After incubation for 2 days, RAW264.7 cells were pretreated with

| In vivo mouse calvarial osteolysis model
The

| Statistical analysis
All the data were analysed using SPSS 17.0 software and expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Student's t tests were used to evaluate the significance of differences. P < .05 or P < .01 was considered significantly different.

| Curcumin attenuates the differentiation and formation of osteoclastic precursor cells without cytotoxicity
To assess the cell toxicity of curcumin, we performed CCK-8 assays

| Curcumin interfered with the function of osteoclasts
Previous studied have indicated that typical F-actin rings indicate the functional state of osteoclasts and reflect the cytoskeletal integrity of osteoclasts. 21 Thus, we performed fluorescent staining to evaluate the potential effects of curcumin on F-actin rings and osteoclast fusion.
The results showed that curcumin markedly attenuated the formation

| Curcumin ameliorated the up-regulation of osteoclastic-related genes in vitro
The expression of osteoclastic-related genes such as c-fos, NFATc1,

| Immunomodulatory effect of curcumin on macrophage polarization in RAW264.7 cells
Recent studies have demonstrated the immunomodulatory effect of macrophage polarization on the regulation of inflammation reactions, leading to alterations in wear debris-induced osteolysis and bone loss. 8,24 Therefore, the immunomodulatory effects of curcumin on RAW264.7 cells were investigated using flow cytometry, ELISA, and immunofluorescence staining. The results of flow cytometry (Figure 4) showed that the M1-type macrophages decreased from 66.06% in the TiPs group to 43.04% in the TiPs + Cur group, whereas the percentage of M2-type macrophages was higher in the TiPs + Cur group (40.63%) than in the TiPs group (17.89%) and control group (7.66%). In addition, the results of immunofluorescence staining were highly consistent with the results of flow cytometry ( Figure 5A). The expression of the M1 marker CCR7 was higher in the TiPs group than in the other groups; however, the M2 marker Arg-1 was significantly induced by curcumin treatment. Furthermore, the secretion levels of proinflammatory and anti-inflammatory cytokines were measured by ELISA. The results indicated that the M1 cytokines TNF-α and IL-6 were markedly stimulated by TiPs treatment without curcumin, whereas the production of M2 cytokines (IL-4 and IL-10) was higher with curcumin intervention than that in the other groups ( Figure 5B-E). Collectively, these results indicated that curcumin has an immunomodulatory ability in RAW264.7 cells to regulate macrophage polarization.

| Curcumin alleviates osteoclast differentiation and functions by inhibiting the Akt and NF-κB pathways
The potential mechanisms of this inhibitory effect on RANKL-  Figure 6B,D). Immunofluorescence staining of p65 further verified this inhibitory effect on the NF-κB pathway ( Figure 6E).

| Curcumin exerted a protective effect on TiPstimulated bone destruction
A calvarial resorption model was established to assess the therapeutic effectiveness of curcumin for treating TiP-stimulated osteolysis in vivo, and the characteristics of TiPs are shown in Figure S2. The reconstruction images from micro-CT are presented in Figure 7A.  Figure S1).
Immunofluorescence staining of CCR-7 and Arg-1 was also performed to assess macrophage polarization in vivo. The results showed that the expression of the M1 marker CCR-7 (green) was clearly increased by TiPs stimulation in the positive control group, whereas the fluorescence intensity of the M2 marker Arg-1 (red) was the lowest in the TiPs group. However, the TiPs + Cur group had a lower proportion of CCR-7 (M1) macrophages and a higher proportion of Arg-1 (M2) macrophages following curcumin intervention ( Figure 8E). Thus, the results of macrophage polarization in mice were very consistent with these results in vitro.

| D ISCUSS I ON
Numerous studies have shown that the balance between osteoblast-induced osteogenesis and osteoclast-stimulated bone destruction is essential for the maintenance of bone homeostasis and that excess osteoclastic activity and activation lead to bone diseases such as osteolysis and osteoporosis. [28][29][30] Thus, it is necessary to inhibit osteoclast activity and formation to treat boneloss-related diseases. Curcumin, a major biologic component of

F I G U R E 6
Curcumin ameliorated the activation of Akt and NF-κB p65 phosphorylation but had no inhibitory effect on the MAPK pathways. (A and B), RAW264.7 cells were pretreated with or without curcumin for 4 h and then with 100 ng/mL RANKL for indicated time periods (0, 5, 15 or 30 min). Then, the cells were collected and lysed for Western blot analysis. C, The relative grey levels corresponding to p-ERK, p-JNK and p-p38 were quantified and were normalized to β-actin using ImageJ software. D, The relative grey levels corresponding to p-Akt, p-p65 and p-IκBα were quantified and normalized to β-actin using ImageJ software. E, RAW264.7 cells were pretreated with or without 20 μmol/L curcumin for 4 h and then stimulated by RANKL for 30 min. The cells were prepared for immunofluorescence staining of p65. F, RAW264.7 cells were cultured in osteoclast induction medium with or without 20 μmol/L curcumin for 1, 3 or 5 d. Cells were then collected and lysed for Western blot analysis. G, The relative grey levels corresponding to c-fos and NFATc1 were quantified and normalized to β-actin using ImageJ software. Data are presented as mean ± SD; *P < .05 and **P < .01 compared with the control group. Scale bar = 100 μm In our study, we demonstrated that curcumin ameliorated the activation of Akt and NF-κB p65 phosphorylation but had no effect on ERK, JNK and p38 phosphorylation, indicating that curcumin treatment had no inhibitory effect on the MAPK pathways.
The decrease in IκBα phosphorylation further confirmed that the NF-κB pathway was blocked following curcumin intervention. In addition, c-fos and NFATc1, two downstream transcription factors, were also markedly decreased at the gene and cellular levels following curcumin treatment.
Because the state of macrophage polarization is critical for the inflammatory microenvironment, the immunomodulatory effect of curcumin was also evaluated. Wang et al reported that probiotic treatment protected against CoCrMo particle stimulated osteolysis in mice by regulating the M1/M2 ratio. 8  and participate in the process of wear particle-mediated osteolysis.
The effectiveness and safety of curcumin in other cells will be investigated in our future studies.
To conclude, our results suggested that curcumin ameliorated the RANKL-mediated differentiation, fusion and maturation of osteoclasts and had an immunomodulatory effect on macrophage F I G U R E 9 Schematic illustration of curcumin has immunomodulatory and inhibitory effects on RANKLinduced osteoclast formation. Curcumin attenuated the up-regulation of Akt and NF-κB p65 phosphorylation and the activation of the downstream transcription factor NFATc1. In addition, curcumin created an immunomodulatory microenvironment and promoted macrophage polarization from the M1type to the M2-type phenotype polarization. Examinations of the potential molecular and cellular mechanisms revealed that this protective effect of curcumin on osteoclastogenesis was mediated by attenuating the up-regulation of Akt and p65 phosphorylation and the activation of the downstream transcription factor NFATc1. Using an in vivo mouse calvarial destruction model, it was further confirmed that curcumin ameliorated TiP-stimulated osteolysis and bone loss, thereby demonstrating its potential therapeutic agent for the treatment of osteoclastic disease.

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

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used to support the findings of this study are available from the corresponding authors' request.