inhibits the differentiation of osteoclasts

Background: Osteoclasts are large terminal-differentiated cells with multiple nuclei and are the only cells with bone resorption activity in the body. The abnormal migration and differentiation of osteoclasts may accelerate bone absorption, a crucial process in the occurrence and development of osteoporosis. Regulating the differentiation and activation of osteoclasts is a breakthrough point for the prevention and treatment of osteoporosis. Coumarin derivative of 3-(4-methoxyl)-1-(2-(4-coumarin) prop)-2-en-1-one (MCPEO) was selected in this study. We aimed to investigate the effects of (MCPEO) on osteoclast differentiation. Methods: Bone marrow mononuclear cells (BM-MNCs) were collected from 6-week-old ICR mice and inoculated in vitro. BM-MNC and RAW264.7 cells were induced by the receptor activator of nuclear factor κ B ligand (RANKL) to differentiate them into osteoclasts. The cells were then added with 0.01, 0.05, 0.1, 0.5, and 1 μM MCPEO. The cell viability of differentiated osteoclasts was analyzed by methylthiazolyldiphenyl-tetrazolium bromide assay. The differentiated osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. Miuse osteoclast activation was investigated by absorptive activity analysis. Filamentous actin (F-actin) staining was employed to identify the formation of F-actin rings in the differentiated mouse osteoclasts. The change level of critical transcription factors related to osteoclast differentiation was determined by Western blot analysis. Results: Data show that MCPEO affected the cell viability of differentiated osteoclasts, inhibited the formation of TRAP-positive polynuclear cells, and decreased the absorption activity and the formation of F-actin rings in the differentiated osteoclasts. Furthermore, MCPEO influenced the change level of crucial transcription factors related to osteoclast differentiation. Conclusions: MCPEO inhibited the differentiation of RANKL-induced BM-MNC and RAW264.7 cells into osteoclasts. TRAP staining into COAS (0.2 mL/well) osteoclast activity were seeded into 12-well (1 mL/well) for actin (F-actin) staining. After 7 0.01, and 1 μM MCPEO for


Background
Bones are not quiescent tissues but persistent metabolic connective tissues. Old bone matrix is constantly replaced by newly formed bone matrix through a continuous process known as bone remodeling, which is critical to maintain bone mass and strength [1]. Osteoclasts are derived from hematopoietic stem cells and are responsible for the absorption of old bones. These cells are derived from mesenchymal stem cells by secreting and mineralizing the bone matrix and can reconstruct the resorbed bone mass. The synergy between these two types of cells maintains bone metabolism balance [2,3]. Disparity in bone metabolism may lead to various skeletal related diseases, such as osteoporosis, rheumatoid arthritis, multiple myeloma, and aseptic prosthesis, which may be caused by the pathological increase or hyperfunction of osteoclasts [4]. Bone marrow stem cells, monocytes, macrophages, and mononuclear macrophages in various normal and abnormal (infection and tumor) tissues in the hematopoietic mononuclear-macrophage system can form osteoclasts and be stimulated by the parathyroid hormone, macrophage colony-stimulating factor, tumor necrosis factor-alpha, lipopolysaccharide, and interleukin. Osteoclasts are large terminal-differentiated cells with multiple nuclei and are the only cells with bone resorption activity in the body [5]. The abnormal migration and differentiation of osteoclasts may accelerate bone absorption, a crucial process in the occurrence and development of osteoporosis. Regulating the differentiation and activation of osteoclasts is a breakthrough point for the prevention and treatment of osteoporosis [6].
Coumarin is commonly found in higher plants, animals, and microbial metabolites and exhibits the advantages of low molecular weight, relatively simple synthesis, and high bioavailability [7]. Coumarins, flavonoids, lignans, and stilbenes are classified as phytoestrogens because their structures and functions are similar to those of animal estrogens. The main biological activities of coumarins include anti-menopausal, anti-osteoporotic, anti-tumor, anti-cardiovascular diseases, anti-dementia, anti-inflammatory, and anti-coagulation activities [8]. However, most of the bioactive mechanisms of coumarins and their derivatives remain unclear and unevaluated. For example, the mechanism of coumarins and their derivatives in inhibiting osteoporosis remains unknown [9]. Hence, scholars must continue to explore the biological activities of the effective ingredients in coumarins and their derivatives and synthesize and screen those with low toxicity for the treatment of several diseases.

MCPEO treatment
Six-week-old ICR mice were sacrificed through cervical dislocation and placed in 75% ethanol to instantaneously sterilize the body surface. The long bones of the limbs were separated, and the attached soft tissues were thoroughly removed. The bones were cleaned twice with aseptic PBS and placed in flat dishes. The joints of both ends of the medullary cavity were cut off, and the marrow cavity was washed repeatedly with serum-free α-MEM medium by using a 10 mL sterile syringe. Cell suspension was collected and centrifuged at 1000 rpm/min for 5 min. The cells were resuspended in a-MEM medium containing 15% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin, inoculated into six-well culture plates, and cultured overnight at 37°C and 5% CO2 under saturated humidity. The cell suspension was regarded as BM-MNCs. BM-MNC density was adjusted to 1×10 6 cells/mL and resuspended in α-MEM medium containing 100 ng/mL RANKL, which contained 15% IU/mL penicillin and 100 μg/mL streptomycin. BM-MNCs were seeded into 96-well culture plates (0.2 mL/well) for MTT and TRAP staining and into COAS (0.2 mL/well) for osteoclast absorption activity detection. BM-MNCs were seeded into 12-well culture plates (1 mL/well) for filamentous actin (F-actin) staining. After 7 days of cultivation, the cells in all groups were added with 0.01, 0.05, 0.1, 0.5, and 1 μM MCPEO and DMSO (solvent control, MCPEO was dissolved in DMSO) and cultured for another day.

RANKL induced osteoclast formation and MCPEO treatment
RAW264.7 cells were re-suspended in α-MEM medium containing 10% FBS, 2 mmol/L L-glutamine, 100 IU/mL penicillin, and 100 μg/mL/mL streptomycin. The cells were inoculated into 96-well culture plate (200 μL / well) for TRAP staining and MTT assay, and the cells were inoculated into six-well culture plate (2mL/ well) for total protein extraction and follow-up Western blot. At 4 h of incubation, RANKL was added into the culture. After another 3 days of induction, the cells in all groups were added with 0.01, 0.05, 0.1, 0.5, and 1 μM MCPEO and DMSO (solvent control, MCPEO was dissolved in DMSO) and cultured for another day.

Cell viability detection (MTT assay)
BM-MNC and RAW264.7 cells were induced by RANKL and treated with various MCPEO concentrations. After incubation, the culture medium was discarded, and the cells were washed three times with PBS. Serum-free DMEM containing 0.05 mg/mL MTT was added into each well, and the samples were incubated at 37°C for 4 h. The liquid was removed, and 150 μL of DMSO was added into each well. After 10 s of shaking, the optical density was recorded at 595 nm by using an enzyme-labeled meter.

Detection of osteoclast formation by TRAP staining
BM-MNC and RAW264.7 cells were induced by RANKL and treated with various MCPEO concentrations. At the end of incubation, the culture medium was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. TRAP staining was performed in accordance with the manufacturer's instructions. Under the inverted microscope, five visual fields were randomly selected from each well to count more than three nuclei TRAP-positive cells labeled as differentiated osteoclasts.

Analysis of the absorption activity of differentiated osteoclasts
The BM-MNCs were induced by RANKL and treated with various MCPEO concentrations. After incubation, the medium was removed, the COAS culture plates were washed with double-distilled water. The cells were cleaned using an ultrasonic cleaning instrument for 3 min. Hypochloric acid (10%) was added into each well of the plate, which was incubated at room temperature for 5 min. The COAS culture plates were washed with double-distilled water twice, air dried at room temperature

RAW264.7 cells were induced by RANKL and treated with various MCPEO
concentrations. At the end of culture, the cells were collected, the total protein was extracted, and the concentration was measured and adjusted. The protein samples were separated by SDS-PAGE and transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skimmed milk at room temperature for 60 min. c-Fos, p-c-Fos, c-Jun, p-c-Jun, NFATc1, and β-actin primary antibodies were added into the nitrocellulose membrane and incubated overnight on ice. The nitrocellulose membrane was then washed with TBST, and the second antibody was added and incubated at room temperature. Enhanced chemiluminescence was performed.

Statistical analysis
All experiments were repeated in triplicate, and data were presented as the mean ± standard error of the mean (SEM). The statistical differences between groups were evaluated by ANOVA, followed by Tukey's post-hoc test using SPSS v.17.0 software. Difference at P < 0.05 was considered statistically significant.

MCPEO affected the F-actin morphology in differentiated osteoclasts
The results of Phalloidin-TRITC staining showed that after 8 days of induction, the intact and regular F-actin rings appeared in the RANKL-stimulated BM-MNCs and in the RANKL + DMSO groups. MCPEO could inhibit the formation of F-actin rings in the differentiated osteoclasts. At specific concentrations (0.5 and 1 μM), this derivative could inhibit osteoclast differentiation and reduce F-actin ring formation (Fig. 4).

Effects of MCPEO on the change level of critical transcription factors related to osteoclast differentiation
During the differentiation of RAW264.7 cells into osteoclasts induced by RANKL, the expression levels of NFATc1, p-c-Jun, p-c-Fos, and c-Fos, which are the key transcription factors of osteoclast differentiation, were upregulated. However, MCPEO could inhibit their expression, and the inhibitory effects became increasingly evident with the increased MCPEO concentrations (Fig.5). Patients with osteoporosis are characterized with decreased bone density and increased fracture risk. Current treatments to promote bone synthesis include the use of estrogen receptor analogues and bisphosphonates as anti-bone resorption agents and specific drugs such as parathyroid hormones. However, given the side effects, some defects occur in the two schemes above [10]. Therefore, developing new anti-osteoporosis drugs is urgent.

Discussion
Coumarin derivatives can influence bone metabolism by regulating the differentiation of osteoblasts or osteoclasts, which show anti-osteoporosis effects. For example, osthole, a coumarin derivative from Chinese herbal medicine, can stimulate the differentiation of rat skull osteoblasts through bone morphogenetic protein (BMP)-2/ p38MAPK/Runx-2/osterix pathway [11]. In addition, osthole considerably promotes the formation of new bones on the surface of mouse skull through the β-catenin-BMP signaling pathway. Bone loss occurs in ovariectomized rats, and osthole can solve this problem by improving bone microstructure, tissue morphological parameters, and biomechanical properties [12]. Imperatorin and bergapten are coumarin derivatives that can increase the expression level of BMP-2 and promote bone formation through the p38 and ERK-dependent signaling pathways [13]. Coumarin-dihydropyridine hybrids can promote the formation of osteoblast in vitro and inhibit bone resorption in ovariectomized animal models. These hybrids can increase bone mineral density and volume, promote the expression of osteogenic gene, and increase bone formation and mineral adhesion rates [14]. Psoralen is a coumarin derivative that is extracted from Chinese herbal medicine, promotes the differentiation of osteoblasts in mouse skull by activating BMP signaling pathways, increases the expression level of osteoblast-specific genes, and enhances the activity of alkaline phosphatase [12]. Coumarin compounds, such as aesculin [15], wedelolactone [16], osthole [17,18], and cinnamoyloxy-mammeisin [19], can inhibit the differentiation and activation of osteoclasts and potentially treat osteoporosis.
The anti-osteoporosis biological activities of coumarin derivatives provide additional options for the treatment of bone metabolic disorders. MCPEO was synthesized in our laboratory, and its biological activity was preliminarily explored.
To provide the bases for the treatments of skeletal metabolic disorders, we determined the effects of MCPEO on osteoclast differentiation.
Osteoclasts have high activity of TRAP enzyme, which is involved in the bone resorption of these cells and can be used as a marker of their activity. The serum TRAP enzyme level is considerably increased due to the decreased estrogen level.
Bone resorption activity is stronger than bone formation in menopausal women. In addition, serum TRAP enzyme level is remarkably increased in patients with primary osteoporosis caused by aging and is negatively correlated with bone density. TRAP enzyme level can be used as a monitoring index for primary osteoporosis. TRAP staining is the simplest direct method to identify osteoclast differentiation. For TRAP staining, the cytoplasm of the osteoclasts is wine red, and the characteristics of multiple nuclei are evident. MCPEO can inhibit the differentiation of BM-MNCs into TRAP positive cells (osteoclasts). The results were consistent with the inhibitory effects of other coumarin derivatives on osteoclast differentiation [17,19].
Mature osteoclast plays a unique role in bone degradation, a physiological process that regulates bone tissue development and bone remodeling. Osteoclast is absorbed and migrates on the bone surface until apoptosis [20]. Sealing zones are first assembled in the osteoclast, which consists of dense podosomes, to form the F-actin rings and immobilize the cell on the surface of mineralized matrix. Podosomes and F-actin rings are rich and evident in normal osteoclasts. In the sealing zone, the plasma membrane of osteoclasts that is in contact with the bone is rearranged into a ruffled border. Proton and chloride ions are secreted from the cells, acidify the extracellular media, and dissolve the hydroxyapatite (inorganic components) in the bone. Acid proteases, such as cathepsin K and MMP9, and phosphatase, such as TRAP, are also produced by osteoclast and further degrade bone matrix proteins (organic components). Osteoclasts form new adhesion structures on the bone surface that are adjacent to the existing resorption lacunae to continuously exert its absorption activity and are repolarized to complete their bone resorption function [21,22].
MCPEO can suppress the bone resorption activity by inhibiting the formation of F-actin rings in osteoclasts. The results were consistent with the inhibitory effects of coumarin derivatives, such as wedelolactone and osthole, on osteoclast activation [16,18].
RANKL binds to the receptor activator of nuclear factor kappa B, which is located on the surface of osteoclast precursor. The intracellular domain of RANK subsequently recruits the TNF receptor-activating factor (TRAF) family, such as TRAF2/6. Activated TRAF stimulates the key transcription factors related to osteoclast differentiation and functions, such as AP-1 family (Fos/Jun) and activated T nuclear factor (NFATc1). These transcription factors induce the expression of osteoclast-related genes. NFATc1 is the master transcription factor in osteoclast differentiation. NFATc1 with AP-1 (Fos/Jun), PU.1, and microphthalmia-associated transcription factor form specific transcription complexes, which then act in the promoter region of genes, such as TRAP, CTR, cathepsin K, β 3 integrin, and OSCAR, which are related to the osteoclast attachment, migration, acidification, and degradation of bone matrix [23,24]. This study indicated that MCPEO affected the expression of NFATc1 and AP1 family proteins during osteoclast differentiation.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Funding
This study was supported by the National Nature Science     are expressed as mean ± SEM **P < 0.01, *P < 0.05 versus RANKL + DMSO (con).