Flavonoids compounds from Tridax procumbens inhibit osteoclast differentiation by down‐regulating c‐Fos activation

Abstract The total flavonoids from Tridax procumbens (TPFs) have been reported significantly to suppress on RANKL‐induced osteoclast differentiation and bone resorption in mouse primary cultured osteoclasts. However, the effects of ethyl ether fraction of Tridax procumbens flavonoids (TPF) on osteoclastogenesis remain unknown. In this study, we investigated the effects of TPF on lipopolysaccharides (LPS)‐induced osteoclast differentiation, actin ring formation, and explored its molecular mechanism in vitro. Matured osteoclast was counted as the number of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells, and activity of osteoclast was assessed by performing the pit formation assays. Real‐time polymerase chain reaction (RT‐PCR) was performed for evaluation of the expression of osteoclast differentiation‐related genes. TPF reduced the TRAP‐positive multinucleated osteoclasts, inhibited TRAP and acid phosphatase (ACP) activities and decreased the expression of osteoclast differentiating genes, including cathepsin K, metalloproteinase‐2 (MMP‐2), MMP‐9, MMP‐13 and osteoclast‐associated receptor (OSCAR). Furthermore, osteoclast‐dependent actin rings formation and resorption pits were dramatically inhibited by the treatment with TPF. TPF markedly decreased the expression levels of transcription factors such as c‐Fos, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and activator protein‐1 (AP‐1). Taken together, our findings indicated that TPF suppressed both osteoclast differentiation and activities. Therefore, TPF might be a promising and emerging drug candidate for the treatment of bone diseases such as osteoporosis.


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AL MAMUN et AL. fracture in bone of the older women. Many pathological bone disorders, including postmenopausal osteoporosis, rheumatoid arthritis (RA), osteoarthritis, lytic bone metastasis and Paget's disease are progressed by osteoclast-induced bone resorption. 4,5 Thus, identification of small molecules that specifically inhibit osteoclastic activity is a promising strategy of the drug discovery for the treatment of osteoporotic bone diseases. 6 In addition, bioactive compounds from plant origin have paid emerging attention due to the important sources of potentially useful new therapeutic drugs having less or no side effect. 7 The Tridax procumbens extracts are well-known phytochemical agents because extracts are used for the treatment of asthma, ulcer, piles and urinary problems. 8 Previously, we demonstrated that the ethyl ether and ethyl acetate fraction of Tridax procumbens flavonoids (TPFs) significantly inhibited osteoclast differentiation and osteoclastic bone resorptive activity. It was reported that the TPFs could inhibit osteoclasts differentiation-related marker genes expression, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metallopeptidase-9 (MMP-9) and MMP-13 in mouse osteoclasts. 9 Another study investigated that the TPFs induced the differentiation and bone-forming activity of osteoblasts by enhancing the levels of osteoblast differentiation-related markers, including alkaline phosphatase (ALP), osteocalcin, type 1 collagen, runt-related transcription factor (Runx2), osterix, bone morphogenetic protein-2 (BMP-2), BMP-4 and BMP-7. 10 Recently, we showed that the only ethyl ether fraction of Tridax procumbens flavonoids (TPF) was significantly induced higher bone mass by increasing bone mineral density and bone mineral content in low calcium diet mice model compared with control. 11 In that report, bone formation parameters, bone volume/tissue volume (BV/TV), number of osteoblast (N.Ob), osteoblast surface/bone surface (Ob.S/BS), mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR) were enhanced in TPF-treated mice compared with control. 11 Moreover, TPF stimulated synergistic effects on BMP-2-induced bone formation in critical-sized calvarial defect mouse model. 12 However, the effect of TPF on differentiation and activity of osteoclast in bone resorption remains uncleared. In the present study, the effects of TPF on lipopolysaccharide (LPS)-induced osteoclastic differentiation and activities in bone resorption have been investigated. The LPS-induced osteoclast formation was counted by the number of TRAP-positive multinucleated cells (two or more nuclei observed under light microscopy). Both TRAP and acid phosphatase (ACP) activities were measured for assessing the role of TPF in osteoclasts differentiation. This is the first evidence in our knowledge that TPF inhibited LPS-induced osteoclastogenesis by down-regulating the expression of osteoclasts differentiating markers including TRAP, ACP, cathepsin K, matrix metallopeptidase-2 (MMP-2), MMP-9, MMP-13, osteoclast-associated receptor (OSCAR), tumour necrosis factor-α (TNF-α, c-fos, RANKL, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and activator protein-1 (AP-1) as well as destructing the actin ring formation.

| Sample preparation
The different parts of Tridax procumbens such as leaf, root and flowers were collected, shade dried and crushed for finely powdered form as detailed explained elsewhere with minor modifications. 8 In brief, finely powdered samples (200 g in each) were extracted with 80% methanol in Soxhlet distillation and filtrates were repeatedly extracted with petroleum ether, ethyl ether and ethyl acetate by using separating funnel. Petroleum ether fraction was discarded as being rich in fatty substances, whereas the ethyl ether fraction that contains free flavonoids and ethyl acetate fraction that contains flavonoids with bounded sugars.
For this present study, we collected the ethyl ether fraction flavonoids from separation funnel, dried and used for osteoclast differentiation.

| Total flavonoids determination
The flavonoid content was estimated by aluminium chloride (AlCl 3 ) method as described elsewhere with some modifications. 8 The dried ethyl ether fraction flavonoids were separately mixed with methanol (1.5 mL), aluminium chloride (0.1 mL of 10% AlCl 3 ), potassium acetate (0.1 mL of 1 mol/L) and distilled water (2.8 mL) and kept at room temperature for 0.5 hour The absorbances were measured with a spectrophotometer (at 415 nm) and compared with quercetin as a standard. Finally, the flavonoids were dissolved with 75% methanol for further use.

| TRAP and ACP activity assays
TRAP and ACP assays for osteoclast differentiation were performed as described elsewhere with some modifications. 9 In brief, the TPF-treated cells were lysed by Trion-X-100 in PBS (0.2%), and the supernatant was measured for TRAP activity using para-Nitrophenylphosphate (pNPP) with a reaction buffer (0.1 mol/L sodium acetate (pH 5.8), 1 mmol/L ascorbic acid, 0.15 mol/L KCl and 10 mmol/L disodium tartrate) and ACP activity using Acid Phosphatase Liquicolor Assays Kit (Sigma-Aldrich) according to the instruction of manufacturer. About 0.3N NaOH solution was used for stopping the reaction, and optimum densities were measured at 405 nm by using microplate spectrophotometer (T60 U, PG Instruments Ltd.).

| Cell viability assay
For colorimetric method, methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich) was used for the viability of cells as described elsewhere with little deviation. 11 Briefly, about 1.4 × 10 7 cells/well of BMCs seeded in 6-well plates. After 4 days, culture cells were treated with TPF at different concentrations (50, 100 µg/mL) for 48 hours.
Then, fresh medium containing 0.5 mg/mL MTT was added to the cultured cells for 4 hours. The cells produced blue formazan products were dissolved in dimethyl sulfoxide (DMSO) and measured at 550 nm under spectrophotometrically (T60 U, PG Instruments Ltd.).

| Osteoclastic bone resorption assay
For assessment, the effects of TPF on osteoclastic bone resorption, pit formation assay in vitro was performed as described previously. 9 Briefly, the BMCs were cultured in dentin slices, α-MEM containing M-CSF (50 ng/mL), LPS (1 ng/mL) and FBS (10%) for 6 days with different concentrations of TPF. Then, the cells were treated with NH 4 OH (1 N) for 5 min and stained with 0.5% toluidine blue. The resorption pit was visualized under a microscope and analysed by image software system (KS400; Carl Zeiss).

| Actin ring formation assay
Osteoclastic actin rings were detected by using phalloidin staining assay. Briefly, the cultured cells were fixed with 3.7% formaldehyde in PBS for 10 min. The fixed cells were permeabilized with 0.1% Triton X-100 for 10 min and stained with phalloidin conjugate solution (Sigma-Aldrich) for 40 min. Then, the fixed cells were washed with PBS and the nuclei were stained with Hoechst 33342 (Sigma-Aldrich). Fluorescence microscope was used for photomicrographs and distributions of actin rings (KS400; Carl Zeiss).

| Real-time Polymerase chain reaction assay
The primary osteoclasts were cultured in 6-well plate for 6 days after different concentration of TPF treatment (50 and 100 μg/ mL). NucleoSpin (Macherey-Nagel) was used for the isolation of the total RNA from each well of cells. RNA aliquots were reversed transcript to complementary DNAs by using reverse transcriptase kit (Fermentas). The cDNA products were subjected to PCR amplification with gene-specific primers (Table 1)

| Western blot analysis
The c-Fos, NFATc1 and AP-1 were detected by Western blotting as described elsewhere with some modification. 9 Briefly, 6 days after the TPF-treated primary osteoclasts were lysed and the protein con-

| Statistical analyses
We analysed all data Student's t test followed by analysis of variance (ANOVA) with F test. P values <.05, .001 and .0001 were considered significant, very significant and strongly significant, respectively.
The data are represented as mean (m) ± standard deviation (SD) values of independent replicates.

| Effect of TPF on LPS-induced osteoclast differentiation
The TRAP-positive multinucleated osteoclasts (two or more nuclei observed under light microscopy) were significantly decreased dose dependently by TPF treatment ( Figure 1A). The TPF-treated osteoclasts at the concentration of 50 μg/mL were exhibited smaller and fewer nuclei compared with the control group ( Figure 1A). Moreover, treatment with TPF at the concentration of 100 μg/mL showed markedly reduced multinucleated osteoclasts in culture compared with the control ( Figure 1A). The TRAP and ACP activities were dose dependently decreased in cultured cells that treated with TPF at the concentrations of 50 and 100 μg/mL compared with control group (Figure 1B,C). The osteoclast surface was markedly decreased in TPF-cultured cells at the concentrations of 50 and 100 μg/mL compared with the control group ( Figure 1D,E). RT-PCR data indicated that TRAP and ACP gene expression levels per well were significantly lower in TPF-treated cells compared with the control group ( Figure 1F,G). Moreover, the TRAP and ACP gene expression levels were substantially lower in cells treated with TPF at higher concentration (100 μg/mL) compared with TA B L E 1 Primer sequences of real-time PCR

Gene
Forward Reverse the cells treated with TPF at 50 μg/mL concentration. So, TPF affected the TRAP and ACP genes expression levels in a dose-dependent manner. Additionally, cell viability results showed that TPF exposure to higher concentration (100 μg/mL) did not affect the death spots of primary osteoclasts ( Figure 1H).

| Effect of TPF on LPS-induced osteoclastic bone resorption
To examine whether TPF inhibited on LPS-induced bone resorption activity of osteoclasts, pit formation assay was performed in vitro.  Figure 2B). TPF also diminished the total resorption areas of active osteoclasts ( Figure 2B,C). These data suggested that TPF inhibited LPS-induced bone resorption activity of osteoclasts.

| Effects of TPF on the expressions of TNF-α RANKL and OPG
To address the molecular mechanism of TPF on osteoclastogenesis,

| Effect of TPF on LPS-induced actin ring formation
We investigated whether TPF could affect on LPS-induced actin ring formation. In the presence of LPS exposure, primary osteoclasts were differentiated into active osteoclasts and appeared distinct actin ring structures ( Figure 4A). However, TPF potentially decreased the number and size of actin rings compared with control group (Figure 4A,B). These results indicated that TPF suppressed on LPS-induced actin rings and bone resorptive activity.

| Effects of TPF on the expressions of c-Fos, NFATc1 and AP-1
It is known that several transcription factors such as c-Fos, NFATc1 and AP-1 are essential for osteoclast differentiation. Therefore, cells

| D ISCUSS I ON
Multinucleated osteoclasts attributed to a bone homeostatic imbalance between bone resorption and bone formation which is common in pathological hallmarks of bone destruction, including osteoporosis, inflammatory joint disorders, rheumatoid arthritis, periodontitis and bone cancer. Therefore, control of osteoclast differentiation is an identified therapeutic strategy to the treatment of osteoclast-specific diseases. 13 The drugs are currently used in osteoclast-specific disease treatment primarily includes steroids, for example oestrogen replacement therapy, selective oestrogen receptor modulators (SERMs) and bisphosphonates, RANKL inhibitors, parathyroid hormone (PTH) peptides, anti-Dickkopf-related protein 1 (DKK1), anti-sclerostin (SOST) antibodies and strontium ranelate (SR) are broadly classified as either bone anabolic or anti-bone resorptive agents involved bone resorption inhibition, which maintain balance between bone remodelling and bone mass by inhibiting osteoclast differentiation. 13,14 However, most of these drugs are associated with side effects, including hypertension, thromboembolism, endometriosis and hypercalcemia. 13 As osteoclasts are responsible for bone destruction-specific diseases, therefore developing new drugs for therapies that have both bone anabolic and anti-bone resorption effects would be more effective in treating bone loss in osteoclast-specific diseases like osteoporosis. Total flavonoids isolated from TPFs are able to inhibit osteoclast differentiation and bone resorption. Previously, we showed that TPFs could suppress RANKL-induced osteoclast differentiating marker genes expressions such as TRAP, cathepsin K, MMP-9 and MMP-13. 9 We have also reported the anabolic potential of TPFs on osteoblast differentiation and bone formation by enhancing expressions of ALP, osteocalcin, type 1 collagen, Runx2, osterix, BMP-2 and BMP-7. 10,11 In the present study, we examined the effects of TPF on LPS-induced osteoclast differentiation and bone resorption in primary mouse osteoclasts. We found that TPF decreased LPS-induced TRAP and ACP activities, and suppressed bone resorption without affecting the osteoclasts viability ( Figure 1A-H Blockade of TRAP and ACP production during LPS-induced osteoclast formation is highly potent protector of osteoclastic bone resorption and cartilage destruction. 18,19 The present data support the concepts that TRAP and ACP are significantly decreased in the TPF-treated osteoclasts compared with the control group ( Figure 1A-G). It was reported that cathepsin K is essential for making initial structures of actin rings as well as activity of osteoclasts. 17 Here, our data suggested that TPF efficiently inhibited mRNA ex- The data were represented as mean ± SD (n = 5) of 5 independent experiments. *P < .05 vs TPF of 100 μg/mL c-Fos-dependent osteoclast-specific gene expression and bone resorption. 26 Another importance, c-Fos pathways crosstalk to activation of inflammatory pathways in osteoclasts, and therefore, TPF may suggest as an important therapy for the treatment of inflammatory bone diseases, like osteoporosis. As suppression of osteoclast function by TPF modulates the disturbance of actin ring production, the expression osteoclast differentiating markers such as cathepsin K, MMP-9, MMP-13, OSCAR and TNF-α are also inhibited by TPF.
However, TPF-mediated osteoclast inhibition needs more further clarification for molecular insight. The present study demonstrated that TPF inhibited osteoclastogenesis by down-regulation c-Fos activation, subsequently suppressed the expression of gene related to osteoclastogenesis and attenuated the activation and nuclear translocation of AP-1 and NFATc1. Our findings suggest that TPF be a potential therapeutic candidate for the treatment of bone diseases associated with excessive bone resorption.

CO N FLI C T O F I NTE R E S T
All of the authors clearly declare that they have no competing and commercial interests.

AUTH O R CO NTR I B UTI O N S
MAAM designed the study, carried out experimental work on biological investigation, choice of assay methods, critically reviewed the manuscript and proofread. MMHA, MAZS and MAAB assisted in data analysis and interpretation, critically reviewed the manuscript and proofread. This manuscript is not under review elsewhere, and all authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sets generated or analysed during the current study are included in the article.