Total saponin from Anemone flaccida Fr. Schmidt abrogates osteoclast differentiation and bone resorption via the inhibition of RANKL-induced NF-κB, JNK and p38 MAPKs activation
© Kong et al.; licensee BioMed Central. 2015
Received: 25 September 2014
Accepted: 17 February 2015
Published: 15 March 2015
Osteoclasts, bone-specialized multinucleated cells, are responsible for bone destructive diseases such as rheumatoid arthritis and osteoporosis. Natural plant-derived products have received substantial attention given their potential therapeutic and preventive activities against bone destructive diseases. In the present study, we investigated the effects of total saponin (TS) from Anemone flaccida Fr. Schmidt, on receptor activator of nuclear factor-κB ligand (RANKL)-induced in vitro osteoclast differentiation. We observed that TS concentration-dependently inhibited RANKL-induced osteoclast formation from RAW 264.7 cell and bone marrow-derived macrophages (BMMs), as well as decreased extent of actin ring formation and lacunar resorption. The RANKL-stimulated expression of osteoclast-related transcription factors were also diminished by TS. Moreover, TS blocked the RANKL-triggered TRAF6 expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and inhibited NF-κB p65 DNA binding activity. Furthermore, TS almost abrogated the nuclear factor of activated T cells (NFATc1) and c-Fos expression. Taken together, our results demonstrated that TS suppresses RANKL-induced osteoclast differentiation and inflammatory bone loss via the down-regulation of TRAF6 level, suppression of JNK and p38 MAPKs and NF-κB activation, and subsequent decreased expression of c-Fos and NFATc1. Therefore, TS may be a potential agent and needs to be more evaluated in vivo or in clinical trials to become a therapeutic for lytic bone diseases.
Bone remodeling depends on the balance between bone resorption and bone formation . It is necessary to repair damaged bone and to maintain mineral homeostasis. An imbalance in favor of bone resorption, most often due to excess osteoclastic activity, leads to bone loss in pathological conditions such as rheumatoid arthritis (RA) and osteoporosis . Especially focal bone destruction within inflamed joints is the most specific hallmark of RA and leads to deformation, laxity, and functional disability .
Osteoclasts are the primary cells responsible for bone resorption and play crucial roles not only in maintaining bone homeostasis but also in development of pathological conditions such as joint destruction in RA [4-6]. Therefore, Osteoclasts become the main targets of current anti-resorptive drugs. The interaction of receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast differentiation and activation . The binding of RANKL and RANK on osteoclast progenitor cells triggers the activation of tumor necrosis factor receptor-associated factor 6 (TRAF6) and subsequently the activation of NF-κB and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) [8-10]. c-Fos and Nuclear factor of activated T cells (NFATc1) are two important downstream transcription factors in the RANKL/RANK signal pathway and play crucial role on the osteoclast differentiation [9,11].
There is a growing interest in the utilization of medicinal plants for prevention and treatment of bone disorders including RA and osteoporosis . The dry root of Anemone flaccida Fr. Schmidt, commonly known as “Di Wu” (Chinese name), is widely used as a folk medicine in the clinical compound prescription for the treatment of rheumatic diseases, external wounds and inflammations in China. Saponins are the characteristic components and also the main active ingredients of A. flaccida , and Diwu Fengshian Capsule with total saponin (TS) as the main active component has entered phase 3 clinical trial in China . It has also been reported that saponins isolated from A. flaccida possess anti-inflammation, immunoregulatory, analgesia and antitumor properties [14,15]. Bone metabolism is tightly associated with immune system and inflammation joint destruction in RA and other forms of arthropathies [16,17]. The anti-inflammation and immune-regulatory properties of TS from A flaccida may prove its role in osteolytic bone diseases. However, to date the direct effects of A. flaccida on bone metabolism have not been studied.
In the present study, we investigated the direct effect of TS, the major bioactive constituents of A. flaccida, on osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, the possible mechanism associated with its inhibitory effect on osteoclast differentiation was also explored.
Materials and methods
Alpha Modified Eagles Medium (a-MEM), fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin, were purchased from Invitrogen Life Technologies. Mouse soluble RANKL was obtained from PeproTech Biotechnology. Antibodies against NFATc1 and TRAF6 were purchased from Santa Cruz Biotechnology. Antibodies against c-Fos, ERK, p-ERK, JNK, p-JNK, p38, p-p38, p65, p-IκB-a and GAPDH were obtained from Cell Signaling Technology. LightShift™ Chemiluminescent EMSA Kit was provided by Pierce Technology.
Preparation of plant material and total saponins
Rhizome of A. flaccida Fr. Schmidt was collected from Jiufeng County of Hubei Province, China. TS isolated from A. flaccida were provided by Dr. Shiwei Jia (GKH Pharmaceutical Ltd.) and prepared as reported [13,15]. TS were dissolved in dimethyl sulfoxide (DMSO) and diluted in PBS for storage in −20°C freezer which is used in all subsequent experiments.
RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). RAW 264.7 cells were grown in a-MEM supplemented with 10% heat inactivated FBS, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin. Incubations were performed at 37°C in 5% CO2, and cultures were fed every 2–3 days.
Cell survival viability assay
Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) method using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA) according to the manufacturer’s instructions. The experiments were carried out 3 times in triplicate measurements.
RAW 264.7 cells were seeded onto a 96 well plate (1 × 104 cells/well) with complete a-MEM containing RANKL (50 ng/mL) in the presence of TS or vehicle control for 6 days at 37°C and 5% CO2. After 6 days, cells were washed with PBS and fixed with 10% formaldehyde for 15 min. Then washed with PBS. Fixed cells were subjected to an assay for Tartrate-resistant acid phosphatase (TRAP) activity. The experiments were carried out 3 times in triplicate measurements.
Preparation of Bone Marrow-derived Macrophages (BMMs) and in vitro osteoclast formation
Sprague -Dawley (SD) rats (4-7-week-old) were housed in Laboratory Animal Center (China Academy of Chinese Medical Sciences, Beijing, China) according to the guidelines for the care and use of laboratory animals of the NIH and China Academy of Chinese Medical Sciences (Beijing, China). All experimental procedures were approved by the Committee for Animal Use of the China Academy of Chinese Medical Sciences.
Total bone marrow cells were collected from tibia and femur of SD rat by flushing the marrow space with a-MEM. After removing the red blood cells (RBCs) with ACK buffer (0.01 mM EDTA, 0.011 M KHCO3, and 0.155 M NH4Cl, pH 7.3), cells were cultured for 1 day in a-MEM containing 10% fetal bovine serum (FBS). Nonadherent cells were collected and further cultured with 20 ng/mL M-CSF in a-MEM containing 10% FBS. After 3 days, culture medium was removed and adherent cells were used for osteoclast differentiation. BMMs were cultured for 6 days in medium containing 20 ng/mL M-CSF and 50 ng/mL RANKL to induce osteoclast formation.
TRAP staining kit was obtained from Sigma Alrich (USA). TRAP staining was carried out according to the manufacturer’s protocol. The images were taken with a digital camera attached to the microscope. TRAP positive multinucleated cells (≥3 nuclei) were scored as osteoclast-like (OCL) cells. The number of TRAP-positive cells was counted using an eyepiece graticule at a magnification of 100 and the results expressed as the number of cells per field of vision.
For F-actin ring staining, RAW 264.7 cells were stimulated with RANKL (50 ng/mL) for 6 days to induce osteoclast formation in the presence or absence of TS (0.1, 0.5 and 2.5 μg/mL). For NF-κB-p65 nuclear translocation experiment, RAW 264.7 cells were pretreated in the presence of TS or vehicle control overnight, then added RANKL (50 ng/mL) for 30 min. After incubation, the cultures were fixed in 4% paraformaldehyde in PBS and cells permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells were then stained for F-actin by incubation in 1 mg/ml TRITC-conjugated phalloidin or by anti-NF-κB-p65 for 2 h at 37°C. The preparations were then washed and mounted in Vectashield mounting medium with Hoechest 33258 (Vector Laboratories, Peterborough, UK). Cells were inspected with an Olympus x41 microscope. The experiments were carried out 3 times in triplicate measurements.
Bone resorption pit assay
To study the effect of TS on osteoclastic bone resorption, RAW 264.7 cells were seeded onto 50 um thick bovine bone slices and cultured with complete a-MEM containing RANKL (50 ng/mL) in the presence of TS or vehicle control at 37°C and 5% CO2. After 7 days, cells were then removed by sonication and the bovine bone slices were stained with toluidine blue to identify resorption pits. Also resorption lacunae were visualized by a Hitachi S-3400 N scanning electron microscope.
Enzyme Linked Immunosorbent Assay (ELISA)
RAW 264.7 cells were suspended in a-MEM containing 10% FBS and plated at a concentration of 1 × 105 cells/well into a 24-well culture dish (Corning). Then, they were treated with either TS or vehicle for 24 h before adding RANKL (50 ng/mL) to stimulate TNF-a secretion for 16 h. At the end of culture, medium was collected and analyzed for TNF-a using the ELISA kit according to the manufacturer’s instructions.
RAW 264.7 cells were seeded onto 75 cm2 flask pretreated with TS or vehicle control overnight at 37°C and 5% CO2. Then RANKL (50 ng/mL) was added for 30 min. Western blot was performed according to our previously described protocol . The primary antibodies used in the experiment included anti-ERK1/2(1:1000), anti-phospho-ERK1/2 (1:200), anti-Jun N-terminal kinase (JNK) (1:200), anti-phospho-JNK(1:200), anti-p38(1:500), anti-phospho-p38(1:200), anti-IκBα-p (1:250), anti-c-Fos (1:1000), anti-TRAF6 (1:1000), anti-GAPDH (1:1000). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies (1:5000) were used as secondary antibodies. The membranes were then visualized using the ECL system (Pierce, USA). GAPDH (internal control) was used to confirm equal protein loading.
Electrophoretic Mobility Shift Assay (EMSA)
RAW 264.7 cells were incubated with or without various concentrations of TS (0.1, 0.5, 2.5 μg/mL) for 12 h. The cells were then treated with RANKL (50 ng/mL) for 30 min. The nuclear protein extracted from the cells using a NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce, USA). EMSA were performed according to protocol of the LightShiftTM Chemiluminescent EMSA Kit. The synthetic biotinlabeled double-stranded NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′) were used to measure the effect of the TS on NF-κB nuclear protein–DNA binding activity. Briefly, nuclear proteins (30 μg), poly (dI-dC), and biotin-labeled double-stranded NF-κB oligonucleotide were mixed with the binding buffer. In addition, the excess amounts of unlabeled oligonucleotide were used for the competition assay to confirm specificity of binding. The DNA/protein complex was electrophoresed on 5% non-denaturing polyacrylamide gel. The DNA mobility shift due to binding of NF-κB complex was detected with enhanced chemiluminescence (ECL) reagent (Pierce, USA).
All raw data were processed by authorized software SPSS 13.0. The data were analyzed by One-way ANOVA followed by Dunnett’s t-test to assess the statistical significance of the differences between the study groups. Differences were considered statistically significant when P was less than 0.05.
TS suppresses RANKL-induced osteoclast differentiation
The effects of TS were further confirmed using primary BMMs. TS inhibited the multinucleated osteoclast formation induced by RANKL and M-CSF in BMMs (Figure 1C).
TS attenuates RANKL-stimulated F-actin rings formation and osteoclastic bone resorption
The bone resorption function of osteoclasts depends on dynamic regulation of the actin cytoskeleton. Actin ring structure is a characteristic cytoskeletal feature of functional osteoclasts . Therefore, we next examined whether TS affects actin ring structure of mature osteoclasts. OCLs were double-stained with phallotoxins and Hochest 33258 to allow for visualization of the cytoskeleton and nuclei respectively. In mature osteoclasts on tissue culture plates, F-actin was arranged into a ring-like structure (actin ring) at the cell periphery. Treatment of with TS (0.1-2.5 μg/mL) caused both shrinkage of osteoclasts and disruption of actin ring structure in a dose-dependent manner (Figure 2C and F).
To examine whether the above suppressive effect of TS was due to its cytotoxicity, MTS assays were carried out to test cell survival viability. As shown in Figure 2G and Additional file 1: Figure S1, TS from 0.1 to 2.5 μg/mL did not shown any cytotoxicity on RAW 264.7 cells and BMMs under the experimental conditions used in the present studies. No cytotoxicity of TS was also confirmed by a trypan blue dye exclusion test (Additional file 2: Figure S2). Furthermore, apoptosis of RAW 264.7 was also unaffected by these anti-osteoclastic TS concentrations (Additional file 3: Figure S3), suggesting that the observed reduction of osteoclast formation and activity was a result of osteoclast differentiation inhibition by TS.
TS inhibits RANKL-stimulated secretion of inflammatory cytokines
TS abrogates RANKL-induced TRAF6 expression
TS inhibits RANKL-induced MAPKs phosphorylation
TS suppresses RANKL-induced Activation of NF-κB signaling pathway
TS down-regulates RANKL-induced expression of NFATc1 and c-Fos
The root of A. flaccida has long been used for the treatment of arthritis and traumatic injury in folk medicine of China. TS is the main active constituent of this herb medicine and has been previously demonstrated to exhibit beneficial effects on adjuvant-induced arthritis by its immunomodulatory and anti-inflammation effects [14,23]. However, the effects of TS on the bone resorption remain unclear. The results in this study showed that TS inhibits osteoclast formation and bone resorption via the suppression of RANKL-induced activation of NF-κB and MAPKs signaling pathway. These data provide the mechanistic explanation, at least in part for the protective effect of TS against lytic bone diseases, such as in RA.
Recent studies have identified osteoclasts as the principal cell type responsible for bone destruction . Osteoclasts are unique multinuclear giant cells, which are derived from monocyte-macrophage lineage cells. Their differentiation from precursors can be induced by RANKL, a key factor that also controls the function and survival of mature osteoclasts . As a ligand, RANKL interacts to its receptor RANK, and induces them to differentiate into osteoclasts. Notably, until now, no other endogenous factors that can lead to osteoclast formation without RANKL participation have been found. Therefore, RANKL is used as a convincing inducer in studies on differentiation and function of osteoclasts. In the present study, we adopted the RANKL-induced osteoclast differentiation platform from RAW 264.7 cells, and our results revealed that TS significantly reduced the osteoclast differentiation. It is generally accepted that the formation of bone resorption pits occurs in conjunction with the process of osteoclast differentiation. In addition, the results in our study showed that TS dramatically attenuates osteoclastic bone resorption as detected by bone resorption pit assay. These data confirmed that TS inhibits both the formation and the resorptive function of osteoclasts.
Inflammatory cytokines, such as TNF-a, induce pre-osteoclast fusion, support the survival of mature osteoclasts, and stimulate osteoclastic bone resorption . It was recently reported that the RANK–RANKL pathway is involved in TNF-a-induced osteoclast differentiation [26-29]. Triterpenoid saponins from A flaccida have previously been reported to inhibit LPS-induced cyclooxygenase-2, and PGE2 expression in BEL-7402 and HepG2 hepatoma cell . In the present study, TS strongly suppressed RANKL-induced TNF-a secretion by RAW 264.7 cells, which partly explains the application of TS in diseases with inflammatory bone destruction.
RANKL stimulates osteoclast precursors to commit to the osteoclastic phenotype by binding to its receptor RANK on the surface of osteoclast precursors . The binding of RANKL with its receptor RANK interacts and induces the trimerization and activation of signaling adaptor molecule TRAF6, which is an essential initiating step during osteoclast differentiation . Our data showed that TS significantly inhibited TRAF6 expression. As downstream of RANK signaling, three major subfamilies of MAPKs (ERK 1/2, JNK and p38 MAPK) have been implicated as key regulators of various cellular responses, including cell proliferation, differentiation and apoptosis [30,31]. These MAPKs also play pivotal roles in the development of osteoclasts [32-34], and are thus key molecular targets for therapeutic application in inflammatory bone diseases . Indeed, specific inhibitors for ERK1/2, JNK and p38 MAPK could disturb RANKL-stimulated osteoclast differentiation [22,36-38]. In the present study, we demonstrated that TS could notably inhibit the RANKL-induced phosphorylation of JNK and p38 MAPK, suggesting that TS targets JNK and p38 MAPK cascades.
Genetic studies have demonstrated that NF-κB signaling pathway also plays a crucial role in osteoclast differentiation . NF-κB knockout mice showed the defects of osteoclast differentiation and severe osteopetrosis, indicating that NF-κB is a crucial factor in osteoclast differentiation . The classical NF-κB signaling pathway involves the activation of the IKK complex, which phosphorylates IκBα and targets them for ubiquitin-dependent degradation . In the alternative IκB-independent pathway, direct phosphorylation of NF-κB subunit p65 by IKK also modulates NF-κB transcription activity . In this study, TS inhibited RANKL-induced NF-κB activation by inhibiting p-IκBα and p65 nuclei translocation and DNA binding activity, which indicates that inhibiting the activation of NF-κB pathway contributed to the suppression of TS against osteoclast differentiation.
Following the activation of the MAPK and NF-κB signaling pathways caused by RANKL, multiple osteoclastogenic transcription factors, such as c-Fos, NFATc1 and PU.1, should be induced to mediate the formation of mature and functionally active osteoclasts . When BMMs are stimulated by RANKL, the expression of NFATc1 is induced through c-Fos and auto amplification by NFATc1 [41,42]. NFATc1-deficient embryonic stem cells do not form mature osteoclasts by RANKL treatment and overexpression of ectopic ca-NFATc1 in BMMs appropriately induces osteoclast differentiation from BMMs even in the absence of RANKL [41,43]. c-Fos, a major component of the transcription factor AP-1, can induce the expression of NFATc1 to regulate osteoclast differentiation after RANKL stimulation. Mice lacking c-Fos develop osteopetrosis as a result of a complete ablation of osteoclast formation. Our results revealed that RANKL stimulation markedly up-regulated the expression of both c-Fos and NFATc1, which is dramatically suppressed by TS treatment.
This study was supported by grants from the major projects of national science and technology (2011ZX09101-006-03,2013ZX09301307), natural science foundation of Beijing (No. 7112096), and fundamental research funds for the central public welfare research institutes (ZZ070836).
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