Background
Bone remodeling is a regulated, keenly balanced process affected by delicate changes in inhibitory cytokines and proinflammatory factors. These occur predominantly through the TNF-family molecule RANKL (Receptor Activator of NF-kappaB Ligand, a.k.a., OPGL, TRANCE, ODF, and TNFSF11) and its receptor, RANK (TNFRSF11A), which are vital regulators of bone remodeling and essential for the development and stimulation of osteoclasts [
1,
2]. Such growth factors and cytokines are associated with the inflammatory processes in rheumatic disease. Various osteopenic disorders, such as Rheumatoid Arthritis, involve amplified osteoclast activity, which can cause the increase in bone resorption, and ultimately crippling damage to bone. Inhibition of RANKL function via Osteoprotegerin (OPG), a natural decoy receptor, can be useful in treating osteoporosis and arthritis. The proposed method establishes an unexpected molecular paradigm that connects bone morphogenesis; variations of these methods create the opportunity to conceive innovative therapies that will impede the bone loss associated with arthritis and osteoporosis [
3‐
6]. Furthermore, apart from the above cytokines (RANKL and M-CSF), recent studies have determined that osteoclastogenesis may also be negatively or positively influenced by many other cytokines or recombinant proteins [
6‐
10].
Radix Paeoniae Rubra (RPR), the dried root of either
Paeonia lactiflora Pall. or
Paeonia veitchii Lynch, is a traditional Chinese medicine commonly used for treatment of various diseases in China. RPR has been frequently used to enhance blood circulation, dissipate stasis, and protect the liver [
11,
12]. RPR contains triterpenes [
13], flavonoids [
13], polyphenols [
14], and glycoside compounds, such as paeoniflorin, paeonin, benzoylpaeoniflorin, albiflorin, paeonol, and oxypaeoniflorin [
15‐
17], some of which were demonstrated to have some pharmacological effects, including anti-oxidative, anti-atherosclerosis, and anti-inflammatory effects [
14,
18].
For thousands of years, RPR has been used to treat various diseases, including hepatitis, diabetes, obesity, traumatic injuries, dementia, and arthritis [
19,
20]. The herb contains monoterpene glycosides, galloyl glucoses, and phenolic compounds, and many researchers suggest that it has immuno-regulatory, anti-oxidant, anti-allergic, and anti-inflammatory effects [
21‐
23]. Recently, it has shown that monoterpene glycosides perform a wide variety of biological activities.
Methods
RPR (Chishao) was obtained from the GMP pharmaceutical company (Sun Ten Pharmaceutical Co., Taipei, Taiwan). The herb powder (20 g) was extracted with five folds (volume) of distilled water and sterilized by autoclaving (121 °C and 1 atm) for 15 min. Then, the supernatant was collected and further dried under vacuum (76 mmHg, 25 °C). Distilled water dissolved the dried powder (1 mg/ml) before used. The outgoing quality control profiling of Radix Paeoniae Rubra was identified and analysis according to the Taiwan Herbal Pharmacopoeia. The voucher specimen has been deposited in the Institute of Chinese Pharmaceutical Sciences, China Medical University.
Cell culture
We used the RAW264.7 murine monocytic/macrophagic cell line human peripheral blood mononuclear cells (PBMCs) for the model system of osteoclastogenesis. Cells were maintained as previously described [
10]. RAW264.7 cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). Human PBMCs from healthy donors were separated by gradient centrifugation with Ficoll-Hypaque reagent and were re-suspended in α-MEM supplemented with 10% heat-inactivated FBS.
Osteoclast differentiation
For osteoclastic differentiation, cells were cultured in the presence of 25 ng/ml murine M-CSF and 50 ng/ml murine RANKL (for RAW264.7, from PeproTech, USA), human M-CSF and human RANKL (for PBMCs, from Peprotech, USA), and RPR. In some experiments, cells were pre-incubated for 40 mins with pharmacologic inhibitor p38/MAPK (SB203580; 10 ng/ml), ERK1/2 (PD98059; 20 μM), and JNK (SP600125; 10 ng/ml) (all from Calbiochem, La Jolla, CA) before RPR was added. In other experiments, cells were pre-incubated for 30 mins with an NFκB inhibitor (NF-κB SN50, cell-permeable inhibitor peptides; Calbiochem, San Diego, CA); caspase-3 specific inhibitor, Z-DEVD-FMK (R&D Systems, Inc., USA); caspase-9 specific inhibitor, Z-LEHD-FMK (BD Biosciences, San Diego, CA); or the general caspase inhibitor, Z-VAD-fmk (Bachem, Bubendorf, Sweden) at a concentration of 20 μM before RPR treatment.
MTT assay
Cell viability was determined by MTT assay following the procedure described previously [
24]. Briefly, cell cultures were treated with varying concentrations of RPR for different set periods. After an incubation with 0.5 mg/ml MTT for 4 h at 37 °C, MTT Formosan was dissolved with the addition of an equivalent cell culture volume of 0.04 N HCl. An ELISA plate reader determined that the absorbance value was 570 nm. The following equation: [OD of solvent-treated cells-OD of compound-treated cells/OD of solvent-treated cells] × 100% was performed to determine cell viability (%).
TRAP staining
Tartrate-resistant acid phosphatase (TRAP) staining of mature osteoclasts was done as previously described [
10]. Briefly, cells were stained with TRAP (Acid Phosphatase Kit 387-A; Sigma-Aldrich, St. Louis, MO) for 30 s, naphthol AS-BI phosphate and tartrate solution for 1 h at 37 °C and then counterstained with a hematoxylin solution. In order to control phosphatase activity that may occur in the background, we opted to use a greater dilution of 1 M tartrate (final 20 mM) and not the tartrate supplied as part of the kit [
10]. Intensifying the tartrate dilution suppressed control cells’ staining to allow the RPR and RANKL/M-CSF-treated cells to be positive. The total of TRAP-positive cells and nuclei per TRAP-positive cell in every individual well were respectively counted, followed by the photographing of the morphological features of the osteoclasts.
Bone resorption assay
The bone resorption assay was conducted as previously described [
10]. Cells were seeded into 24-well plates covered by artificial bone slides (BD BioCoat™ Osteologic™ Bone Cell Culture System). After 7 or 14 days of culture(for RAW264.7 or PBMCs, respectively), the wells were washed with DPBS, and the cells within were detached with incubation of 5% sodium hypochlorite for 5 min. The pits that remained in each well were calculated using a microscope and photographed.
Transfection and luciferase assay
Transfection and luciferase assays were performed based on a previously described method [
10]. RAW264.7 cells were seeded into 24-well plates at a density of 7 × 10
4 cells/well 1 day prior to transfection. Plasmid DNA, lipofectamine reagent and lipofectamine plus reagent were all mixed together in serum free DMEM and moved into the cells according to the recommended procedure of the manufacturer. 20 h after transfection, the cultures were then treated with RANKL (100 ng/ml) or RPR (100 ng/ml) for 12–16 h. The cells were rinsed two times with PBS and lysed in a reporter lysis buffer (Promega, Madison, WI). Following the instructions of the manufacturer, a dual-luciferase reporter assay system (Promega, Madison, WI) then measured the results of luciferase activity.
Western blot
The Western blot procedure was performed as described previously [
10]. Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.5% Tween-20 (TBST) for 1 h at room temperature and then probed with p-p38, p-ERK, p-JNK, p38, ERK, JNK (Cell signaling, USA), anti-c-fos, anti-NFATc1 (Santa Cruz Biotechnology, Dallas, TX) for 1 h at room temperature. After three washes, he blots were washed in TBST and developed with horseradish peroxidase conjugated in an anti-mouse antibody (Santa Cruz Biotechnology, Dallas, TX) (diluted in 1:5000) for 1 h in ambient temperature. After another wash, the membrane was subjected to film treated with a chemiluminescence reagent with ECL plus Western blotting reagents (Amersham). Every individual blot was then stripped and re-probed with anti-β actin antibodies to allow the standardization of expression amongst samples. This experiment was replicated three times to corroborate the results of this assay.
Preparing RNA and real-time RT-PCR
Total RNA and actual RT-PCR were prepared using the SYBR Green incorporation method [
10,
25]. A comparative cycle threshold technique using β-actin as the housekeeping gene was used to measure relative gene expression. The primers for NFATc1 were 5′-CGAGCCGTCATTGACTGTGC-3′ (sense) and 5′-GAGCGCTGGGAGCATTCGAT-3′ (anti-sense); 5′- GGTGGAACAGTTATCTCCAG-3′ (sense) and 5′-TGTCTCCGCTTGGAGTGTAT-3′ (anti-sense) for c-Fos; 5′-CGTGCTGACTTCACACCAACAGC-3′ (sense) and 5′-CACTTTTGAAGAGTGCAAACCGCC -3′ (anti-sense) for OSCAR; 5′- CTGTCCTGGCTCAAGAAACAG-3′ (sense) and 5′-CATAGTGGAAGCGCAGATAGC-3′ (anti-sense) for TRAcP; and 5′- GCGGTGGTATTATCTCTTGG-3′ (sense) and 5′-TTCCCTCATTTTGGTCACAAG -3′ (anti-sense) for calcitonin receptors.
Statistical analysis
Statistical analyses were conducted as previously described [
10]. All of the experiments were done in duplicate and the results were averaged. The results were expressed as mean ± SD of averages obtained in at least three experiments. The Student’s
t-test was used to evaluate variations in the means. A
p < 0.05 was considered statistically significant.
Discussion
RPR (Chishao) was obtained from the GMP pharmaceutical company. The outgoing quality control profiling of Radix Paeoniae Rubra was identified and analysis according to the Taiwan Herbal Pharmacopoeia. One of the preliminary exclusions of animal drugs and toxic drugs. And there are not potential side effects with human in recent research. RPR is used to encourage blood circulation, eliminate blood stasis, reduce fever, cool blood, eliminate stagnant blood, and minimize swelling. Furthermore, RPR also has anti-inflammatory and immune-regulatory usages. The bioactive elements of the plant’s root include a variety of monoterpene glycosides, galloyl glucoses, and phenolic compounds [
27,
28]. Furthermore, RPR also has anti-inflammatory and immuno-regulatory usages. RPR-induced K562 tumor cells developed apoptosis because caspase-3 mRNA and caspase-9 mRNA were increased [
26]. However, it is unclear if the signaling pathways are an integral part of RPR-induced osteoclast differentiation.
RPR is already known to activate RAW264.7 macrophages and human PBMCs to differentiate into osteoclasts. However, the concentration facilitating optimal differentiation remains unknown. A dose-dependent design (Fig.
1c and
d) was used to demonstrate RPR’s novel and unique behavior in osteoclastogenesis, and offer new understanding of the molecular system correlating osteo-immunology with the immune response associated with osteoclasts.
Activation of MAP kinase is a vital step in osteoclastogenesis [
29]. In this study, we found that RPR stimulates osteoclast differentiation through a signaling pathway of MAP kinases, which is the same mechanism as TRAIL [
30] or ribosome-inactivating protein B-chain [
10].
This experiment further employed inhibitors to verify the connection between JNK, ERK, and p38 MAP kinase regarding the behavior of RPR. The differentiation of murine RAW264.7 cells into TRAP-positive multi-nuclear cells was impeded by these kinase inhibitors. The data revealed that NF-κB, p38 MAP kinase, ERK, and JNK signaling pathways are vital to RPR’s osteoclastogenic impact. Similar stimulation of MAP kinases caused the same patterns of expression of vital transcription elements, namely, NFATc1 and c-fos. These can help clarify that RPR function is similar to RANKL in osteoclast differentiation.
The use of an in vitro culture system provides evidence that RPR is an innovative effector molecule that improves the development of osteoclast-like cells. This study also defines the vital function of NF-κB, p38 MAP kinase, ERK, and JNK signaling in inducing osteoclastogenesis, and is the first to test RPR in osteoclast differentiation.
Using MS/MS, we identified the major components from aqueous RPR extract (Additional file
1: Figure S1). gallic acid (1), oxypaeoniflorin (2), albiflorin (3), paeoniflorin (4), benzoic acid (5) and benzoylpaeoniflorin (6) were identified from the aqueous extract of RPR (Additional file
2: Figure S2). However, galloylpaeoniflorin was not found in either paeoniflorin or benzoic acid, based on the retention time under the same solvent conditions [
31]. In future studies, we would like to evaluate the osteoclastogenesis properties of the above components.
Acknowledgements
The authors would like to thank the Proteomics Core Laboratory, Department of Medical Research, and China Medical University Hospital for obtaining the MS data.