Preparation of a water extract of M. verticillata seeds
M. verticillata seeds (Yeongcheon herb, Korea) were authenticated by Prof. K.H. Bae (Chungnam National University, Korea). A voucher specimen with registration number 221 is maintained in the herbarium of the KM Application Center at the Korea Institute of Oriental Medicine. Dried M. verticillata seeds (50 g) were extracted by boiling in distilled water (1 L) for 3 h as described previously [19]. The water extract of M. verticillata seeds (WEMV) was filtrated using standard sieves (150 μm), concentrated by lyophilization, and stored at –20 °C until use. A WEMV stock solution was prepared by dissolving the lyophilized powder in distilled water followed by filtering through a 0.2 μm syringe filter. The yield of dried extract from the staring materials was approximately 8.96 % (w/w).
Gas chromatography/ mass spectrometry analysis
The lyophilized water extract of M. verticillata seeds was submitted to separation through gas chromatography with detection by mass spectrometry (GC/MS). The analysis was performed in a GC/MS system (Agilent Technologies, Atlanta, GA, USA) using DB-5 MS capillary column (30 m × 0.25 mm × 0.25 μm). Chromatographic conditions were as follows: the extract (1 μL) was injected in split mode with a ratio of 1/20 at 350 °C, oven initial temperature was 70 °C during 1 min, followed by heating at a rate of 5 °C/min at 300 °C, and totally run for 67 min. The mass analyzer was set to scan from 10 to 800 atomic mass unit. β-sitosterol was used as standard marker for GC/MS analysis. Peak identification was carried out comparison of the experimental mass spectrum in the National Institute of Standards and Technology (NIST) and Wiley GC-MS libraries. Fourteen components, including 1,3-dihydroxyacetone dimer, d-alanine, 5-hydroxymethyl furfural, 2-hydroxy-gamma-butyrolactone, palmitic acid, oleamide, and β-sitosterol, were identified in WEMV (data not shown).
Cell culture
Mouse bone marrow macrophages (BMMs) were obtained from mouse bone marrow cells (BMCs) as described previously [20]. For osteoclast differentiation, BMMs (1 × 104 cells/well, 96-well plate) were cultured in α-MEM differentiation medium containing 60 ng/mL of macrophage colony stimulating factor (M-CSF) and 100 ng/mL of RANKL for 4 days. Tartrate-resistant acid phosphatase (TRAP)-positive stained cells with more than three nuclei were counted as multinuclear osteoclasts [TRAP (+) MNCs]. For osteoblast differentiation, C2C12 cells (ATCC, USA) were cultured (1 × 104 cells/well, 96-well plate) in α-MEM differentiation medium containing 100 ng/mL of BMP-2 for 3 days. Primary calvarial osteoblasts (5 × 104 cells/well, 48-well plate) obtained as described previously [21] were cultured in α-MEM differentiation medium containing 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate for 5 days. BMMs were cultured in α-MEM proliferation medium, and C2C12 cells and osteoblasts were cultured in DMEM proliferation medium containing various concentrations of WEMV for 2 days to examine cytotoxicity using a CCK-8 assay (Dojindo Molecular Technologies Inc., Tokyo, Japan). All media contained 10 % fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) and were replaced at 3-days intervals.
Enzyme assay
TRAP and alkaline phosphatase (ALP) activity assay were performed as described previously [20]. Briefly, cells were fixed in 10 % formalin, permeabilized with 0.1 % Triton X-100, and incubated with TRAP assay buffer (50 mM sodium tartrate, 0.12 M sodium acetate, pH 5.2) with p-nitrophenyl phosphate (1 mg/mL) or incubated with ALP assay buffer [100 mM sodium carbonate (pH 10), 1 mM MgCl2, 50 mM Na2CO3] with naphthol AS-BI phosphate. After a 10-min incubation at 37 °C, the reaction was stopped with stop solution and the absorbance was measured. MNCs were stained with TRAP staining buffer containing naphthol AS-MX phosphate and Fast Red Violet LB (Sigma-Aldrich).
Western blot analysis
Cells were washed twice with phosphate-buffered saline (PBS) and total cell lysate was obtained using Radioimmunoprecipitation assay (RIPA) buffer (Millipore, MA, USA) containing protease and phosphatase inhibitors. Protein concentration was determined using a bicinchoninic acid assay (BCA) Assay Kit (Thermo Scientific, Rockford, IL, USA). Total protein (30 μg) was separated by 12.5 % SDS-PAGE gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membrane, and immunoblotted with specific antibody. Antibodies for MAPK, nuclear factor-kappaB (NF-kB), or phospholipase C (PLC) signaling proteins (Cell Singling Technology, MA, USA) or c-Fos and NFATc1 transcription factors (Santa Cruz Biotechnology, CA, USA) were used in this study. Chemiluminescent signals were detected using a ChemiDoc imaging system (Bio-Rad Laboratories, CA, USA) and a chemiluminescence reagent (Thermo Scientific).
Real-time quantitative polymerase chain reaction (qRT-PCR)
Total RNA was isolated using an RNAspin total RNA extraction kit (Intron, Daejeon, Korea). cDNA was synthesized from 1 μg of total RNA using AccuPower RT-PreMix (Bioneer, Daejeon, Korea) according to the manufacturer’s protocol. qRT-PCR analysis was performed using a CFX96 Touch Real-Time PCR System (Bio-Rad, CA, USA) and AccuPower GreenStar qPCR Master Mix (Bioneer, Daejeon, Korea). The qRT-PCR reactions comprised 40 cycles at 94 °C for 20 s and 60 °C for 40 s. All reactions were performed in triplicate. The relative expression levels of target genes were normalized to hypoxanthine phosphoribosyltransferase (HPRT) expression and analyzed using Bio-Rad CFX Manage 3.1 software (Bio-Rad, CA, USA).
Retrovirus preparation and infection
A pMX-puro-green fluorescent protein (GFP) retrovirus vector (provided by Dr. T. Kitamura, University of Tokyo) and a pMX-Ca-NFATc1 vector encoding HA-tagged Ca-NFATc1, a constitutively expressed form of NFATc1 provided by Dr. N.A. Clipstone (Northwestern University), were used for retrovirus preparation as described previously [20]. Briefly, to generate retroviral stocks, the vector was transfected into Plat-E retroviral packaging cells. BMMs (3 × 106 cells, 100-mm dish) were infected with retrovirus soup from the packaging cells by incubation with polybrene (6 μg/mL) for 8 h. Infected BMMs were selected with puromycin (2 μg/mL, Sigma-Aldrich) for 5 days. The selected BMMs were cultured in differentiation medium with WEMV for 4 days and then stained with TRAP-staining solution.
Bone resorption assay
Osteoclasts obtained from a BMCs-osteoblasts coculture system were cultured on OsteoAssay Surface plates (Corning, MA, USA). Osteoclasts were preincubated with WEMV for 3 h and further cultured in differentiation medium for an additional 16 h. After removing cells, stained osteoclasts and resorbed areas were observed under an inverted microscope (40× magnification). The resorption areas were analyzed in three randomly selected fields of each well using Image J software.
Statistical analysis
Statistical significance of the differences in TRAP activity, MNC number, and mRNA levels of genes was analyzed using Student’s t-test. Data are presented as means ± the standard deviation of three independent experiments. Differences are considered significant at p < 0.05.