Apigenin was purchased from Enzo Life Science (Plymouth, PA, USA). α-Modified minimum essential medium (α-MEM), RPMI 1640 medium, and penicillin/streptomycin antibiotic mixture were obtained from Life Technologies, Inc. (Grand Island, NY, USA). Fetal bovine serum was obtained from Moregate BioTech (Bulimba, Australia). Recombinant murine M-CSF and recombinant human soluble RANKL (sRANKL) were from R&D Systems (Minneapolis, MN, USA) and Pepro Tech EC., Ltd. (London, UK), respectively.
Osteoblastic cell cultures
Preosteoblastic MC3T3-E1 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). Cells were maintained in a 55-cm2 dish in α-MEM, supplemented with 10 % fetal bovine serum, 50 units mL−1 penicillin and 50 μg mL−1 streptomycin, in a humidified atmosphere of 5 % CO2 in air at 37 °C. After reaching 70 % confluence, cells were detached by treatment with 0.05 % trypsin, replated in either 55-cm2 dishes or 12-well plates (area of each well, 3.8 cm2) at a density of 1 × 104 cells cm−2, and grown in α-MEM supplemented with 10 % fetal bovine serum, 50 units mL−1 penicillin, 50 μg mL−1 streptomycin, 5 mM β-glycerophosphate (Sigma-Aldrich, Tokyo, Japan), and 50 μg mL−1 ascorbic acid (Sigma-Aldrich). Fresh medium and apigenin were supplied to cells at 2-day intervals. Apigenin at 1, 5, and 10 μM was added to medium according to previous study (Bandyopadhyay et al. 2006). MC3T3-E1 cells formed nodules, and mineralization of nodules was observed after cultivation for 2–3 weeks.
Formation of osteoclastic cells
Multinucleated osteoclastic cells formed from spleen cells by adding osteoclast differentiating factors, RANKL and M-CSF. Spleen cells were collected from the splenic tissues of 6-week-old male ddY mice (Sankyo Laboservice, Tokyo, Japan). Erythrocytes contaminating the spleen cell fraction were eliminated by adding 0.83 % ammonium chloride in 10 mM Tris–HCl (pH 7.4) to the cell pellet. Mouse spleen cells (2.4 × 105 cells/well) in 96-well plates (0.32 cm2/well) were cultured with 50 ng mL−1 human sRANKL and 30 ng mL−1 M-CSF for 7 days. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air. Fresh medium and apigenin at 1, 5, 10 μM were supplied at 2-day intervals. Multinucleated osteoclastic cells formed were fixed on the well in 3.7 % formaldehyde for 5 min and then in a mixture of ethanol and acetone (1:1; v:v) for 1 min. These were then stained for tartrate resistant acid phosphatase (TRAP) activity, one of osteoclastic differentiate markers. TRAP-positive multinucleated cells (five or more nuclei) were counted under a microscope (IX70; Olympus, Tokyo, Japan).
The Institutional Animal Care and Use Committee of Toin University of Yokohama approved all animal protocols and procedures.
Toxicity of apigenin for cells
MC3T3-E1 cells were replated in 96-well plates (area of each well, 0.32 cm2) at a density of 1 × 103 cells cm−2 and grown in α-MEM supplemented with 10 % fetal bovine serum, 50 units mL−1 penicillin, 50 μg mL−1 streptomycin, and apigenin at various concentrations. After subculture for 53 or 74 h, the cell layers were washed with RPMI 1640 medium. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; DOJINDO, Kumamoto, Japan) reagent (0.5 mg mL−1 RPMI 1640) was added to each well, followed by incubation for 4 h for formazan formation. After the medium was removed, dimethyl sulfoxide was added to each well to dissolve the formazan, and absorbance was measured at 570 nm. The absorbance of the solution at 570 nm was related to the number of live cells.
Sirius Red staining of collagen
Collagen was stained with Sirius Red (Tullberg-Reinert and Jundt, 1999). Sirius Red F3BA was purchased from Polysciences, Inc. (Wamington, PA, USA). Dye was dissolved in saturated aqueous picric acid at a concentration of 100 mg dL−1. Cells were plated in 24-well plates (2.0 cm2/well) at a density of 1 × 104 cells cm−2 and subcultured with apigenin for 3 days. Cell layers were air-dried overnight on a sterile bench and fixed with 1 mL of Bouin’s solution (Sigma, St. Louis, MO, USA) for 1 h. Fixation fluid was removed by suction and culture plates were washed by immersion in running tap water for 15 min. Culture plates were air-dried, and 1 mL of Sirius Red dye reagent was added. Cells were stained for 1 h with shaking on a plate shaker. Stained cell layers were washed with 0.01 N hydrochloric acid to remove all non-bound dye, and stained material was dissolved in 0.5 mL of 0.1 N sodium hydroxide by using a plate shaker for 30 min at room temperature, after which the absorbance was measured at 550 nm.
Measurement of alkaline phosphatase activity
MC3T3-E1 cells were subcultured in 12-well plates (3.8 cm2/well) in α-MEM containing 10 % fetal bovine serum, 5 mM β-glycerophosphate, and 50 μg mL−1 ascorbic acid. After the cells had reached confluence (day 3), apigenin was added to cultures at various concentrations for 12 days. Cells were washed with 10 mM Tris–HCl, pH 7.2, and were sonicated in 1 mL of 50 mM Tris–HCl (pH 7.2) containing 0.1 % Triton X-100 and 2 mM MgCl2 for 15 s with a sonicator (Ultrasonic Disruptor UD-201; Tomy Co., Tokyo, Japan). Alkaline phosphatase activity was determined using an established technique with p-nitrophenyl phosphate as the substrate (Hagiwara et al. 2011). Protein concentrations were determined using BCA protein assay reagent (Pierce Chemical Co., Rockford, IL, USA) with bovine serum albumin as a standard.
Quantitation of calcium deposition
MC3T3-E1 cells were subcultured in α-MEM containing 10 % fetal bovine serum, 5 mM β-glycerophosphate, and 50 μg mL−1 ascorbic acid. After the cells had reached confluence (day 3), apigenin was added at various concentrations to the culture medium and cells were subcultured for 15 days. The amount of calcium, deposited as hydroxyapatite in the cell layer, was measured as follows: Layers of cells in 12-well plates (3.8 cm2/well) were washed with PBS and incubated overnight with 1 mL of 2 N HCl with gentle shaking. Ca2+ ions in the samples were quantitated by the o-cresolphthalein complexone method with a Calcium C kit (Wako Pure Chemical Industries, Osaka, Japan). This kit is specific for Ca2+ ions and has a detection limit of 1 μg mL−1. The solution of Ca2+ ions (20 mg dL−1) provided in the kit was used as the standard solution.
Analysis of bone mineral density of OVX mice treated with apigenin
BALB/c female mice (6-weeks old) were purchased from Sankyo Laboservice (Tokyo, Japan) and were housed individually at 24 °C with a 12 h light–dark cycle. The mice underwent a sham-operation (n = 5) or were surgically ovariectomized (OVX; n = 10) under anesthesia with Nembutal® injection (Dainippon Sumitomo Pharma, Tokyo, Japan). The mice were assigned to three groups (n = 5 for each): (1) untreated (Sham: sham-operated controls); (2) untreated (OVX controls); and (3) OVX administered intraperitoneally with apigenin (10 mg kg−1 body weight) at 2-day intervals. Apigenin was dissolved in ethanol and was diluted tenfold with saline before injection. The volume ingested was 50 μL. After the 28-day experimental period, the left and right femurs were surgically obtained from the anesthetized mice. The success of ovariectomy was confirmed by uterine atrophy in OVX mice.
The bone mineral density of the femurs was assessed using an X-ray CT System (LA Theata LCT-100; Aloka, Tokyo, Japan). We monitored the bone mineral density of femurs at 0.3 mm intervals and separately analyzed each trabecular and cortical bone. Values at 2.1 and 2.4 mm from the epiphysis of the femur are shown in the figures. Animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of the Toin University of Yokohama.
Numerical data have been expressed as mean ± SD values of the results from three to four cultures, and the significance of differences was analyzed by ANOVA (Dunnett’s test). Statistical significance was set at P < 0.05. Experiments were repeated independently in triplicate and the results were qualitatively identical in every case. Results from representative experiments are shown.