2.1 Preparation of geraniin
Geraniin (purity > 99%) was kindly provided by Professor Ji-kai Liu (Kunming Institute of Botany, Chinese Academy of Sciences, P.R China). In the in vivo experiments, geraniin was suspended in 0.5% sodium carboxymethyl cellulose before use. The compound was dissolved in DMSO and then diluted with phosphate-buffered saline (PBS) before the in vitro experiments. The final concentration of DMSO did not exceed 0.05%, a concentration that is not toxic to osteoclasts.
2.2 Isolation and culture of primary rat osteoclasts
Osteoclasts were isolated mechanically from the long bone of rats by a method described previously by Lakkakorpi et al14. Briefly, one-day-old rats were euthanized via cervical dislocation, and their femora were separated. The bones were then dissected away from adherent soft tissue under sterile conditions. The medullary contents were scraped from the bone cylinders in HEPES-buffered medium 199 supplemented with 15% foetal calf serum, 100 U/ml benzylpenicillin, and 100 U/ml streptomycin. The medullary bones were homogenized gently, and the cell suspension was collected in a centrifuge tube. The cell suspension was allowed to settle for 10 s before the supernatant was dropped onto the plastic substrate. This osteoclast-rich supernatant was collected again and filtered through a nylon mesh (70 µm). Following centrifugation, the cells were cultured under a humidified atmosphere of 5% CO2 at 37°C for 30 min to allow the osteoclasts to subculture in 24-well plates directly. The cell culture plates were washed two times with a pre-warmed PBS solution to remove nonadherent cells, and the remaining adherent cells on the plates were continuously cultured. The cells were identified by their staining positive for TRAP and by their ability to form resorption pits on bovine bone slices. Osteoclasts were cultured and divided into six groups as follows (n = 4 per group): blank control group administered the complete culture media with no added substances (control group); geraniin groups treated with graded concentrations of geraniin (10-7 mol/L, 10-8 mol/L, 10-9 mol/L, and 10-10 mol/L) in osteoclasts (geraniin groups); positive control group treated with 10-6 mol/L of E-64 (Sigma Chemical Co., St. Louis, MO, USA) in osteoclasts (E-64 group).
2.3 In Situ Hybridization Assay
The sequences of probes against CatK mRNA were 5’- TGGAA GAAGA CCCAC GGGAA GCAGT ACAAC AGCAA -3’; 5’- CATAC GTATG AGCTG GCCAT GAATC ACCTG GGAGA -3’; and 5’- TATAT GACCA CTGCC TTCCA ATATG TGCAG CAGAA -3’. Hybridization was performed using a CatK mRNA kit (Boster Bioengineering Company, Wuhan, P.R. China) according to the instructions provided by the manufacturer. The endogenous peroxidase was inactivated by covering the slices with 0.6% hydrogen peroxide (v/v) in absolute methanol for 30 min at room temperature. mRNA fragments were exposed to pepsin diluted with 3% citric acid for digestion at 37°C for 2 min. After sufficient washing, 20 μL of prehybridization solution was added to the slices in a humidified chamber with 20% glycerol at 38-42 °C for 3 h. Then, 20 μL of the probe was added to the slices, and hybridization was performed at 38-42 °C for 18 h. The non-specifically bound probe was washed off with decreasing concentrations of standard saline citrate (SSC) starting at 2×SSC and ending with 0.2×SSC at 37 °C. Confining liquid was added to the slices that were then stored at 37 °C for 30 min. Anti-digoxin antibodies were pipetted onto slices, and the slices were incubated at 37 °C for 60 min followed by sufficient rinsing. Afterward, the slices were incubated with SABC peroxidase at 37 °C for 20 min. Then, the slices were treated with biotinylated-peroxidase and DAB in turn. After DAB staining for 20-30 min, the slices were dehydrated with alcohol, cleared with dimethylbenzene, sealed, and observed under a microscope (Olympus, Japan). The values of integral optical density (IOD) and mean optical density (MOD, equal to IOD divided by area of staining) of positive cells in the visual field were detected with a graphic analysis system (HPIAS-1000, Wuhan Champion Image Engineering Company of Tongji Medical University, Wuhan, P.R., China).
2.4 Immunocytochemical analysis
Immunocytochemistry analysis was conducted using a commercial kit (Zhongshan Golden Bridge Biological Company, Beijing, P.R., China) according to the manufacturer's directions. Briefly, the slices were coated with 0.1% poly-L-Lysine, and then one slice was put in each well of a 24-well plate. The cells were cultured in the 24-well plates with poly-L-Lysine-coated slices for 24 h. The slices were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, and treated with 3% H2O2 for 10 min to inactivate endogenous peroxidase. The slices were incubated overnight using an anti-cathepsin K antibody at a 1:500 dilution and were finally stained with DAB. The slices were observed under a confocal microscope. The values of IOD and MOD for positive cells with brown nuclei were calculated as above.
2.5 Statistical analysis
Data are expressed as the mean ± standard deviation (S.D.). Statistical comparisons were made by analysis of variance (ANOVA) and Student's t-test. Differences were considered statistically significant when P was less than 0.05.