Human OCs were prepared as reported by Mitsuzaki et al. 52 with slight modifications. Peripheral blood was collected from healthy normal volunteers after informed consent. Mononuclear cells (PBMCs) were prepared from diluted peripheral blood (1:2 in Hanks Balanced Salt Solution), which was layered over Histopaque 1077 (Sigma, St. Louis, MO, USA) solution, centrifuged (400 g), washed and suspended in D-MEM/10% FCS. 3 × 10⁶ PBMCs/cm² were plated in 24-well plates or in chamber slides and allowed to settled for 2 hours. Wells were then rinsed to remove non-adherent cells. Monocytes were maintained at 37°C, in 5% CO₂, in medium supplemented with 10% FCS and cultured for 14 days in the presence of human M-CSF (25 ng/ml), RANKL (30 ng/ml) and 10^-7 M PHT. Culture media were replenished with fresh media every 3–4 days. Cells were used for the described experiments when mature multinuclear cells were predominant in the cultures.

TRAP staining of the cells was performed as reported by Villanova et al. 53. Cells were fixed in 3% para-formaldehyde with 0.1 M cacodilic buffer, pH 7.2 (0.1 M Sodium cacodilate, 0.0025% CaCl₂) for 15 min, extensively washed in the same buffer, and stained for TRAP (Acid Phosphatase Kit n. 386 – Sigma, St. Louis, MO, USA). After washing with distilled water and drying, mature TRAP positive multinucleated cells containing more than three nuclei were considered as osteoclasts.

The dried fruits of Emblica officinalis were extracted with absolute ethanol and the yield was 9.33%. The chemical composition has been determined by GC/MS and was reported elsewhere 5455.

Electrophoretic mobility shift assay (EMSA) was performed by using double stranded ³²P-labelled oligonucleotides as target DNA 51. Binding reactions were set up as described elsewhere in binding buffer (10% glycerol, 0.05% NP-40, 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5 mM DTT, 10 mM MgCl2), in the presence of poly(dI:dC).poly(dI:dC) (Pharmacia, Uppsala, Sweden), 2 μg of crude nuclear extracts and 0.25 ng of labelled oligonucleotide, in a total volume of 20 μl 28. After 30 min binding at room temperature, samples were electrophoresed at constant voltage (200 V for 1 hr) through a low ionic strength (0.25 × TBE buffer) (1 × TBE = 0.089 M Tris-borate, 0.002 M EDTA) on 6% polyacrylamide gels until the tracking dye (bromophenol blue) reached the end of a 16 cm slab. Gels were dried and exposed for autoradiography with intensifying screens at -80°C. In these experiments, DNA/protein complexes migrate through the gel with slower efficiency. In studies on the inhibitors of protein/DNA interactions, the addition of the reagents was as follows: (a) poly(dI:dC).poly(dI:dC); (b) labelled oligonucleotides mimicking the binding sites for transcription factors to be analyzed; (c) plant extracts; (d) binding buffer; (e) nuclear factors. The nucleotide sequences of double-stranded target DNA utilized in these experiments were 5'-CGC TGG GGA CTT TCC ACG G-3' (sense strand, HIV-NF-kB binding site), and 5'-CTG ATT TCC CCG AAA TGA CGG-3' (sense strand, STAT-3 binding site).

After 14 days of cell culture and 2–3 days of incubations with E. officinalis extracts, the cells were rinsed twice with PBS solution and fixed for 25 min in 4% paraformaldehyde at room temperature. Apoptotic cells were detected by the DeadEnd Colorimetric Apoptosis Detection System (Promega) according to the manufacturer's instructions. Measurement of apoptosis was calculated as a percentage of apoptotic nuclei (dark brown nuclei) versus total nuclei of multinucleated TRAP positive cells, evaluated in three independent measurements. A dark brown DAB signal indicates positive staining, while shades of blue-green to greenish tan indicate a nonreactive cell 4647.

Immunocytochemistry analysis was performed employing the streptavidin-biotin method using Ultraystain Polyvalent-HRP Immunostaining Kit. OCs grown in multichamber slides were fixed in 100% cold methanol, and permeabilized with (v/v) Triton X-100 (Sigma) in TBS (Tris-buffered saline). Cells were incubated in 3% H₂O₂ and blocked with Super Block reagent (Ultraystain Polyvalent-HRP Immunostaining Kit). After the reaction with the primary antibodies, rabbit polyclonal antibodies of human origin (Santa Cruz Biotech) against MMP9, FAS receptor, IL-6, and NF-kB (2 mg/ml) were used accordingly to the manufacturer's protocols, at 1:500 (MMP9), 1:100 (FAS receptor), 1:800 (IL-6) and 1:800 (NF-kB) dilutions. Incubation was carried out at 4°C for 16 hr. Cells were then incubated at room temperature with anti-polyvalent Biotinylated Antibody (Ultraystain Polyvalent-HRP Immunostaining Kit). After rinsing in TBS, Streptavidin HRP (Ultraystain Polyvalent-HRP Immunostaining Kit) was applied, followed by the addition of Substrate-chromogen mix (AEC Cromogeno kit). After washing, cells were mounted in glycerol/TBS 9:1 and observed using a Leitz microscope 4647.

The cytoxicity analysis was determined on in vitro cultured human OCs. PBMCs were plated in 96-well plates and, after 14 days, OCs were incubated with E. officinalis plant extracts for 3 days. Determinations of viable cells were performed after colorimetric assay with MTT (thiazolyl blue). The assay, based on the conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells 56, provides a quantitative determination of viable cells. After 72 hr of treatments in triplicate, 200 μL of MTT was added to each well of cells, and the plate was incubated for 2 hr at 37°. The medium was removed, and the MTT crystals were solubilized with 50% DMF. Spectrophotometric absorbance of each sample was then measured at 570 nm.

Data are presented as the mean ± SEM from at least three independent experiments. Statistical analysis was performed by one-way analysis of variance followed by the Student's t-test. A P value < 0.005 was considered statistically significant.

Human primary osteoclasts were obtained from peripheral blood and cultured in complete D-MEM plus MCSF, PTH and RANKL for 14 days. OCs differentiation was tested by tartrate-acid resistant acid phosphatase (TRAP)-positivity (Fig. 1) and metalloproteinase-9 (MMP-9) expression (data not shown). In order to test the effect of E. officinalis extracts on osteoclast differentiation, mature OCs (Fig. 1A) or monocytes during the two weeks of induction (Fig. 1B) were exposed to 0.5, 5, 50 μg/ml of plant extracts. The conditions used for these experiments correspond to the concentrations of E. officinalis extracts leading to 50% of inhibition (IC₅₀ value) of cell growth, previously analyzed in different cell lines 55. As reported in Figure 1, the presence of comparable levels of TRAP-positive cells cultured both in presence and in absence of E. officinalis extracts did not affect the process of osteoclastogenesis, at the concentrations employed. Quantitative data from three independent experiments are presented in the lower sides of Figure 1, demonstrating that treatment of the cultures with E. officinalis extracts does not have inhibitory effects on the development of TRAP-positive OCs. Cytotoxic effects of E. officinalis extracts were then analyzed. Human primary OCs were treated with increasing amount of E. officinalis extracts (0.5–500 μg/ml) for 72 hours and the viability of the cells was examined by the colorimetric MTT assay 56. As shown in Figure 2, 0.5, 5 and 50 μg/ml of E. officinalis extracts did not cause any cytotoxic effect on the total cell population (1–5% of which is constituted by OCs). Only E. officinalis extracts used at 500 μg/ml were found to induce a slight but not significant decrease of viability.

In a previous study we demonstrated that in different cell lines (K562, B-lymphoid Raji, T-lymphoid Jurkat and HEL cells) E. officinalis extracts retain an antiproliferative effect 55. In the present paper we investigated osteoclasts in terms of apoptosis. To this aim, TUNEL test was performed on OCs after exposure, up to 60 hours, to 0.5, 5, and 50 μg/ml of E. officinalis extracts. As shown in the representative experiment reported in Figure 3 (panels A and B), a low but significant level of apoptosis (20%) was induced by 0.5 μg/ml of extract; at 5 and 50 μg/ml, a dramatic increase (respectively 50% and 98%) in TUNEL-positive nuclei was observed. Table 1 reports summary data from three independent experiments, confirming the observation that 5 and 50 μg/ml of E. officinalis extracts consistently induce high levels of apoptosis of osteoclasts. Times of exposure shorter than 60 hours were also tested (24 and 48 hours) without obtaining significant differences from untreated cells (data not shown). These results were confirmed by immunocytochemical analysis of FAS receptor, a well known apoptosis-related protein 57, whose expression increased, as shown in Figure 3C, in OCs treated with E. officinalis extracts at all the concentrations used. When extracts from different medicinal plants, such as Satureja montana and Satureia hortensis were employed, no OCs apoptosis was induced (data not shown).

The ability of E. officinalis extracts to interfere with NF-kB binding to DNA was investigated, given that the transcription factor NF-kB plays a critical role in OCs activities by regulating the expression of a large number of OCs specific genes 484950. E. officinalis extracts were incubated in presence of 5 μg of nuclear extracts from K562 cells with an oligonucleotide containing a cis element of the LTR of HIV-1 representing the DNA binding site for NF-kB. DNA-protein interactions were then analyzed by EMSA 51. As reported in Figure 4, a dose dependent effect was observed, indicating the ability of E. officinalis extracts to completely inhibit NF-kB interaction with its cis element, when used at 100, 50, and 25 μg/reaction. On the contrary, 100 μg of E. officinalis extracts were not able to abolish the DNA-protein interactions of the transcription factor STAT-3 with its cis element (right side of Fig. 4) indicating a selectivity of the effects of E. officinalis extracts for NF-kB/DNA interactions. The sensitivity of NF-kB/DNA interactions to E. officinalis extracts was demonstrated to be related to the type of plant extracts and not to the extracting buffers, since no inhibitory effects were observed (a) with the extracting buffer and (b) other extracts from medicinal plants, such as Oroxylum indicum, Cuscuta reflexa, Paederia foetida, Hygrophilla auriculata, Ocimum sanctum (data not shown and Lampronti et al.) 57.

In order to determine whether E. officinalis extracts affect NF-kB dependent biological activity in OCs, we have evaluated the effects of the plant extracts on the expression of IL-6, a target gene of NF-kB transcription factors 5859. Immunocytochemical analysis, reported in Figure 5, clearly shows a significant decrease of IL-6 levels in OCs treated with 5–50 μg/ml of E. officinalis extracts after comparison to control untreated cells.