Fig 1.
Effects of OCLI-023 on osteoclast differentiation in RANKL-stimulated BMMs.
(A) Chemical structure of OCLI-023. (B) BMMs were incubated for 3 days with M-CSF (10 ng/mL) in the presence or absence of 1 μM or 5 μM OCLI-023. Cell viability was evaluated by the MTT assay. (C) BMMs were cultured with M-CSF (10 ng/mL) and RANKL (20 ng/mL) either with or without 1 μM or 5 μM OCLI-023. After 4 or 5 days, the cells were fixed and stained with TRAP. Magnification; 50X. (D) TRAP-positive multinucleated cells with ≥3 nuclei were scored. **p < 0.01 versus the vehicle-treated control. (E) BMMs were cultured on glass coverslips for 4 days with M-CSF (10 ng/mL) and RANKL (20 ng/mL) in the presence or absence of OCLI-023 (5 μM). The cells were stained with rhodamine-conjugated phalloidin and DAPI to visualize actin rings and nuclei, respectively. (F) The number of actin rings was analyzed. **p < 0.01 versus the vehicle-treated control.
Fig 2.
Effects of OCLI-023 on RANKL-induced mRNA expression during osteoclast differentiation and resorption pit formation.
(A) BMMs were cultured in the presence of M-CSF (10 ng/mL) and RANKL (20 ng/mL) with vehicle or OCLI-023 (5 μM). The mRNA expression of TRAP (Acp5), cathepsin K (Ctsk), DC-STAMP (Dcstamp), and NFATc1 (Nfatc1) was analyzed using real-time quantitative PCR. (B) BMMs were seeded on bone slices and incubated with M-CSF (10 ng/mL) and RANKL (20 ng/mL) to induce osteoclast differentiation. After 3 days, the cells were incubated with or without OCLI-023 (5 μM) for an additional 2 days. Resorption pits were observed by hematoxylin staining (upper) and scanning electron microscopy (lower). **p < 0.01 versus the vehicle-treated control.
Fig 3.
Effects of OCLI-023 on RANKL-stimulated signaling.
BMMs were serum-starved for 5 h, then pretreated with OCLI-023 (5 μM) or vehicle for 1 h before RANKL (50 ng/mL) stimulation for the indicated times. Phosphorylation of JNK and IκBα was assessed by western blot. JNK or β-actin was used as the loading control.
Fig 4.
Micro-CT analysis of alveolar bones in mice with experimental periodontitis.
(A) Both nonligated and ligated mice were injected with either vehicle or OCLI-023. Palatal (upper) and buccal (middle) sides of maxilla and sagittal (lower) sectional microCT images of the second molar exhibit the alveolar bone loss. NL + V, nonligated with the vehicle; NL + OCLI-023, nonligated with OCLI-023; L + V, ligated with the vehicle; L + OCLI-023, ligated with OCLI-023. (B) The linear distance from the CEJ to the ABC of the second molar was analyzed. n = 5 in each group. *p < 0.05, **p < 0.01 versus the ligated, vehicle-treated group.
Fig 5.
Histological analysis of alveolar bones in an experimental periodontitis model.
(A) Both nonligated and ligated mice were injected with either vehicle or OCLI-023 as described in Fig 4. The fixed maxillae were decalcified, sectioned, and stained with H&E (upper and middle) and with TRAP (lower). Scale bars, 100 μm. P, pulp; D, dentin; IDS, interdental septum of the alveolar process; IRS, interradicular septum of the alveolar process. (B) The osteoclast number per bone surface was assessed. n = 5 in each group. **p < 0.01 versus the ligated, vehicle-treated group.