Effects of H₂O₂ on mRNA and protein expression of Tβ4 in PDLCs.

<p>Cells were incubated for 48 hours with the indicated times with 200 μM H₂O₂ (A) and the indicated concentrations of H₂O₂ (B) for 48 hours. The mRNA and protein expressions were examined by RT-PCR and Western blotting, respectively. Data were representative of three independent experiments. The bar graph shows the fold increase in protein or mRNA expression compared with control cells. * Statistically significant differences compared with the control, <i>p</i><0.05.</p

Effect of Tβ4 peptide on H₂O₂-induced osteoclastogenic cytokines (A) and osteoclastogenic factors (B) in PDLCs.

<p>Cells were pretreated with indicated concentrations of Tβ4 peptide for 2 hours and then incubated with 200 μM H₂O₂ for 48 hours (A, B). The mRNAs expression was examined by RT-PCR analysis. This data were representative of three independent experiments. The bar graph shows the fold increase in mRNA expression compared with control cells. * Statistically significant differences compared with the control, <i>p</i><0.05.</p

Effects of H₂O₂ on the expression of Wnt5a and its cell surface receptors in PDLCs.

<p>The mRNA and protein expressions were determined by PCR analysis (A) and Western blot analysis (B), respectively. The bar graph shows the fold increase in protein or mRNA expression compared with control cells * Statistically significant differences compared with the control, <i>p</i><0.05. The data presented were representative of three independent experiments.</p

Effect of H₂O₂-induced MAPK and NF-κB signaling pathways (A) and effect of Tβ4 peptide on H₂O₂-induced MAPK and NF-κB activation (B) in PDLCs.

<p>Cells were incubated with 200 μM H₂O₂ for indicated times (A). Cells were pretreated with indicated concentrations of Tβ4 peptide (0.1–5 μg/mL) for 2 hours and then incubated with 200 μM H₂O₂ for 60 minutes (B). Data were representative of three independent experiments. The bar graph shows the fold increase in protein expression compared with control cells * Statistically significant differences compared with the control, <i>p</i><0.05. # Statistically significant difference compared with the H₂O₂—treated group.</p

Effects Wnt5a siRNA and Wnt5a peptide on the Tβ4 peptide-mediated RANKL-induced osteoclastogenesis in mouse BMMs (A-C).

<p>The BMM cells were incubated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) or CM collected from PDLCs. * Statistically significant differences compared with the control, <i>p</i><0.05. # Statistically significant difference compared with the each group. The data presented were representative of three independent experiments.</p

Effect of Tβ4 peptide on H₂O₂-induced cytotoxicity (A), Tβ4, inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expressions (B, C), NO and prostaglandin E₂ (PGE₂) secretion (D, E) in PDLCs.

<p>Cells were pretreated with indicated concentrations of Tβ4 peptide for 2 hours and then incubated with 200 μM H₂O₂ for 48 hours (A-E). Cell viability was measured by MTT assay (A). Protein and mRNA expressions were assessed by RT-PCR (B) and Western blot analysis (C), respectively. The production of NO (D) and PGE₂ (E) were measured by Griess reaction and ELISA, respectively. Data replicated the quantifications of cytotoxicity, NO, and PGE₂ with the standard deviation of at least three experiments (n = 4). The bar graph shows the fold increase in protein or mRNA expression compared with control cells. * Statistically significant differences compared with the control, <i>p</i><0.05. # Statistically significant difference compared with the H₂O₂—treated group.</p

Anti-inflammatory effect of Tβ4 peptide in gingival fibroblasts.

<p>Cells were pretreated with indicated concentrations of Tβ4 peptide for 2 hours and then incubated with 200 μM H₂O₂ for 48 hours (A-C). Protein expressions were assessed by Western blot analysis (A). The production of NO (B) and PGE₂ (C) were measured by Griess reaction and ELISA, respectively. Data replicated the quantifications of NO and PGE₂ with the standard deviation of at least three experiments (n = 4). The bar graph shows the fold increase in protein expression compared with control cells. * Statistically significant differences compared with the control, <i>p</i><0.05. # Statistically significant difference compared with the H₂O₂—treated group.</p

Effects Wnt5a siRNA and Wnt5a peptide on the Tβ4 peptide-mediated inhibition of iNOS and COX-2 expressions (A), NO and PGE₂ secretions (B, C), pro-inflammatory cytokines production (D) and osteoclastogenic factors (E) in PDLCs.

<p>The PDLCs were pre-treated with Wnt5a siRNA (30 nM) or Wnt5 peptide (500 ng/mL) for 2 hours, post-incubated with Tβ4 peptide (1 μg/mL) and 200 μM H₂O₂ for 48 hours (A-E), and then conditioned medium (CM) was collected. The bar graph shows the fold increase in protein or mRNA expression compared with control. * Statistically significant differences compared with the control, <i>p</i><0.05. # Statistically significant difference compared with the H₂O₂-treated group. The data presented were representative of three independent experiments.</p

Indirect effects of Tβ4 peptide on RANKL-induced osteoclastogenesis in mouse BMMs (A-C).

<p>Cells were pretreated with indicated concentrations of Tβ4 peptide for 2 hours, post-incubated with 200 μM H₂O₂ for 48 hours, and then conditioned medium (CM) was collected. Mouse BMMs were cultured with CM in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL), as described in Materials and methods. After 5 days, cells were fixed and stained for TRAP as a marker of osteoclasts (A), and the number of TRAP-positive multinucleated cells (MNCs) was scored (B). TRAP osteoclast activity was assayed using the TRAP cytochemical stain technique (C). * Statistically significant differences compared with the control, <i>p</i><0.05. The data presented were representative of three independent experiments.</p

Inhibition of Osteoclast Differentiation by Gold Nanoparticles Functionalized with Cyclodextrin Curcumin Complexes

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under <i>in vitro</i> conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both <i>in vitro</i> and <i>in vivo</i>. We prepared β-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR–CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR–CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR–CGNPs. In addition, the CUR–CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR–CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the <i>in vivo</i> results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR–CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR–CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis