Curcumin inhibits osteoclastogenic potential in PBMCs from rheumatoid arthritis patients via the suppression of MAPK/RANK/c-Fos/NFATc1 signaling pathways

Abstract

The aim of the present study was to determine the effects of curcumin on the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs) obtained from patients with rheumatoid arthritis (RA), and to investigate the underlying molecular mechanisms. PBMCs from patients with RA (n=12) and healthy controls (n=10) were cultured to assess osteoclastogenic potential. The number of tartrate‑resistant acid phosphatase‑positive osteoclasts differentiated from PBMCs isolated from patients with RA was significantly increased compared with that of the healthy controls. In addition, the osteoclast number in patients with RA was correlated with the clinical indicators, Sharp score (r=0.810; P=0.001) and lumbar T‑score (r=‑0.685; P=0.014). Furthermore, the resorption area was increased in the RA group compared with the healthy controls. The mRNA and protein expression levels in PBMC‑derived osteoclasts treated with curcumin were measured by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. Curcumin inhibited the osteoclastogenic potential of PBMCs, potentially by suppressing activation of extracellular signal‑regulated kinases 1 and 2, p38 and c‑Jun N‑terminal kinase, and inhibiting receptor activator of nuclear factor κB (RANK), c‑Fos and nuclear factor of activated T cells (NFATc1) expression. The results of the present study demonstrated that curcumin may inhibit the osteoclastogenic potential of PBMCs from patients with RA through the suppression of the mitogen‑activated protein kinase/RANK/c‑Fos/NFATc1 signaling pathways, and that curcumin may be a potential novel therapeutic agent for the treatment of bone deterioration in inflammatory diseases such as RA.

Figure 1

Enhanced osteoclastogenic potential of PBMCs from patients with RA. (A) The morphology of PBMCs following culture with 50 ng/ml M-CSF and 100 ng/ml RANKL. Following three days in culture, PBMCs were adhesive and narrowly elongated, following six days fibroblast-like cells were significantly increased, and PBMCs were in cell condensation following nine days of culture. Polynuclear giant cells were observed at day 14. (B) Observation of osteoclast morphology in PBMCs from RA patients using TRAP staining following 14 days in culture. Cells were fixed in 4% formalin, permeabilized with 0.1% Triton X-100 and stained with TRAP solution. (C) PBMCs isolated from RA patients and healthy controls were cultured with 50 ng/ml M-CSF and 100 ng/ml RANKL for 14 days and TRAP staining performed. The number of osteoclasts (TRAP-positive cells containing ≥3 nuclei/cell counted in 10 fields of each sample) differentiated from PBMCs from patients with RA was significantly increased compared with healthy controls. Data are expressed as the mean ± standard deviation, n=10 per group. PBMCs, peripheral blood mononuclear cells; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor κB ligand; TRAP, tartrate-resistant acid phosphatase; RA, rheumatoid arthritis; OC, osteoclasts.

Figure 2

Enhanced osteoclastic bone-resorption potential in PBMCs from patients with RA. PBMCs seeded on bovine bone slices were incubated with 50 ng/ml M-CSF and 100 ng/ml RANKL for 21 days. PBMCs were then removed, and the bone slices were (A) stained using toluidine blue or (B) analyzed by scanning electron microscopy, to identify resorption pits. The lacunar number and area of bone resorption were increased in the RA group compared with the healthy control group. PBMCs, peripheral blood mononuclear cells; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor κB ligand; RA, rheumatoid arthritis.

Figure 3

Osteoclastogenic potential in PBMCs from RA patients was closely associated with the clinical parameters, Sharp score and T-score, in RA patients. (A) Linear correlation plot of the osteoclast number and Sharp score in patients with RA. The Sharp score was calculated by assessing articulation in the two hands of the patients with RA and correlating this value with the number of osteoclasts counted in TRAP-stained PBMCs isolated from patients with RA and healthy controls. The Sharp score was significantly positively correlated with the number of osteoclasts in patients with RA. (B) Linear correlation plot of the osteoclast number and lumbar T-score in patients with RA. A significant negative correlation was detected between the osteoclast number and the lumbar T-score in patients with RA. RA, rheumatoid arthritis; TRAP, tartrate-resistant acid phosphatase; PBMCs, peripheral blood mononuclear cells; OC, osteoclasts.

Figure 4

Curcumin inhibits the osteoclastogenic potential of PBMCs from patients with RA. (A) PBMCs from healthy controls were incubated with various concentrations of curcumin (0–40 µM) in the presence of M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 48 h, and the cell viability was examined using the Cell Counting kit-8 assay. Cell viability was not affected by <20 µM curcumin. PBMCs from patients with RA were incubated with various concentrations of curcumin (0–10 µM) in the presence of 50 ng/ml M-CSF and 100 ng/ml RANKL for 14 days. (B) The osteoclast differentiation was measured by TRAP staining and the number of osteoclasts (TRAP-positive cells containing ≥3 nuclei/cell) was counted in 10 fields of each sample. Curcumin treatment inhibited the number of osteoclasts generated in a dose-dependent manner. The (C) mRNA and (D) protein expression levels of RANK were measured by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively, and were reduced by curcumin treatment. (E) The protein expression levels of (E) c-Fos and NFATc1, and (F) p-ERK1/2, ERK1/2, p-p38, P-38, p-JNK and JNK were measured and normalized to β-actin. Curcumin treatment decreased the protein expression levels of c-Fos, NFATc1, p-ERK1/2, p-p38 and p-JNK in a dose-dependent manner. Data are expressed as the mean ± standard deviation, n=3 per group. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. 0 µmol/l curcumin treatment. PBMCs, peripheral blood mononuclear cells; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor κB ligand; RA, rheumatoid arthritis; RANK, receptor activator of nuclear factor κB; TRAP, tartrate-resistant acid phosphatase; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; p, phosphorylated; ERK1/2, extracellular signal-regulated kinases 1 and 2; JNK, c-Jun N-terminal kinase.