Curcumin Attenuation of Wear Particle-Induced Osteolysis via RANKL Signaling Pathway Suppression in Mouse Calvarial Model

Abstract

Wear particle-induced chronic inflammation and osteoclastogenesis are two critical factors in the osteolytic process. Curcumin (CUR) is an active compound of the medicinal herb Curcuma longa and has anti-inflammatory and antiosteoclastogenic properties. Our study tested the hypothesis that CUR might attenuate polymethylmethacrylate- (PMMA-) induced inflammatory osteolysis using mouse calvaria osteolysis model in vivo and in vitro. The mice were divided into four groups: phosphate-buffered saline group, CUR, PMMA, and PMMA + CUR groups. Three days before PMMA particle implantation, the mice were intraperitoneally injected with CUR (25 mg/kg/day). Ten days after the operation, the mouse calvaria was harvested for microcomputed tomography, histomorphometry, and molecular biology analysis. As expected, CUR markedly reduced the secretion of tumor necrosis factor-α, interleukin- (IL-) 1β, and IL-6 in the calvarial organ culture. Moreover, CUR suppressed osteoclastogenesis and decreased bone resorption in vivo compared with PMMA-stimulated calvaria. Furthermore, CUR downregulated the osteoclast-specific gene expression and reversed the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin messenger RNA and protein ratio in PMMA particle-stimulated mice. These results suggest that CUR attenuated PMMA particle-induced inflammatory osteolysis by suppressing the RANKL signaling pathway in the murine calvarium, which could be a candidate compound to prevent and treat AL.

Figure 1

Curcumin prevented PMMA particle-induced mouse calvarial osteolysis. (a) Representative 3D μCT reconstructed images of the calvaria in each group (N = 6/group). Measured (b) BMD, (c) trabecular BV/TV ratio, (d) number of pores, and (e) the percentage of total porosity within the ROI from each sample (N = 6/group; ^∗∗p > 0.05 versus the PBS group; ^∗p < 0.001 versus the PBS group; ^#p < 0.01 versus the PMMA group).

Figure 2

Curcumin prevented PMMA particle-induced mouse calvarial osteolysis. (a) Representative hematoxylin and eosin (H&E) staining of calvarial sections in each group (N = 6/group). (b) Representative Masson trichrome staining of calvarial sections in each group (N = 6/group).

Figure 3

Curcumin prevented PMMA particle-induced osteoclastogenesis. Histomorphometric analysis of (a) the ratio of remaining area of bone (%), (b) the eroded surface area (mm²), (c) the number of TRAP-positive multinucleated osteoclasts, and (d) the percentage of osteoclast surface per bone surface (OcS/BS, %) within the ROI in each group (N = 6/group; ^∗∗p > 0.05 versus the PBS group; ^∗p < 0.001 versus the PBS group; ^#p < 0.01 versus the PMMA group).

Figure 4

The mRNA levels of TRAP, CTR, CK, NFATc1, OPG, and RANKL were analyzed by RT-PCR, and the results were normalized to the expression in the PBS groups (N = 6/group; ^∗∗p > 0.05 versus the PBS group; ^##p < 0.05 versus the PBS group; ^∗p < 0.001 versus the PBS group; ^#p < 0.01 versus the PMMA group).

Figure 5

ELISA results for TNF-α, IL-1β, IL-6, OPG, RANKL protein levels, and the RANKL/OPG ratio in the supernatants of cultured calvaria (N = 6/group; ^∗∗p > 0.05 versus the PBS group; ^##p < 0.05 versus the PBS group; ^∗p < 0.001 versus the PBS group; ^#p < 0.01 versus the PMMA group).

Figure 6

Schematic representation of the molecular mechanisms of curcumin action in particle-induced osteolysis. CUR markedly reduced the secretion of inflammatory cytokines. Furthermore, CUR downregulated the osteoclast-specific gene expression and reversed the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin ratio in PMMA particle-stimulated mice.