Curculigoside Protects against Titanium Particle-Induced Osteolysis through the Enhancement of Osteoblast Differentiation and Reduction of Osteoclast Formation

Abstract

Wear particle-induced periprosthetic osteolysis is mainly responsible for joint replacement failure and revision surgery. Curculigoside is reported to have bone-protective potential, but whether curculigoside attenuates wear particle-induced osteolysis remains unclear. In this study, titanium particles (Ti) were used to stimulate osteoblastic MC3T3-E1 cells in the presence or absence of curculigoside, to determine their effect on osteoblast differentiation. Rat osteoclastic bone marrow stromal cells (BMSCs) were cocultured with Ti in the presence or absence of curculigoside, to evaluate its effect on osteoclast formation in vitro. Ti was also used to stimulate mouse calvaria to induce an osteolysis model, and curculigoside was administrated to evaluate its effect in the osteolysis model by micro-CT imaging and histopathological analyses. As the results indicated, in MC3T3-E1 cells, curculigoside treatment attenuated the Ti-induced inhibition on cell differentiation and apoptosis, increased alkaline phosphatase activity (ALP) and cell mineralization, and inhibited TNF-α, IL-1β, and IL-6 production and ROS generation. In BMSCs, curculigoside treatment suppressed the Ti-induced cell formation and suppressed the TNF-α, IL-1β, and IL-6 production and F-actin ring formation. In vivo, curculigoside attenuated Ti-induced bone loss and histological damage in murine calvaria. Curculigoside treatment also reversed the RANK/RANKL/OPG and NF-κB signaling pathways, by suppressing the RANKL and NF-κB expression, while activating the OPG expression. Our study demonstrated that curculigoside treatment was able to attenuate wear particle-induced periprosthetic osteolysis in in vivo and in vitro experiments, promoted osteoblastic MC3T3-E1 cell differentiation, and inhibited osteoclast BMSC formation. It suggests that curculigoside may be a potential pharmaceutical agent for wear particle-stimulated osteolysis therapy.

Figure 1

Curculigoside treatment attenuated Ti-induced inhibition of osteoblastic differentiation in MC3T3-E1 cells. (a) After being intervened with Ti and treated with curculigoside at different concentrations (25, 50, and 100 μg/ml) for 24 h, 48 h, and 72 h, MC3T3-E1 cell viability was detected by the CCK-8 assay (n = 6). (b, d) After being intervened with Ti and treated with curculigoside, cell apoptosis was detected by flow cytometry (n = 3). (c, e) Mitochondrial membrane potential of MC3T3-E1 cells cultured under Ti and different concentrations of curculigoside was assayed by flow cytometry using JC-1 assay kits, and the decrease in the MMP index in each group cell was quantified (n = 3). Results are represented as the mean ± SD. ^▲P < 0.05, compared to the Con group; ^▲▲P < 0.01, compared to the Con group; ^∗P < 0.05, compared to the Ti group; ^∗∗P < 0.01, compared to the Ti group. Ti: titanium particle; Con: control; MMP: mitochondrial membrane potential.

Figure 2

Curculigoside treatment alleviated Ti-induced inhibition on ALP activity, mineralization, and ROS generation in MC3T3-E1 cells. (a) MC3T3-E1 cells were detected with ALP staining, and ALP activity was quantified. (b, d) MC3T3-E1 cells were assayed by Alizarin Red staining, and the mineralization in each group cell was quantified. (c, e) Intracellular ROS level of MC3T3-E1 cells after Ti intervention and curculigoside treatment was assayed by flow cytometry, and ROS production in each group cell was quantified (n = 3); results are represented as the mean ± SD. ^▲P < 0.05, compared to the Con group; ^▲▲P < 0.01, compared to the Con group; ^∗P < 0.05, compared to the Ti group; ^∗∗P < 0.01, compared to the Ti group. Ti: titanium particle; Con: control; ALP: alkaline phosphatase.

Figure 3

The levels of TNF-α, IL-1β, IL-6, RANKL, and OPG in MC3T3-E1 cells were detected using ELISA kits (n = 3); results are represented as the mean ± SD. ^▲P < 0.05, compared to the Con group; ^▲▲P < 0.01, compared to the Con group; ^∗P < 0.05, compared to the Ti group; ^∗∗P < 0.01, compared to the Ti group. Ti: titanium particle; Con: control.

Figure 4

Curculigoside treatment promoted the osteogenic-associated gene expression in Ti-induced in MC3T3-E1 cells. (a) qRT-PCR analysis was performed to detect the relative mRNA expression of Runx2, osterix, and OCN in MC3T3-E1 cells. (b) Western blot analysis was performed to detect the relative protein expression of casepase-3, casepase-9, SIRT1, BMP-2, RANKL, and OPG in MC3T3-E1 cells (n = 3); results are represented as the mean ± SD. ^▲P < 0.05, compared to the Con group; ^▲▲P < 0.01, compared to the Con group; ^∗P < 0.05, compared to the Ti group; ^∗∗P < 0.01, compared to the Ti group. Ti: titanium particle; Con: control.

Figure 5

Curculigoside treatment inhibited Ti-induced activation of osteoclast activity in BMSCs. (a) After being intervened with Ti and treated with curculigoside at different concentrations (25, 50, and 100 μg/ml) for 48 h, BMSC viability was detected by the CCK-8 assay (n = 6). (b) The levels of TNF-α, IL-1β, and IL-6 in BMSCs were detected using ELISA kits. (c) Cellular immunofluorescence of F-actin in BMSCs (n = 3); results are represented as the mean ± SD. ^▲P < 0.05, compared to the Con group; ^▲▲P < 0.01, compared to the Con group; ^∗P < 0.05, compared to the Ti group; ^∗∗P < 0.01, compared to the Ti group. BMSCs: bone marrow stromal cells; Ti: titanium particles; Con: control.

Figure 6

Curculigoside treatment alleviated osteoclast-associated gene expression in Ti-induced BMSCs. (a) qRT-PCR analysis was performed to detect the relative mRNA expression of cathepsin K, TRAP, NFATc1, and MMP-9 in BMSCs. (b) Western blot analysis was performed to detect the relative protein expression of IκBα, p-IκBα, p65, p-p65, IKKα, p-IKKα, NFATc1, and cathepsin K in BMSCs (n = 3); results are represented as the mean ± SD. ^▲P < 0.05, compared to the Con group; ^▲▲P < 0.01, compared to the Con group; ^∗P < 0.05, compared to the Ti group; ^∗∗P < 0.01, compared to the Ti group. BMSCs: bone marrow stromal cells; Ti: titanium particles; Con: control.

Figure 7

Curculigoside treatment attenuated Ti-induced osteolysis in a mouse calvaria model. (a) Micro-CT reconstruction images of the calvaria in each group of mice. (b) BMC, BMD, BV/TV, and Tb.Th were determined (n = 3); results are represented as the mean ± SD. ^▲P < 0.05, compared to the sham group; ^▲▲P < 0.01, compared to the sham group; ^∗P < 0.05, compared to the Ti group; ^∗∗P < 0.01, compared to the Ti group. Ti: titanium particle; BMC: bone mineral content; BMD: bone mineral density; BV/TV: bone volume to tissue volume; Tb.Th: trabecular thickness.

Figure 8

Curculigoside treatment attenuated Ti-induced osteolysis in histological analysis of calvaria sections: (a) HE staining; (b) TRAP staining. Ti: titanium particle.

Figure 9

Immunohistochemical analysis for RANKL, OPG, TNF-α, IL-1β, and IL-6 in calvaria sections of each group of mice. Ti: titanium particle. Magnification, ×200.