Licochalcone D inhibits osteoclast differentiation and postmenopausal osteoporosis by inactivating the NF-κB signaling pathway

Abstract

Background: Osteoporosis is prevalent among postmenopausal women and is characterized by excessive bone resorption primarily mediated by osteoclasts. This study aimed to investigate the effects of the natural compound Licochalcone D (Lico D) on osteoclast differentiation and its therapeutic potential in ovariectomized (OVX) mouse models of osteoporosis.

Methods: The cytotoxicity of various doses of Lico D on mouse bone marrow-derived macrophages (BMMs) was evaluated using CCK-8 assays. The differentiation of BMMs into osteoclasts was induced by RANKL treatment, followed by exposure to Lico D at doses of 2, 4, and 8 µg/ml. Additionally, 10 µM BAY 11-7821 (an NF-κB inhibitor) was used to inhibit NF-κB signaling in RANKL-stimulated BMMs. TRAP staining was conducted to measure osteoblast cell number. Western blot analysis was performed to measure protein levels of osteoclast differentiation markers and NF-κB-related factors. RT-qPCR was performed to assess the mRNA levels of downstream genes in the NF-κB pathway. In animal experiments, OVX mice received intraperitoneal injections of Lico D at doses of 10 or 50 mg/kg. Subsequently, femurs were harvested for histopathological examination.

Results: Lico D at doses of 2-8 µg/ml showed no significant cytotoxicity toward BMMs. In addition, Lico D inhibited RANKL-induced osteoclast formation and downregulated protein levels of osteoclast-specific genes (mmp9, ctsk, c-Fos and nfatc1). Moreover, Lico D suppressed the phosphorylation of NF-κB p65 and IκBα in RANKL-treated BMMs. Importantly, the suppressive effects of Lico D, especially at 8 µg/ml, on osteoclast cell number and osteoclast-specific markers were comparable to BAY 11-7821. Moreover, Lico D inhibited OVX-induced bone loss and restored dysregulated bone parameters in mice.

Conclusion: Lico D inhibits RANKL-induced osteoclast differentiation and alleviates postmenopausal osteoporosis in mice by suppressing the NF-κB signaling pathway.

Keywords: BAY 11-7821; Bone marrow-derived macrophages; Licochalcone D; NF-κB signaling; Osteoporosis.

Fig. 1

Licochalcone D (Lico D) at doses of 2–8 µg/ml has no significant toxicity to BMMs. A. The chemical structure of Lico D (C₂₁H₂₂O₅) is shown. B-C. Cell counting kit-8 (CCK-8) assays were performed to evaluate the viability of bone marrow-derived macrophages (BMMs) exposed to different doses of Lico D for 48 h (B) or 5 days (C). ^*p < 0.05, ^***p < 0.001 versus Lico D (0 µg/mL) group

Fig. 2

Lico D inactivates NF-κB signaling in RANKL-induced osteoclasts. (A) The timeline of RANKL, Lico D, and BAY 11-7821 treatments. (B) Western blot analysis was conducted to measure protein levels of factors related to the NF-κB pathway. The ratios of p-IκBα/IκBα (C) and p-p65/p65 (D) were quantified. E-G. Relative mRNA levels of NF-κB downstream genes, including iNOS (E), TNF-α (F), and MCP-1 (G), were assessed by RT-qPCR. ^###p < 0.001 versus control group, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus RANKL + Lico D (0 µg/mL) group

Fig. 3

Lico D inhibits osteoclast formation in vitro. (A) TRAP staining was performed to measure the number of osteoclasts in control, RANKL + Lico D, and RANKL + BAY 11-7821 groups. (B) Quantification of TRAP-positive cells in RANKL + Lico D groups and RANKL + BAY 11-7821 group. ^###p < 0.001 versus control group. ^**p < 0.01, ^***p < 0.001 versus RANKL + Lico D (0 µg/mL) group

Fig. 4

Licochalcone D (Lico D) downregulates osteoclast-specific genes in context of RANKL stimulation. A. Expression levels of proteins related to osteoclast differentiation were measured using western blot analysis. Relative protein levels of mmp9 (B), ctsk (C), c-fos (D), and nfatc1 (E) in response to Lico D and BAY 11–782 treatment were quantified in RANKL-induced osteoclasts and normalized to β-actin. ^##p < 0.01, ^###p < 0.001 versus control group. ^**p < 0.01, ^***p < 0.001 versus RANKL + Lico D (0 µg/mL) group

Fig. 5

Lico D plays a protective role against ovariectomized (OVX)-induced bone loss in mouse models of osteoporosis. A. H&E staining was performed to evaluate bone loss in the sham, model, model + Lico D-L, and model + Lico D-H groups. Red arrows indicate that trabeculae are thinner, fractured, and disconnected, causing the loss of the vertebral body’s “railing-like” structure. B-F. Micro-CT analysis was performed to measure structural parameters of distal femurs of mice, including bone mineral density (BMD) (B), bone volume/total volume (BV/TV) (C), bone surface/total volume (BS/TV) (D), trabecular number (Tb.N) (E), and trabecular separation (Tb.Sp) (F). ^***p < 0.001 versus sham group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus model group