Sophorae Flos extract inhibits RANKL-induced osteoclast differentiation by suppressing the NF-κB/NFATc1 pathway in mouse bone marrow cells

Abstract

Background: Sophorae Flos (SF) is a composite of flowers and buds of Styphnolobium japonicum (L.) Schott and has been used in traditional Korean and Chinese medicine for the treatment of hemostasis and inflammation. Previous studies reported that SF possesses anti-obesity properties, as well as anti-allergic, anti-proliferative, and anti-inflammatory activities. However, the effect of SF in bone resorption has not been studies. In this study, we examined the potential of SF extract (SFE) to inhibit receptor activator of NF-κB ligand (RANKL) -induced osteoclast differentiation in cultured mouse-derived bone marrow macrophages (BMMs).

Methods: BMMs, that act as osteoclast precursors, were cultured with M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 4 days to generate osteoclasts. Osteoclast differentiation was measured by tartrate-resistant acidic phosphatase (TRAP) staining and the TRAP solution assay. Osteoclast differentiation marker genes were analyzed by the quantitative real-time polymerase chain reaction analysis. RANKLs signaling pathways were confirmed through western blotting.

Results: SFE significantly decreased osteoclast differentiation in a dose-dependent manner. SFE inhibited RANKL-induced osteoclastogenesis by suppressing NF-κB activation. By contrast, SFE did not affect phospholipase C gamma 2 or subsequent cAMP response element binding activation. SFE inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1).

Conclusions: SFE attenuated the RANKL-mediated induction of NF-κB through inhibition of IκBα phosphorylation, which contributed to inhibiting of RANKL-induced osteoclast differentiation through downregulation of NFATc1.

Keywords: NF-κB; NFATc1; Osteoclast; PLCγ2; Sophorae Flos (SF).

Fig. 1

Effects of Sophorae Flos extract (SFE) on bone marrow marcrophages. a BMMs were cultured with various concentrations of SFE for 1 day. b BMMs were cultured with or without (control) 100 μg/ml SFE for 4 days. Cell viability was measured as described in methods. Data from three independent experiments are expressed as relative proliferation (% of control) ± SD

Fig. 2

Effects of Sophorae Flos extract (SFE) on osteoclast differentiation. Bone marrow macrophages were cultured with various concentrations of SFE and treated with RANKL (100 μg/ml) and M-CSF (50 μg/ml) for 4 days. a Osteoclasts stained for tartrate-resistant acidic phosphatase (TRAP). b TRAP⁺ multinuclear cells (MNCs) with more than three nuclei were considered to be mature osteoclasts. c Total TRAP activity from TRAP⁺ mono-, di-, and multinuclear cells was quantified as described in methods. Data from three independent experiments are expressed as mean ± SD. *P < 0.05, ^# P < 0.01 versus control (0 μg/ml SFE)

Fig. 3

Effects of Sophorae Flos extract (SFE) on the expression of osteoclast differentiation marker genes. Bone marrow macrophages were cultured with RANKL and M-CSF treatment in the presence or absence of SFE (100 μg/ml) for 4 days. The expression of marker genes of osteoclast differentiation was measured by real-time quantitative PCR. Target gene mRNA levels were normalized to GAPDH and are presented as fold change from control (0 μg/ml SFE). Data are expressed as mean ± SD and are representative of at least three independent experiments. *P < 0.05, #P < 0.01 versus control

Fig. 4

Effects of Sophorae Flos extract (SFE) on RANKL-induced intracellular signaling and expression of c-fos and NFATc1 in osteoclasts. Bone marrow macrophages were cultured with RANKL and MCSF in the presence or absence of SFE (100 μg/ml) for 4 days. Protein expression levels were evaluated by western blot analysis. a Activation of MAPKs and NF-κB measured using by their respective antibodies. Expression of (b) c-fos and (c) NFATc1 detected by the respective antibodies. All data are representative of at least three independent experiments

Fig. 5

Sophorae Flos extract (SFE) does not inhibit RANKL-mediated activation of PLCγ2 and CREB in osteoclasts. Bone marrow macrophages were cultured with M-CSF and RANKL in presence or absence of SFE (100 μg/ml) for 4 days. Lysate (30 μg protein) was subjected to SDS-PAGE and the phosphorylation of (a) PLCγ2 and (b) CREB analyzed by western blotting. All data are representative of at least three independent experiments