Rhusflavone Modulates Osteoclastogenesis Through RANKL-Induced AKT Signaling in Bone Marrow-Derived Macrophages

Abstract

Osteoclast differentiation inhibition is a viable treatment strategy for osteoporosis because osteoclasts play a vital role in disease progression. Rhusflavone (Rhus), a biflavonoid, exhibits a sedative-hypnotic effect via the positive allosteric modulation of GABA(A) receptors. Although several biflavonoids possess activities that help prevent bone loss, the potential effects of Rhus on osteoclastogenesis have not been reported yet. In this study, we investigated the effects and underlying biological mechanisms of Rhus isolated from the dried roots of Rhus succedanea on osteoclastogenesis in primary cultured bone marrow-derived macrophages. No cytotoxicity was observed in bone marrow macrophages (BMMs) or during osteoclast differentiation. However, Rhus reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis. The results of F-actin ring formation demonstrated that Rhus suppresses the bone resorption activity of osteoclasts. Additionally, Rhus inhibits the expression of osteoclast differentiation marker proteins, specifically c-Fos and NF-ATc1. Western blot analysis revealed that Rhus primarily attenuated RANKL-mediated key signaling pathways, particularly the AKT signaling pathway. Furthermore, we found that the AKT activator and inhibitor pharmacologically abolished and enhanced the inhibitory effects of Rhus on osteoclast differentiation, respectively. Taken together, our findings provide evidence that Rhus is a promising biologically active compound that regulates osteoclast differentiation by inhibiting the AKT signaling pathway, which may contribute to future drug development.

Keywords: AKT; BMM; RANKL; Rhusflavone; bone; osteoclast.

Figure 1

Extractoin of Rhusflavone (Rhus) from the dried roots of Rhus succedanean and its cytotoxicity effects. (A) Procedure for the isolation of Rhus. (B,C) ¹H-NMR (500 MHz, CD3OD) spectrum (B) and ¹³C-NMR (125 MHz, CD3OD) spectrum (C) of Rhus. (D) HPLC analysis of the isolated Rhus. The inset shows the chemical structure, purity, and molecular formula. (E,F) Bone marrow macrophages (BMMs) were seeded onto 96-well plates and treated with the indicated Rhus concentration for 24 h (E). The BMMs were differentiated into osteoclasts using 30 ng/mL macrophage colony-stimulating factor (M-CSF) and 100 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) for 3 days, and the cells were subsequently treated with the indicated Rhus concentration (F). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. OCs: osteoclasts. The data presented are derived from three separate experiments and are expressed as the mean ± standard deviation (SD).

Figure 2

Effects of Rhusflavone (Rhus) on osteoclastogenesis and bone-resorbing activity. (A,B) Bone marrow macrophages (BMMs) were seeded into 48-well plates and cultured for 5 days in a medium containing 100 ng/mL of receptor activator of nuclear factor kappa B ligand (RANKL) and 30 ng/mL of macrophage colony-stimulating factor (M-CSF) with 1–30 μM of Rhus. Mature osteoclasts were detected with tartrate-resistant acid phosphatase (TRAP) staining (A). TRAP-positive multinucleated osteoclasts (MNCs) with over three nuclei were considered as mature osteoclasts and counted under a microscope (B). A quantity of 3–10 nuclei (left), 10< nuclei (right). Scale bar: 100 μm. (C,D) F-actin ring (red) formation was detected using a fluorescence microscope after staining with TRITC–phalloidin (red) and DAPI (blue) (C). The number of ring formations greater than 1 mm² was analyzed (D). Scale bar: 100 μm. *, p < 0.05 compared to RANKL treatment alone. The data presented are derived from three separate experiments and are expressed as the mean ± standard deviation (SD).

Figure 3

Effects of Rhusflavone (Rhus) on osteoclast-specific transcription factors. (A,B) Bone marrow macrophages (BMMs) were seeded in 6-well plates and differentiated for 1 day (A) or 2 days (B). Western blot analysis was used to assess c-Fos and β-actin (A); NF-ATc1 and β-actin (B) expression levels. The relative level (%) is presented as a bar graph. (C) After treating Rhus for 24 h, c-Fos (green) was immunostained. Subsequently, DAPI (blue) was used to stain the nucleus. Relative fluorescence intensity fold changes are presented as bar graphs. *, p < 0.05 compared to RANKL treatment alone. The data presented are derived from three separate experiments and are expressed as the mean ± standard deviation (SD).

Figure 4

Effects of Rhusflavone (Rhus) on receptor activator of nuclear factor kappa B ligand (RANKL)-induced intracellular signaling pathways. (A–C) Bone marrow macrophages (BMMs) were seeded on 6-well plates and stimulated in 100 ng/mL RANKL with 10 μM Rhus for the indicated time. AKT, p-AKT, and β-actin (A); ERK, p-ERK, JNK, p-JNK, p38, and p-p38 (B); IkB and β-actin (C) expression were investigated using western blot analysis. The relative level (%) is presented as a bar graph. *, p < 0.05 compared to RANKL treatment alone at 0 min. #, p < 0.05 compared between RANKL alone and RANKL + Rhus at each min time point. The data presented are derived from three separate experiments and are expressed as the mean ± standard deviation (SD).

Figure 5

Effects of Rhus on the inhibition of receptor activator of nuclear factor kappa B ligand (RANKL)-induced AKT signaling pathway in osteoclastogenesis. (A) Bone marrow macrophages (BMMs) were differentiated with 10 μM Rhus, in the presence or absence of 1 μM SC79 or LY294002, for 1 day. Western blot analysis was used to assess c-Fos and β-actin expression levels. The relative level (%) is presented as a bar graph. (B,C) The BMMs were cultured in 30 ng/mL M-CSF and 100 ng/mL RANKL with 10 μM Rhus, in the presence or absence of 1 μM SC79 or LY294002, for 5 days. Mature osteoclasts were detected with tartrate-resistant acid phosphatase (TRAP) staining (pink) (B). TRAP-positive multinucleated osteoclasts (MNCs) were counted under a microscope (C). A quantity of 3–10 nuclei (left), 10 < nuclei (right). Scale bar: 100 μm. *, p < 0.05 compared with RANKL alone. #, p < 0.05 compared with RANKL + Rhus. The data presented are derived from three separate experiments and are expressed as the mean ± standard deviation (SD).