Triterpenoid Saponin W3 from Anemone flaccida Suppresses Osteoclast Differentiation through Inhibiting Activation of MAPKs and NF-κB Pathways

Abstract

Excessive bone resorption by osteoclasts within inflamed joints is the most specific hallmark of rheumatoid arthritis. A. flaccida has long been used for the treatment of arthritis in folk medicine of China; however, the active ingredients responsible for the anti-arthritis effects of A. flaccida are still elusive. In this study, W3, a saponin isolated from the extract of A. flaccida was identified as the major active ingredient by using an osteoclast formation model induced by receptor activator of nuclear factor kappa-B ligand (RANKL). W3 dose-dependently suppressed the actin ring formation and lacunar resorption. Mechanistic investigation revealed that W3 inhibited the RANKL-induced TRAF6 expression, decreased phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and suppressed NF-κB p65 DNA binding activity. Furthermore, W3 almost abrogated the expression of c-Fos and nuclear factor of activated T cells (NFATc1). Therefore, our results suggest that W3 is a potential agent for treating lytic bone diseases although further evaluation in vivo and in clinical trials is needed.

Keywords: Anemone flaccida; Bone resorption; Osteoclasts; RANKL.

Fig 1

Analysis of the phytochemical profile of TS. (A) Phytochemical characteristics of TS were analysed by high performance liquid chromatography (HPLC). (B) Structures of the triterpenoid saponin examined. 1: W1; 2: Saponin R; 3: Saponin F; 4: W2; and 5: W3.

Fig 2

TS and its triterpenoid saponin constituents inhibit RANKL-induced osteoclast differentiation. (A) RAW 264.7 cells were treated with different concentrations of TS and its triterpenoid saponin constituents (0.1, 0.5 and 2.5 μg/mL) in the presence of RANKL. Six days post-culture, cells were fixed with 4% paraformadehyde followed by TRAP staining. Representative images of TRAP staining of osteoclast from one of the three experiments are shown. (B) Quantitative analysis shows the mean number of TRAP-positive osteoclast. (C) Confluent RAW264.7 cells were pretreated with TS and its triterpenoid saponin constituents (0.1, 0.5 and 2.5 μg/mL) or solvent for 2 h, then induced with RANKL for another 24 h. The supernatants were collected and analyzed for TNF-α by enzyme-linked immunosorbnent assay (ELISA). All bar graphs represent mean ± SD of three independent experiments. ^###P < 0.001 significantly different from Control. *P < 0.05, **P<0.01 and ***P<0.001 significantly different from RANKL only group.

Fig 3

W3 suppresses osteoclast resorptive activity. RAW 264.7 cells were treated with different concentrations of W3 (0.1, 0.5 and 2.5 μg/mL) in the presence of RANKL. After 6 days, performed TRAP staining (A), or cells were removed and the bone slices stained by toluidine blue (B) or by scanning electron microscopy (SEM) to identify resorption pits (C). Quantitative analysis shows the percentage of bovine bone slice surface occupied by resorption lacunae (D-F for TRAP staining, G for toluidine blue staining and H for SEM). All bar graphs represent mean ± SD of three independent experiments. ^###P < 0.001 significantly different from Control. *P < 0.05, **P<0.01 and ***P<0.001 significantly different from RANKL only group.

Fig 4

W3 inhibits the F-actin rings formation of osteoclast. RAW 264.7 cells were cultured in the presence of RANKL with different concentrations of W3 (0.1, 0.5 and 2.5 μg/mL). After 6 days, double stained with Honest 33258 (nuclear staining) and rhodamine-phalloidin (F-actin structure) and visualized by fluorescence microscopy. Quantitative analysis shows percentage of osteoclasts forming F-actin rings. All bar graphs represent mean ± SD of three independent experiments. ^###P < 0.001 significantly different from Control. *P < 0.05, **P<0.01 and ***P<0.001 significantly different from RANKL only group.

Fig 5

W3 inhibits RANKL-induced osteoclast differentiation from BMM. BMMs were cultured in the presence of RANKL with different concentrations of W3 (0.1, 0.5 and 2.5 μg/mL). Six days post-culture, cells were fixed with 4% paraformadehyde followed by TRAP staining (A). And quantitative analysis of the mean number of TRAP-positive OCLs was performed in this system (B). Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) method when treated with W3 for 48 h (C). All bar graphs represent mean ± SD of three independent experiments. *P < 0.05, **P<0.01 and ***P<0.001 significantly different from RANKL only group.

Fig 6

W3 inhibits the expression of TRAF6 in RAW 264.7 cells treated with RANKL. (A) Time course of the expression of TRAF6 in RANKL-treated RAW 264.7 cells. Cells were treated with RANKL (50 ng/mL) at various time points as indicated. Expression of TRAF6 was determined by Western blot analysis. (B) RAW 264.7 cells were pretreated with W3 or solvent for 2 h, and then treated with RANKL for 24 h. The cell extracts were subjected to Western blot analysis. Relative amounts of each protein were determined by densitometric analysis. One of three experiments with similar results is shown. Data are represented as the mean ± SD. ^###P<0.001 significantly different from control. *P<0.05, **P<0.01 and ***P<0.001 significantly different from RANKL only group.

Fig 7

W3 suppresses MAPKs activation in RAW 264.7 cells induced by RANKL. (A) RAW 264.7 cells were pre-incubated with or without of W3 for 2 h, following treatment with 50 ng/mL RANKL for 30 min. Total protein was extracted and subjected to Western blot analysis. (B) Relative amounts of each protein were determined by densitometric analysis. One of three experiments with similar results is shown. Data are represented as the mean ± SD. ^###P<0.001 significantly different from control. *P<0.05, **P<0.01 and ***P<0.001 significantly different from RANKL only group.

Fig 8

W3 suppresses NF-κB activation in RANKL-induced RAW 264.7 cells. (A) Confluent RAW 264.7 cells were pre-treated with various concentrations of W3 (0.1-2.5 μg/mL) or vehicle for 2 h followed by RANKL (50 ng/mL) for 0.5 h. The harvested cells from triplicate tests were subjected to western blot analysis for p-IκB as described in the text. (B) Confluent RAW 264.7 cells were treated as described above, cells were analyzed for detection of DNA binding of NF-κB by EMSA. Relative NF-κB activity was calculated by densitometric analysis. Data represent the mean ± SD of three measurements. ^###P<0.001 significantly different from basal. *P<0.05, and ***P<0.001 significantly different from RANKL only group.

Fig 9

W3 down-regulates the expression of NFTAc1 and c-Fos in RANKL-treated RAW 264.7 cells. (A) RAW 264.7 cells were treated with indicated concentrations of W3 or solvent for 2 h, followed by RANKL for 24 h. The harvested cells from triplicate tests were subjected to Western blot analysis. (B) Relative amounts of each protein were determined by densitometric analysis. Data represent the mean ± SD of three measurements. ^###P<0.001 significantly different from basal. *P<0.05, and ***P<0.001 significantly different from RANKL only group.

Fig 10

Schematic illustration showing putative-signaling pathways involved in osteoclast differentiation regulated by W3. The figure summarizes the results presented in this study. Red X mark indicates that an inhibition or a down regulation in osteoclast was observed.