Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression

Abstract

Aim: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro.

Methods: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis.

Results: Aconine (0.125, 0.25 μmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, β3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion.

Conclusion: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Figure 1

Effect of aconine on the viability of RAW264.7 cells. (A) The chemical structure of aconine. (B) Effect of aconine on the viability of RAW264.7 cells. The cytotoxic effect of aconine was evaluated using the CCK-8 method. RAW264.7 cells were cultured for 8 h, 24 h, 48 h, 5 d or 7 d in the presence or absence of varying concentrations of aconine. Optical density was measured at 450 nm. Cell viability was expressed as a percentage of the control. Mean±SEM. n=3. ^bP<0.05 vs control.

Figure 2

Aconine inhibits RANKL-induced osteoclast differentiation. (A) RAW264.7 cells were cultured for 4 d with RANKL (100 ng/mL) in the presence of varying concentrations of aconine and then stained for TRAP activity. Representative photomicrographs were taken under a light microscope (magnification ×100). (B) TRAP-positive cells containing more than three nuclei were counted as osteoclasts. Mean±SEM. n=3. ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs only RANKL-treated cells.

Figure 3

Aconine inhibits RANKL-induced osteoclastic bone resorption. RAW264.7 cells were seeded in Osteo Assay Surface 96-well plates overnight and treated with RANKL (100 ng/mL) in the presence of the indicated concentrations of aconine for 7 d. Cells attached to the plates were removed. (A) Resorption pits on the plates were captured using a light microscope (IX71; Olympus) (magnification ×100). (B) Resorption areas were quantified using Image-Pro Plus 6 software. Mean±SEM. n=3. ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs only RANKL-treated cells.

Figure 4

Aconine inhibits the RANKL-induced mRNA expression of osteoclast-specific genes. RAW264.7 cells were pretreated with varying concentrations of aconine for 2 h and then treated with RANKL (100 ng/mL) for 24 h. The mRNA expression levels of osteoclast-specific genes were analyzed using real-time PCR. Mean±SEM. n=3. ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs only RANKL-treated cells.

Figure 5

Aconine inhibits RANKL-induced NF-κB activation. (A) RAW264.7 cells that were stably transfected with a NF-κB luciferase reporter construct were pretreated with varying concentrations of aconine and the NF-κB inhibitor, BAY11-7082, for 30 min and then treated with RANKL (100 ng/mL) for 8 h. (B) RAW264.7 cells were pretreated with aconine for 30 min prior to RANKL (100 ng/mL) stimulation for 30 min. Then, whole cytoplasmic and nuclear proteins were extracted as described in the methods. The expression levels of IκB-α, NF-κB p65 in the cytoplasmic and NF-κB p65 in the nuclear extracts were determined using Western blot analysis. Subcellular fraction purity and the equality of sample loading were evaluated by analyzing the levels of β-actin and Lamin B1. Protein levels were quantified using densitometry. Mean±SEM. n=3. ^bP<0.05, ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs only RANKL-treated cells.

Figure 6

Aconine inhibits RANKL-induced NFATc1 activation. (A) RAW264.7 cells were pretreated with varying concentrations of aconine for 2 h and then treated with RANKL (100 ng/mL) for 24 h. NFATc1 mRNA levels were analyzed using real-time PCR. (B) RAW264.7 cells that were stably transfected with an NFATc1 luciferase reporter construct were pretreated with varying concentrations of aconine and the NFAT inhibitor, CsA, for 30 min and then treated with RANKL (100 ng/mL) for 8 h. Mean±SEM. n=3. ^bP<0.05, ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs only RANKL-treated cells.

Figure 7

Aconine decreases the expression level of the fusion molecule DC-STAMP. RAW264.7 cells were pretreated with varying concentrations of aconine for 2 h and then treated with RANKL (100 ng/mL) for 24 h. (A) DC-STAMP mRNA levels were analyzed using real-time PCR. (B) The protein levels of DC-STAMP were determined using Western blot analysis. Protein levels were quantified using densitometry. Mean±SEM. n=3. ^bP<0.05, ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs only RANKL-treated cells.