Asiatic Acid Attenuates Osteoporotic Bone Loss in Ovariectomized Mice Through Inhibiting NF-kappaB/MAPK/ Protein Kinase B Signaling Pathway

Abstract

Osteoporosis is a condition associated with osteolytic bone disease that is primarily characterized by inordinate osteoclast activation. Protein kinase B (Akt) pathways activated by receptor activator of nuclear factor kappa-B ligand (RANKL) are essential for osteoclastogenesis. Asiatic acid (AA) is a natural pentacyclic triterpenoid compound extracted from a traditional Chinese herb that exhibits a wide range of biological activities. AA has been found to alleviate the hypertrophic and fibrotic phenotype of chondrocytes via the Akt signaling pathway. In this study, we investigated whether AA alleviated bone loss by inhibiting the Akt signaling pathway during osteoclastogenesis and its effect on osteoblasts. The effect of AA cytotoxicity on mouse bone marrow-derived macrophages/monocytes (BMMs) was evaluated in vitro using a Cell Counting Kit-8 assay. The effects of AA on osteoclast differentiation and function were detected using tartrate-resistant acid phosphatase (TRAP) staining and a pit formation assay. A Western blot and qRT-PCR were conducted to evaluate the expression of osteoclast-specific genes and protein signaling molecules. In addition, alkaline phosphatase and alizarin red staining were performed to assess osteoblast differentiation and mineralization. The bone protective effect of AA was investigated in vivo using ovariectomized mice. we found that AA could dose-dependently inhibit RANKL-induced osteoclastogenesis. Moreover, the pit formation assay revealed that osteoclast function was suppressed by treatment with AA. Moreover, the expression of osteoclast-specific genes was found to be substantially decreased during osteoclastogenesis. Analysis of the molecular mechanisms showed that AA could inhibit NF-kappaB/MAPK/Akt signaling pathway, as well as the downstream factors of NFATc1 in the osteoclast signaling pathway activated by RANKL. However, AA did not significantly promote osteoblast differentiation and mineralization. The in vivo experiments suggested that AA could alleviate ovariectomy-induced bone loss in ovariectomized mice. Our results demonstrate that AA can inhibit osteoclastogenesis and prevent ovariectomy-induced bone loss by inhibiting the NF-kappaB/MAPK/Akt signaling pathway. The discovery of the new molecular mechanism that AA inhibits osteoclastogenesis provides essential evidence to support the use of AA as a potential drug for the treatment of osteoclast-related diseases.

Keywords: asiatic acid; osteoblast; osteoclastogenesis; osteoporosis; protein kinase B.

FIGURE 1

Asiatic acid suppresses the differentiation and bone resorption of osteoclasts. (A) The structural formula for asiatic acid. (B) Drug toxicity of asiatic acid on BMMs. After BMMs were treated with asiatic acid for 48 h, and the absorbance value of each well was measured with a CCK8 kit (n = 4). (C) Representative images of asiatic acid inhibition of osteoclast differentiation. BMMs were cultured in a-MEM containing 30 ng/mL M-CSF and 50 ng/ml RANKL with or without asiatic acid. After 7 days, the cells were fixed in 4% paraformaldehyde and stained with TRAP (n = 3). (D,E) Quantitative analysis of the number and area proportion of TRAP-positive osteoclasts (≥3 nuclei) (n = 3). (F) Representative images of asiatic acid inhibition of bone resorption. (G) Quantification analyses of the resorbed area per well (n = 3). All values are presented as the mean ± SD (n = 3). *p < .05; **p < .01; ***p < .001; ****p < .0001.

FIGURE 2

Asiatic acid inhibits RANKL-induced osteoclast-related gene expression. (A–F) BMMs were exposed to M-CSF and RANKL for 7 days with or without AA. The expression of osteoclast-specific genes (e.g., NFATc1, c-fos, CTSK, Acp5, DC-stamp, and V-ATpase-D2) were detected with fluorescence quantitative RT-PCR. All values are presented as the mean ± SD (n = 3). *p < .05; **p < .01; ***p < .001; ****p < .0001.

FIGURE 3

Asiatic acid regulates NFATc1 expression by inhibiting the NF-κB/MAPK/Akt signaling pathway. (A) Representative Western Blot images of the effect of asiatic acid on the MAPK pathway. After 2 h of starvation, BMMs were stimulated for 2 h with or without asiatic acid (20 μM). BMMs were stimulated with RANKL for 0, 5, 10, 20, 30, and 60 min (B–D) Quantitative analyses of the expression of P-p38, P-ERK, P-JNK, p38, ERK and JNK (n = 3). (E) Representative Western Blot images of the effect of asiatic acid on the NF-κB pathway. (F,H) Quantitative analyses of the expression of P-p65, p65 and IκBα (n = 3). (G) Representative Western Blot images of the effect of asiatic acid on the Akt pathway. (K) Quantitative analyses of the expression of P-Akt and Akt (n = 3). (I) Representative Western Blot images of the effect of asiatic acid on NFATc1. BMMs were stimulated with RANKL for 0, 1, 3, and 5 days in the presence or absence of asiatic acid (20 μM). (J) Quantitative analyses of the expression of NFATc1 (n = 3). All values are presented as the mean ± SD (n = 3). *p < .05; **p < .01 compared with the control group (without AA treatment).

FIGURE 4

Asiatic acid could not significantly enhance osteoblast differentiation and mineralization. (A) Representative images of alkaline phosphatase staining showing that the effect of asiatic acid on osteogenic differentiation. After 7 days of osteogenic induction with or without asiatic acid, osteoblasts were observed by alkaline phosphatase staining kit (n = 3). (B) Following 21 days of osteogenic induction, mineralized nodules were evaluated by alizarin red staining kit (n = 3). (C) Quantitative analysis of the percentage of ALP-positive osteoblasts. (D) Quantitative analysis of the percentage of mineralized nodules. All values are presented as the mean ± SD (n = 3).

FIGURE 5

Asiatic acid suppresses the progression of osteoporosis in ovariectomized mice. (A) Three-dimensional reconstructed representative image of the mouse tibia. (B) Representative images of HE-stained tibia in mice. (C) Representative images of Trap staining of mice tibia. (D–F) Quantitative analysis of bone volume/tissue volume (BV/TV), bone trabecular number (TB. N), and bone trabecular separation (TB. SP) of mice (n = 3). (G) Quantitative histological analysis of TRAP-positive cell percentage per bone surface in each group (n = 3). All values are presented as the mean ± SD (n = 3). *p < .05; **p < .01; ***p < .001; ****p < .0001.

FIGURE 6

Proposed scheme for mechanism by which asiatic acid inhibits osteoclastogenesis. After binding to RANKL, the RANK can recruit and activate the TRAF6, the NF-κB/MAPK/Akt signaling pathway are activated, resulting in the self-amplification of NFATc1. NFATc1 regulates the expression of osteoclast specific genes, including c-fos, CTSK, Acp5, DC-stamp, and V-ATpase-D2. Our results demonstrate that AA can inhibit osteoclastogenesis by inhibiting the NF-κB/MAPK/Akt signaling pathway.