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.
Highlights
Osteoporosis is a metabolic bone disease with systemic bone degeneration characterized by reduced bone mass and destruction of the bone microstructure
Alpha modification of Eagle’s medium (α-MEM), fetal bovine serum (FBS), streptomycin, and penicillin were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, United States), and Receptor activator of nuclear factor kappa-B ligand (RANKL) and M-CSF were purchased by R&D Systems (Minneapolis, MN, United States)
Following exposure to RANKL and macrophage colony-stimulating factor (MCSF), the effect of asiatic acid on osteoclast differentiation was evaluated by the number and area of Tartrate resistant acid phosphatase (TRAP)-positive cells
Summary
Osteoporosis is a metabolic bone disease with systemic bone degeneration characterized by reduced bone mass and destruction of the bone microstructure. Osteoclasts derived from bone marrow hematopoietic stem cells are multinucleated giant cells formed by the fusion of multiple mononuclear macrophages (Yahara et al, 2020). It has been well-established that both macrophage colony-stimulating factor (MCSF) and nuclear factor kappa-B ligand (RANKL) are two necessary cytokines responsible for osteoclast formation (Boyce, 2013). TRAF6 regulates osteoclast bone resorption and differentiation through activated downstream signal transduction cascades (e.g., NF-κB/MAPK/Akt signaling pathway) (Boyle et al, 2003; Novack, 2011). Akt participates in osteoclast formation primarily by regulating the glycogen synthase kinase 3β (GSK3β)/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway (Moon et al, 2012). NFATc1 is a vital transcription factor that regulates the expression of osteoclast specific genes [e.g., cathepsin K (CTSK) and dendritic cellspecific transmembrane protein (DC-stamp)] (Takayanagi et al, 2002; Kim et al, 2008; Balkan et al, 2009), impacting the formation of multinucleated mature osteoclasts
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