Myrislignan Targets Extracellular Signal-regulated Kinase (ERK) and Modulates Mitochondrial Function to Dampen Osteoclastogenesis and Ovariectomy-i nduced Osteoporosis

Background: Numerous studies have confirmed that activated osteoclasts cause excessive bone resorption, disrupting bone homeostasis and leading to osteoporosis. Moreover, ERK signaling is the classical pathway related to osteoclast differentiation. Besides, reactive oxygen species (ROS) is mainly from mitochondria, which is closely associated with the differentiation of osteoclasts. Myrislignan (MRL), a natural product derived from nutmeg, has various pharmacological activities. However, its effect on the treatment of osteoporosis is unclear. Therefore, this study mainly investigated whether MRL could inhibit osteoclastogenesis and bone mass loss in ovariectomy (OVX) mice via suppressing mitochondrial function and ERK signaling. Methods: Tartrate-resistant and phosphatase (TRAP) assay and bone resorption assay were used to observe the effect of MRL on osteoclastogenesis. Furthermore, we added MitoSOX RED and tetramethyl rhodamine methyl ester (TMRM) staining to test the inhibitory effect of MRL on mitochondria. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay detected whether MRL suppressed the expression of specific genes in osteoclasts. The impact of MRL on mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) related proteins was evaluated by western blotting. Besides, a specific ERK agonist LM22B-10 (LM), was added to revalidate the inhibitory effect of MRL on ERK. Moreover, we established an OVX mouse model to assess the therapeutic effect of MRL on osteoporosis in vivo . Results: MRL was proven to press osteoclast differentiation and bone resorption function, significantly reducing osteoclastic gene expression. Mechanistically, MRL inhibited the phosphorylation of ERK by suppressing the role of mitochondria, causing the downregulation of nuclear factor of activated T cells 1 (NFATc1) signaling. The experiment result of adding LM further clarified the targeted inhibition effect of MRL on ERK. The results of microscopic computed tomography (Micro-CT) and histology sections of the tibia in vivo indicated that OVX mice had lower bone mass and higher expression of ERK. However, after the MRL application, these results were significantly reversed, suggesting that MRL had a decent anti-osteoporosis effect. Conclusion: We saw for the first time that MRL could inhibit ERK signaling by suppressing mitochondrial function, thus reducing OVX-induced osteoporosis. This novel finding could provide a broad prospect for the treatment of osteoporosis.