Dual suppression of AKT/MAPK signaling and activation of Nrf2 by methylnissolin attenuates osteoclastogenesis and prevents postmenopausal osteoporosis.

Abstract

BACKGROUND: Osteoporosis is a widespread metabolic bone disorder characterized by reduced bone mass and increased fracture risk, particularly in postmenopausal women due to estrogen deficiency. This hormonal decline enhances osteoclast activity, leading to excessive bone resorption. Current therapies are limited by long-term safety concerns, necessitating alternative treatments. Methylnissolin (Astrapterocarpan), a bioactive flavonoid from Astragalus membranaceus, has known anti-inflammatory and antioxidant properties, but its role in bone metabolism remains poorly defined. PURPOSE: This study aimed to elucidate the molecular mechanisms by which Methylnissolin regulates osteoclastogenesis and bone resorption, and to evaluate its therapeutic potential in osteoporosis. METHODS: Network pharmacology identified MAPK1 and AKT1 as potential molecular targets of Methylnissolin in osteoporosis. In vitro assays were performed to assess osteoclast formation and bone resorption in response to Methylnissolin. PCR and Western blotting were used to evaluate its effects on signaling pathways and gene expression. ROS levels and antioxidant enzyme expression were also measured. An ovariectomized (OVX) mouse model was used to assess in vivo efficacy and safety. RESULTS: Methylnissolin inhibited osteoclast differentiation and resorptive activity in a dose- and time-dependent manner. It suppressed MAPK1 and AKT1 phosphorylation, reduced ROS levels, and upregulated antioxidant enzymes. These effects led to downregulation of key osteoclastogenic markers at both gene and protein levels. In OVX mice, Methylnissolin significantly improved trabecular bone parameters without detectable toxicity. CONCLUSION: Methylnissolin is a novel osteoclast inhibitor that acts via coordinated suppression of MAPK/AKT signaling and oxidative stress. These findings support its potential as a safe and effective therapeutic candidate for postmenopausal osteoporosis.