Osteoporosis (OP) is a prevalent chronic metabolic bone disease linked to estrogen deficiency and lacks effective therapies with minimal side effects. While N6-methyladenosine (m6A) methylation plays a crucial role in OP progression, its critical effect on osteoclastogenesis during OP remains unclear. In this study, we demonstrate that METTL3 promotes osteoclast differentiation and bone erosion in OP, and its expression positively correlated with bone loss. METTL3 silencing abolished osteoclast formation, while its overexpression exacerbated bone resorption. Through molecular docking, dynamics simulation, cellular thermal shift assay and surface plasmon resonance assay, we identify the natural compound Nimbolide (Nim) as a direct METTL3 inhibitor that disrupts its methyltransferase activity. Nim treatment suppressed RANKL-induced osteoclastogenesis and attenuated ovariectomy-induced OP in mice. Mechanistically, multi-omics integration (RNA-seq/MeRIP-seq/RIP-seq) combined with in vitro molecular validation revealed that METTL3 installs m6A on Lrp1 mRNA, recruiting the reader YTHDF2 to degrade transcripts and decrease the anti-osteoclastogenic protein LRP1. Nim restored LRP1 by blocking METTL3-mediated m6A methylation, thereby inhibiting osteoclast hyperactivity. Critically, AAV9-driven METTL3 overexpression reversed Nim’s efficacy in OVX mice. Our work identifies Nim as a novel METTL3 inhibitor that protects against OP via the METTL3-m6A-YTHDF2-LRP1 axis, providing novel insights into targeting METTL3-dependent m6A signaling as a promising therapeutic strategy for OP.
