NuanXin Formula inhibits bone resorption to combat osteoporosis by attenuating osteoclast oxidative phosphorylation

Ethnopharmacological relevance: Osteoporosis is a chronic metabolic bone disorder characterized by excessive bone resorption. The NuanXin Formula (NX) is a classical traditional Chinese medicine formula that can warm and tonify kidney Yang, as well as replenish Qi and blood, which are essential for maintaining bone health and regulating bone metabolism. Nevertheless, the functions and mechanisms of NX in osteoporosis therapy remain unclear. Aim of the study: This study aims to evaluate the effects and mechanisms of NX on osteoclastogenesis and to investigate its potential in combating osteoporosis. Materials and methods: The inhibitory effects of NX on RANKL-induced osteoclastogenesis were evaluated using Western blotting, quantitative PCR (Q-PCR), TRAP staining, and pit-formation assays. Bone mass and structure were assessed through micro-CT, biomechanical testing, TRAP staining, IHC staining, and H&E staining. The mechanism of action of NX on osteoclasts was investigated using RNA sequencing, ROS staining, ATP measurement, and mitochondrial membrane potential assays. Results: The in vitro findings demonstrated that NX treatment significantly inhibited osteoclast differentiation and bone resorption activity. Q-PCR and WB analyses indicated that NX substantially downregulates the expression levels of key osteoclast markers, including Nfatc1, Ctsk, Mmp9, and Trap. In vivo experiments revealed that intragastric administration of NX effectively suppressed bone loss and bone resorption, while enhancing the biomechanical properties of bone in ovariectomized (OVX) mice. Mechanistically, NX inhibits oxidative phosphorylation (OXPHOS), decreases mitochondrial membrane potential, and reduces ATP production and reactive oxygen species generation, thereby impeding osteoclast differentiation and activity. Conclusion: NX mitigates osteoporosis by modulating OXPHOS to inhibit osteoclast differentiation and activity, thus offering a potential therapeutic approach for osteoporosis management. However, the study has limitations that require further investigation. NX did not show a clear dose-dependent effect in animal tests, suggesting a need for improved dosing designs. Although we emphasize NX's therapeutic potential, more research is necessary to clarify its mechanism. Variability in plant materials and ingredient ratios might influence NX's