Liuwei Dihuang Wan Inhibits Osteoclastogenesis and Ovariectomy-Induced Bone Loss by Suppressing NF-κB Signaling

Liuwei Dihuang Wan (LWDHW), a classic traditional Chinese medicine formula, is widely used to treat various diseases associated with kidney deficiency and has shown satisfactory clinical efficacy in postmenopausal osteoporosis (PMOP). However, its pharmacological mechanisms remain unclear. To address this, we first analyzed the chemical components of LWDHW using LC-MS/MS, which identified 1,615 components. Network pharmacology then predicted that inflammation and the NF-κB signaling pathway are potential therapeutic mechanisms underlying LWDHW's effects on PMOP. Subsequently, 27 female C57BL/6J mice were randomly assigned to the Sham, Model, and LWDHW groups. Mice in the latter two groups underwent bilateral ovariectomy (OVX), and those in the LWDHW group received oral LWDHW at 9.75 g/kg/day for 8 weeks. Serum and femur samples were collected for Enzyme-Linked Immunosorbent Assay (ELISA), Micro-Computed Tomography (µCT), histological examination, and immunohistochemistry (IHC). Primary bone marrow-derived macrophages (BMMs) were treated with LWDHW and then subjected to TRAP staining and qRT-PCR. In OVX mice, LWDHW significantly alleviated bone loss and deterioration of bone microarchitecture, accompanied by reduced serum β‑CTX levels and decreased osteoclast numbers in femoral tissues. IHC staining revealed notably lower expression of TNF-α, IL-1β, and p-P65 in femoral tissues of LWDHW-treated mice compared to the model group. In vitro, TRAP staining demonstrated that LWDHW inhibited RANKL/M-CSF-induced osteoclast differentiation of BMMs and downregulated the expression of Ctsk, MMP9, and Nfatc1. Western blot analysis further showed that protein levels of P65, p-P65, IκBα, and p-IκBα were significantly decreased in BMMs following LWDHW treatment. Collectively, LWDHW alleviates postmenopausal osteoporosis, possibly by suppressing NF-κB signaling‑mediated osteoclastogenesis.