Integrating Network Pharmacology, IPA, and Molecular Docking to Reveal the Anti-Osteoporosis Effects of EA and EB via the FAK Pathway Abstract：

Ziheng Wei^1,2Fei You³Henghui Li³Si Wu⁴Fen Tang³Xiangyang Wan³Huizhong Dong³Wenxuan Huang³Songyan Gao³Bo Cai⁵Xiongsheng Chen^2*Xin Dong^3*

• ¹School of Medicine, Shanghai Jiao Tong University, Shanghai, China
• ²Department of Orthopaedics, Shanghai General Hospital, Shanghai, China
• ³Shanghai University, Shanghai, Shanghai Municipality, China
• ⁴Departments of Genetics, School of Medicine, Stanford University, Stanford, California, United States
• ⁵Shanghai baoshan Center for Disease Control and Prevention, Shanghai, China

Osteoporosis, a prevalent condition in the elderly and postmenopausal women, elevates fracture risk and diminishes quality of life. Epimedium, a traditional Chinese herb, is widely used for osteoporosis treatment but contains complex compounds requiring isolation of active monomers to enhance efficacy and safety. Using network pharmacology and literature review, we identified five novel anti-osteoporotic monomers: Epimedin A (EA), Epimedin B (EB), Epimedoside A (EPA), 4-Hydroxybenzaldehyde (PHBA), and Baohuoside VI. In vitro assays revealed that EA, EB, and EPA dose-dependently inhibited osteoclast differentiation in bone marrow-derived macrophages (BMMs) via reduced TRAP-positive cells and osteoclast-related gene expression. Conversely, EA and EB promoted osteoblast differentiation in cranial preosteoblasts, evidenced by elevated ALP activity, mineralization, and osteogenic gene markers.In ovariectomized (OVX) mice, EA and EB (5–20 mg/kg) significantly increased bone mineral density (BMD) and trabecular bone number versus OVX controls (P < 0.05), with no pharmacological toxicity observed in organ histopathology. Mechanistically, NF-κB and MAPK (ERK/JNK) pathways were unaffected, as shown by luciferase reporter assays and Western blotting. Target prediction via PharmMapper, SwissTargetPrediction, and SuperPred identified focal adhesion kinase (FAK) as a key mediator. Molecular docking confirmed strong binding of EA and EB to FAK1 (ΔG: -13.012 and -14.0164 kJ/mol) and FAK2 (ΔG: -5.815 and -6.4852 kJ/mol).qPCR validated that EA and EB suppressed FAK1 and FAK2 expression in osteoclasts but upregulated them in osteoblasts at high doses. These findings suggest that EA and EB mitigate osteoporosis by modulating FAK signaling to inhibit bone resorption and enhance formation. This study highlights FAK as a therapeutic target and provides novel insights into Epimedium-derived monomers for precision osteoporosis treatment. Further research is warranted to delineate FAK-dependent mechanisms in vivo.