Hydroxygenkwanin Identified in Rehmanniae Radix Praeparata–Corni Fructus Ameliorates Postmenopausal Osteoporosis via Modulation of the AGE-RAGE Signaling Pathway

Songlin Liang,1 Dezhi Tang,2 Hui Wang,3 Shang Gao,1 Zenghui Tian,1 Xintian Qu,1 Luming Kong,1 Xin Xin,1 Jiatong Li,1 Nianhu Li1,41First College of Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan, 250014, People’s Republic of China; 2Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, People’s Republic of China; 3Institute for Chinese Medicine Innovation, Shandong University of Traditional Chinese Medicine, Jinan, 250300, People’s Republic of China; 4Department of Orthopedics, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, 250014, People’s Republic of ChinaCorrespondence: Songlin Liang, First College of Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan, 250355, People’s Republic of China, Email [email protected]: Osteoporosis (OP) is a metabolic bone disease marked by reduced bone mass and compromised microarchitecture, increasing fragility fracture risk. The traditional Chinese herbal pair Rehmanniae Radix Praeparata–Corni Fructus (RRP-CF) is frequently used for OP, but its mechanism remains unclear.Methods: Network pharmacology, UHPLC-HRMS, molecular docking, and molecular dynamics simulations were integrated to identify the potential active components of RRP-CF and to predict the potential targets and related signaling pathways underlying its anti-osteoporotic effects. These predictions were further validated through in vitro (using 25 and 50 μM hydroxygenkwanin (HGK)) and in vivo experiments in ovariectomized (OVX) rats (using 10 and 20 mg/kg HGK).Results: UHPLC-HRMS identified HGK as one of the detectable components of RRP-CF. A total of 66 common targets between RRP-CF and OP were screened out by network pharmacology; Protein-Protein Interaction (PPI) network analysis showed that estrogen receptor 1 (ESR1), tumor protein p53 (TP53), tumor necrosis factor (TNF), signal transducer and activator of transcription 3 (STAT3), and prostaglandin-endoperoxide synthase 2 (PTGS2) were the key targets. Gene Ontology enrichment suggested that RRP-CF-associated targets were mainly involved in inflammatory response, and the advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE) signaling pathway was identified as a critical pathway. In vivo experiments confirmed that RRP-CF could improve bone microstructure and collagen content, as evidenced by increased BMD (from 106.58 ± 11.09 mg/cm3 in the OVX group to 188.35 ± 10.18 mg/cm3 in the 10 mg/kg HGK-treated group) and bone volume/tissue volume (BV/TV) (from 0.13 ± 0.02 to 0.29 ± 0.05). Mechanistically, HGK treatment downregulated RAGE expression in OVX rat bone tissue (P = 0.0208) and receptor activator of nuclear factor-κB ligand (RANKL)-induced RAW264.7 cells (P = 0.0005), and suppressed osteoclast-related proteins including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) (P = 0.0061 in cells) and cathepsin K (CTSK) (P < 0.0001 in rats; P = 0.0006 in cells). Furthermore, rescue experiments using the RAGE inhibitor FPS-ZM1 suggested that the effects of HGK were associated with modulation of RAGE-related signaling.Conclusion: Our findings indicate that RRP-CF can inhibit bone loss in OVX rats, and suggest that HGK, as a potential active constituent of RRP-CF, may exert anti-osteoporotic effects through modulation of the AGE-RAGE pathway and downregulation of osteoclast-related proteins including p53, p-AKT, NFATc1 and CTSK. These findings provide a preliminary mechanistic basis for developing novel OP therapeutic regimens, though further pharmacokinetic and safety studies are warranted.Keywords: osteoporosis, Rehmanniae Radix Praeparata–Corni Fructus, hydroxygenkwanin, osteoclast differentiation, AGE-RAGE signaling pathway