Mulberry polyphenols (ABRU) promote bone formation and alleviate bone loss via dual regulation of bone metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Mulberry (Morus alba L.) has a longstanding history of use in Traditional Chinese Medicine (TCM). It is recorded in the classic Compendium of Materia Medica (Ben Cao Gang Mu) that mulberry possesses the efficacy to "strengthen muscles and bones". This traditional indication suggests a potential role in skeletal health, yet its scientific and pharmacological basis has not been fully elucidated. The present study provides the first experimental evidence that a standardized polyphenol extract from mulberry (ABRU) bidirectionally regulates bone metabolism, simultaneously promoting bone formation and inhibiting bone loss. This work not only validates a centuries-old ethnopharmacological claim with modern molecular and functional evidence but also identifies mulberry polyphenols as a promising candidate for developing natural product-based therapies for bone metabolic disorders like osteoporosis.

AIM OF THE STUDY: This study aims to investigate the dual regulatory effects and mechanisms of mulberry polyphenols (ABRU) on bone metabolism.

METHODS: MC3T3 cells and Bone marrow-derived macrophages (BMDMs) were used to evaluate the effects of various mulberry extracts on osteoblastic mineralization and osteoclastogenesis. Active extracts were analyzed for their chemical composition using liquid chromatography-mass spectrometry (LC-MS). Network pharmacology, molecular docking, ROC analysis, and thermal shift assays were employed to identify the molecular targets of ABRU. The dual regulation of bone metabolism by ABRU was further assessed in vivo using subcutaneous bone formation assays and a D-galactose-induced mouse model of bone loss.

RESULTS: In vitro experiments demonstrated that mulberry polyphenol (ABRU) significantly enhanced osteoblast proliferation and differentiation. After 24 h, osteoblast proliferation doubled, after 14 days, mineralization (measured by Alizarin Red staining) and alkaline phosphatase activity both increased two-fold. ABRU also inhibited osteoclast function, achieving 50 % suppression within 6 days, indicating a dual regulatory effect on bone metabolism-promoting bone formation while inhibiting resorption. Further investigation using Pparg knockdown revealed that silencing Pparg reduced SOST expression and upregulated osteogenic markers ALP, OCN, RUNX2. Crucially, Pparg suppression activated the Wnt signaling pathway, evidenced by increased LRP5/TCF expression and enhanced nuclear accumulation of β-catenin.In vivo, oral administration of ABRU dose-dependently increased subcutaneous bone formation in nude mice by 2-4 fold. In the D-galactose-induced aging bone loss model, ABRU supplementation improved bone mineral density by ∼20 %, increased trabecular bone area by 24.5 %, and reduced osteoclast number by 58.4 %. Mechanistically, the core polyphenolic components of ABRU inhibit PPARG expression, leading to downregulation of sclerostin (SOST) activity. This molecular pathway supports the dual regulation of bone metabolism by ABRU.

CONCLUSION: This study is the first, through both in vitro and in vivo experiments, to demonstrate that mulberry polyphenols (ABRU) promote bone formation and alleviate age-related bone loss. It also reveals the dual regulatory effects of ABRU on osteoblast differentiation and osteoclast function, mediated via the PPARG/SOST signaling axis.