Bu-Sui-Dan promotes angiogenesis to mitigate age-related osteoporosis via the ZEB1/Notch1 axis

Bu-Sui-Dan (BSD) is a traditional Chinese medicine formulation first recorded in the Song Dynasty text Shi Zhai Bai Yi Xuan Fang for the treatment of "Gu-Wei" (bone flaccidity), a condition clinically corresponding to osteoporosis. Although BSD demonstrates definite anti-osteoporotic effects, the underlying mechanisms remain largely unclear.

To explore the therapeutic mechanisms of BSD in age-related osteoporosis.

Herein, we utilized LC-MS/MS to analyze the chemical composition of BSD. Naturally aged mice were utilized to establish the age-related osteoporosis model. Therapeutic effects were evaluated utilizing micro-CT, histomorphometry, and immunofluorescence. Interactions between BSD components and zinc finger E-box-binding homeobox 1 (ZEB1) were analyzed via molecular docking and microscale thermophoresis (MST). Epigenetic regulation of Delta-like ligand 4 (Dll4)/Notch1 signaling was assessed using quantitative real-time PCR, Western blotting, and chromatin immunoprecipitation. ZEB1's function was validated in vivo via short hairpin RNA (shRNA) knockdown.

We identified 576 bioactive constituents within BSD. Docking and MST assays indicated that BSD active compounds interacted with ZEB1, exhibiting low-micromolar affinities. In vivo, BSD dose-dependently ameliorated osteoporosis, significantly improving bone mineral density, trabecular microarchitecture, and type H vessel formation. Mechanistically, BSD upregulated ZEB1 in endothelial cells, promoting its interaction with CREB-binding protein (CBP). This enhanced histone H3 (H3K4ac, H3K14ac, and H3K18ac) acetylation at Dll4 and Notch1 promoters, activating their transcription. Crucially, targeted ZEB1 knockdown in vivo abolished the BSD-induced histone acetylation, angiogenesis, and bone protection.

The present study uncovered that BSD could effectively improve bone mass and type H vessels in age-related osteoporosis through the ZEB1/Dll4/Notch1 signaling pathway, suggesting that BSD could be a promising therapeutic agent for age-related osteoporosis. However, one limitation of this study is that only a single batch of BSD was used. Therefore, future research employing multiple batches is needed to verify the reproducibility of these findings.