Yougui Pills Alleviate Osteoporosis by Inhibiting Mesenchymal Stem Cell ROS Accumulation via the Nrf2/HO-1 Pathway

Osteoporosis (OP) is a prevalent skeletal disorder characterized by progressive bone mass loss and deteriorated microarchitecture, in which oxidative stress-induced mesenchymal stem cell (MSC) dysfunction serves as a core pathogenic mechanism. Yougui Pills (YGPs), a classical traditional Chinese medicine formula, are widely applied in clinical OP management, yet the cellular and molecular mechanisms underlying their anti-osteoporotic effects remain incompletely defined. This study aimed to elucidate the bone-protective role of YGPs in OP and explore the underlying mechanism, focusing on oxidative stress modulation in MSCs and the Nrf2/HO-1 signalling pathway. The bioactive components of YGPs and YGP-containing serum were characterized via UPLC. In vivo, an ovariectomy (OVX) mouse model was established to evaluate YGPs' effects on bone mass, trabecular microstructure and osteogenesis using micro-CT, histological and immunohistochemical assays; public GEO datasets were re-analysed to profile transcriptomic alterations and oxidative stress signatures in OP-derived MSCs. In vitro, H₂O₂ was used to induce ROS accumulation and oxidative injury in MSCs, with assessments of cell proliferation, apoptosis, ROS levels, migration and osteogenic differentiation. Network pharmacology and siRNA-mediated gene silencing were conducted for target prediction and mechanistic validation. In vivo results showed that YGP treatment significantly ameliorated OVX-induced osteopenia, increased bone mineral density, improved trabecular microstructure, and upregulated osteogenic markers (ALP, OCN, Runx2, COL1A1). Transcriptomic re-analysis revealed upregulated oxidative stress markers in OP MSCs, consistent with In vitro findings that YGPs attenuated H₂O₂-triggered ROS overproduction, suppressed apoptosis and ectopic lipid deposition, and enhanced MSC proliferation, migration and osteogenic differentiation. Mechanistically, YGPs activated the Nrf2/HO-1 signalling axis, while Nrf2 knockdown abrogated YGPs' cytoprotective and pro-osteogenic effects. In conclusion, YGPs mitigate oxidative stress-induced MSC dysfunction and promote osteogenesis via the Nrf2/HO-1 pathway, supporting YGPs as a promising therapeutic candidate for OP.