The N-butanol extract of modified Yanghe decoction alleviates ferroptosis in bone marrow mesenchymal stem cells in glucocorticoid-induced osteoporosis by activating the HIF-1α/GPX4 signaling pathway.

BACKGROUND: Glucocorticoid-induced osteoporosis (GIOP) represents the predominant type of secondary bone loss disorder, linked to a substantially elevated incidence of pathological fractures in clinical populations. Modified Yanghe Decoction (MYHD), a classical Chinese medicinal formula, has demonstrated therapeutic potential in managing osteoporotic conditions. Nevertheless, the specific mechanisms underlying MYHD's effectiveness against GIOP require further elucidation, posing challenges for broader clinical implementation. METHODS: We performed comprehensive analysis of bioactive constituents in the MYHD n-butanol (MYHD-NB) fraction through high-performance liquid chromatography, followed by mechanistic exploration of its biological effects across experimental models. Ten-week-old male C57BL/6J mice received dexamethasone (DEX)-induced modeling and were administered either MYHD or varying concentrations of MYHD-NB via oral gavage. Following an 8-week intervention, femoral specimens underwent multimodal evaluation including histopathological examination, micro-computed tomography imaging, and advanced molecular detection techniques (immunohistochemistry/immunofluorescence). Parallel bioinformatics analysis was executed on RNA-seq datasets from osteoporotic fracture patients in the GEO repository. For in vitro experimentation, DEX-treated murine BMSCs were employed to assess MYHD-NB's regulatory effects on cellular proliferation, migratory capacity, osteogenic differentiation potential, and ferroptotic processes. Complementary network pharmacology approaches were utilized to predict potential molecular targets and pathway interactions. RESULTS: MYHD-NB significantly improved the osteoporosis-like phenotype in mice, as indicated by the improvement of bone microstructure and the upregulation of ALP and RUNX2 expression. RNA-seq analysis of the GEO dataset showed that ferroptosis-related genes are significantly upregulated in the femurs of GIOP patients. Both in vivo and in vitro studies confirmed MYHD-NB's capacity to mitigate dexamethasone-induced ferroptosis in BMSCs while enhancing BMSCs proliferation capacity, migratory potential, and differentiation into osteogenic lineages. Network pharmacology predictions identified HIF-1α as the pivotal regulator mediating MYHD-NB's anti-ferroptotic effects. Molecular investigations revealed MYHD-NB's activation of the HIF-1α/GPX4 signaling pathway, with genetic ablation of HIF-1α completely negating the compound's therapeutic efficacy. CONCLUSION: MYHD-NB mitigates ferroptosis triggered by DEX in BMSCs while enhancing osteogenic differentiation via the HIF-1α/GPX4 signaling pathway, elucidating its therapeutic mechanism against GIOP. These results demonstrate MYHD-NB's superior osteogenic potential within the MYHD formulation, positioning it as a promising candidate for alternative GIOP management strategies.