BACKGROUND: Senescence and impaired osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) are pivotal pathogenic drivers in the progression of postmenopausal osteoporosis (PMOP). The Bushentongluo formula (BSTLF) is a traditional Chinese herbal prescription that has shown beneficial clinical outcomes in the treatment of PMOP, yet its underlying mechanisms remain unclear.
PURPOSE: To elucidate the regulatory function and underlying mechanism of BSTLF in mitigating PMOP.
METHODS: The active ingredients of BSTLF were identified by LC-MS/MS. The ovariectomized (OVX)-induced osteoporosis in mice was utilized to evaluate the effects of BSTLF on bone mass, trabecular structure, and bone strength through micro-CT, histopathology, and biomechanical testing. For in vitro studies, oxidative stress-induced senescence in BMSCs was modeled using H₂O₂, and the impacts of BSTLF on BMSCs proliferation, differentiation, and senescence were analyzed. Subsequently, proteomic sequencing of BMSCs samples was conducted to elucidate the underlying mechanisms of BSTLF regulating BMSCs osteogenic differentiation and senescence. Additionally, further mechanistic investigations, including molecular docking, flow cytometry, immunofluorescence co-localization, co-immunoprecipitation (CO-IP), and siRNA-mediated gene silencing were performed to confirm the involvement of critical signaling.
RESULTS: In vivo results verified that BSTLF ameliorates trabecular bone loss, structural failure, and diminishes bone strength in OVX-induced osteoporotic mice. Simultaneously, our findings indicated that BSTLF upregulates the levels of osteogenic-related markers while downregulating those of senescence-associated biomarkers. In vitro, BSTLF alleviated oxidative stress-induced BMSCs senescence, suppressed mitochondrial damage, and enhanced BMSCs osteogenic differentiation. Furthermore, proteomic sequencing and functional gene enrichment analysis revealed that mitophagy might be the key pathway by which BSTLF suppresses BMSCs senescence. Subsequently, both in vitro and in vivo assays confirmed that BSTLF activates the PINK1/Parkin pathway-mediated mitophagy and improves the mitochondrial function. Moreover, immunofluorescence co-localization and CO-IP experiments confirmed that BSTLF prevents SENP1-dependent SUMOylation of SIRT3, leading to enhanced mitophagy and restored mitochondrial homeostasis. Critically, SENP1 knockdown by siRNA promoted SIRT3 SUMOylation and eliminated the beneficial effects of BSTLF on osteogenic differentiation and the suppression of senescence in BMSCs.
CONCLUSION: These findings elucidated that BSTLF could repair mitophagy deficiency via the SENP1-SIRT3 axis and promote osteogenic differentiation of senescent BMSCs, thereby exerting its anti-osteoporosis effect. Our observation provided experimental validation to support the clinical application of BSTLF in PMOP treatment.
