Taraxasterol alleviates osteoporosis by targeting PI3K/AKT/PPARγ signaling axis to suppress necroptosis and reverse osteogenic-adipogenic differentiation imbalance in BMSCs

BACKGROUND: The shift in BMSC differentiation potential toward adipogenesis at the expense of osteogenesis is a critical mechanism underlying reduced bone formation in osteoporosis. Necroptosis has been implicated as a key driver of bone-fat imbalance in osteoporosis, though its mechanisms remain unclear. Taraxasterol (TAX), a bioactive triterpenoid derived from Taraxacum officinale, exhibits antitumor, antioxidant, and immunomodulatory properties. However, its potential role in osteoporosis has not yet been explored. OBJECTIVE: This study aimed to investigate whether TAX alleviates osteoporosis by regulating necroptosis and osteogenic-adipogenic differentiation in BMSCs, and to elucidate the underlying molecular mechanisms. METHODS: An ovariectomized (OVX) mouse model of osteoporosis was established for in vivo evaluation. Treatment groups received daily oral administration of TAX (5 or 20 mg/kg), with estradiol (E₂, 10 μg/kg) serving as a positive control. Bone microarchitecture and therapeutic response were assessed via micro-CT and immunohistochemistry, while femoral necroptosis and differentiation markers were evaluated by immunostaining. For in vitro studies, BMSCs isolated from osteoporosis patients were treated with the necroptosis inducer TSZ (TNF-α, SM-164, Z-VAD-FMK), with or without TAX. Flow cytometry was used to analyze the phenotype of MSCs. To identify molecular targets and mechanisms, an integrated approach combining network pharmacology, RNA sequencing, SPR assays, and molecular docking was employed. Subsequent analyses included western blotting, PI staining, and RNA interference to assess TAX-regulated necroptosis and PI3K/AKT/PPARγ pathway activity. ARS and ORO staining were employed to analyze osteogenic and adipogenic differentiation, respectively, and mitochondrial function was evaluated with TMRE and MitoSOX Red probes. RESULTS: TAX (20 mg/kg) significantly ameliorated bone loss in OVX mice, suppressed femoral necroptotic signaling, and reversed osteogenic-adipogenic imbalance in OVX-derived BMSCs. In vitro, TAX pretreatment attenuated TSZ-induced differentiation imbalance and necroptosis in patient-derived BMSCs and mitigated mitochondrial damage. Integrated network pharmacology and RNA sequencing revealed that TAX targets the PI3K/AKT/PPARγ axis. ​​Immunohistochemical analysis demonstrated upregulation of the PI3K/AKT/PPARγ axis in the femurs of OVX mice following TAX administration. Molecular docking and SPR assays confirmed effective binding between TAX and PI3K. The protective effects of TAX were abolished by the PI3K inhibitor LY294002. Furthermore, TAX enhanced the rebalancing of osteogenic-adipogenic differentiation in PPARγ-silenced BMSCs. CONCLUSION: Our study reveals for the first time that TAX restores osteogenic-adipogenic equilibrium in OP-BMSCs and promotes bone regeneration through PI3K/AKT/PPARγ activation and mitochondrial protection-mediated suppression of necroptosis. These results position TAX as a promising therapeutic candidate for osteoporosis.