Zeng Chenying, Chen Linjian, Lin Jiajie, Xiao Zipeng, Tu Qingqiang, Yu Wenhui, Zhang Weihao, Li Qibo, Wu Yanfeng, Xie Zhongyu, Zhang Ziji
Department of Orthopedics, the First Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen University, Guangzhou 510080, China; Center for Biotherapy, the Eighth Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen University, Shenzhen 518033, China.
Department of Orthopedics, The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou 510630, China.
Phytomedicine. 2025 Dec;149:157277. doi: 10.1016/j.phymed.2025.157277. Epub 2025 Sep 19.
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.
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.
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.
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.
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.
骨髓间充质干细胞（BMSC）分化潜能向脂肪生成方向转变，以骨生成为代价，是骨质疏松症中骨形成减少的关键机制。坏死性凋亡被认为是骨质疏松症中骨-脂肪失衡的关键驱动因素，但其机制仍不清楚。蒲公英甾醇（TAX）是一种从蒲公英中提取的生物活性三萜类化合物，具有抗肿瘤、抗氧化和免疫调节特性。然而，其在骨质疏松症中的潜在作用尚未得到探索。
本研究旨在探讨TAX是否通过调节BMSC中的坏死性凋亡和成骨-脂肪生成分化来减轻骨质疏松症，并阐明其潜在的分子机制。
建立去卵巢（OVX）骨质疏松小鼠模型进行体内评估。治疗组每日口服TAX（5或20mg/kg），雌二醇（E₂，10μg/kg）作为阳性对照。通过显微CT和免疫组织化学评估骨微结构和治疗反应，同时通过免疫染色评估股骨坏死性凋亡和分化标志物。对于体外研究，从骨质疏松症患者中分离的BMSC用坏死性凋亡诱导剂TSZ（TNF-α、SM-164、Z-VAD-FMK）处理，有或没有TAX。流式细胞术用于分析MSC的表型。为了确定分子靶点和机制，采用了网络药理学、RNA测序、表面等离子体共振（SPR）分析和分子对接相结合的综合方法。随后的分析包括蛋白质免疫印迹、PI染色和RNA干扰，以评估TAX调节的坏死性凋亡和PI3K/AKT/PPARγ信号通路活性。分别采用茜素红染色（ARS）和油红O染色（ORO）分析成骨和成脂分化，并使用四甲基罗丹明乙酯（TMRE）和MitoSOX Red探针评估线粒体功能。
TAX（20mg/kg）显著改善了OVX小鼠的骨质流失，抑制了股骨坏死性凋亡信号，并逆转了OVX来源的BMSC中的成骨-脂肪生成失衡。在体外，TAX预处理减轻了TSZ诱导的患者来源BMSC中的分化失衡和坏死性凋亡，并减轻了线粒体损伤。综合网络药理学和RNA测序显示，TAX靶向PI3K/AKT/PPARγ轴。免疫组织化学分析表明，TAX给药后OVX小鼠股骨中PI3K/AKT/PPARγ轴上调。分子对接和SPR分析证实了TAX与PI3K之间的有效结合。PI3K抑制剂LY294002消除了TAX的保护作用。此外，TAX增强了PPARγ沉默的BMSC中成骨-脂肪生成分化的重新平衡。
我们的研究首次揭示，TAX通过PI3K/AKT/PPARγ激活和线粒体保护介导的坏死性凋亡抑制，恢复了骨质疏松症BMSC中的成骨-脂肪生成平衡，并促进骨再生。这些结果使TAX成为一种有前途的骨质疏松症治疗候选药物。
