Shen Fengteng, Chen Yansong, Xu Zhikun, Wang Wei, Chen Guofang, Ye Fusheng
Department of Orthopedics, Tongxiang First People's Hospital, China.
Department of Urology, Affiliated Xiaoshan Hospital, Hangzhou Normal University, Hangzhou City, 311200, China.
Int J Biochem Cell Biol. 2025 Aug;185:106787. doi: 10.1016/j.biocel.2025.106787. Epub 2025 Apr 24.
Heterotopic ossification (HO) is characterized by abnormal bone formation in soft tissues, often following trauma or surgery. Transforming growth factor-beta (TGF-β) signaling and M2 macrophage polarization play critical roles in the recruitment and differentiation of mesenchymal stromal/progenitor cells (MSPCs), promoting HO.
An elbow joint trauma-induced HO mouse model was established, where model mice were treated with dichloromethylene-bisphosphonate (Cl2MBP) liposomes or PBS liposomes to deplete macrophages. In addition, boldine was administered to evaluate its therapeutic effect on HO formation. Bone marrow mesenchymal stem cells (BMSCs) were also extracted for in vitro experiments. Quantitative real-time PCR (qRT-PCR) and Western blot were conducted to assess gene and protein expression. In vivo methods included Micro-Computed Tomography (Micro-CT) to assess bone formation, histological staining to evaluate tissue changes, immunohistochemistry (IHC) and immunofluorescence to analyze macrophage, CD73 and CD105 cells infiltration. In vitro, BMSCs were identified by flow cytometry and treated with interleukin-10 (IL-10) and/or boldine, and assays such as cell viability (Cell Counting Kit 8 (CCK8)), migration (Transwell), immunofluorescence, ALP staining, and Alizarin Red S staining, were conducted to assess osteogenic differentiation.
Boldine treatment significantly reduced HO formation, decreased collagen deposition, and inhibited M2 macrophage infiltration (P < 0.05). In vitro, boldine reduced IL-10-induced cell activity, migration, and osteogenic differentiation of BMSCs and inhibited TGF-β and pSmad2/3/Smad2/3 protein (P < 0.05).
Boldine attenuates HO by inhibiting M2 macrophage-mediated MSPC migration and might involve the TGF-β signaling, suggesting its potential as a therapeutic approach for managing HO.
异位骨化（HO）的特征是软组织中出现异常骨形成，常继发于创伤或手术之后。转化生长因子-β（TGF-β）信号传导和M2巨噬细胞极化在间充质基质/祖细胞（MSPCs）的募集和分化中起关键作用，促进异位骨化形成。
建立肘关节创伤诱导的异位骨化小鼠模型，用二氯亚甲基二膦酸盐（Cl2MBP）脂质体或PBS脂质体处理模型小鼠以清除巨噬细胞。此外，给予去甲乌药碱以评估其对异位骨化形成的治疗效果。还提取骨髓间充质干细胞（BMSCs）用于体外实验。采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法评估基因和蛋白质表达。体内方法包括用微型计算机断层扫描（Micro-CT）评估骨形成，组织学染色评估组织变化，免疫组织化学（IHC）和免疫荧光分析巨噬细胞、CD73和CD105细胞浸润情况。在体外，通过流式细胞术鉴定BMSCs，并用白细胞介素-10（IL-10）和/或去甲乌药碱处理，然后进行诸如细胞活力（细胞计数试剂盒8（CCK8））、迁移（Transwell）、免疫荧光、碱性磷酸酶染色和茜素红S染色等实验，以评估成骨分化情况。
去甲乌药碱治疗显著减少了异位骨化形成，减少了胶原沉积，并抑制了M2巨噬细胞浸润（P<0.05）。在体外，去甲乌药碱降低了IL-10诱导的BMSCs细胞活性、迁移和成骨分化，并抑制了TGF-β和pSmad2/3/Smad2/3蛋白表达（P<0.05）。
去甲乌药碱通过抑制M2巨噬细胞介导的MSPC迁移来减轻异位骨化，可能涉及TGF-β信号传导，提示其作为治疗异位骨化的潜在治疗方法的可能性。
