Department of Orthopedics Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China.
Department of Orthopedics Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China.
Int Immunopharmacol. 2023 Sep;122:110639. doi: 10.1016/j.intimp.2023.110639. Epub 2023 Jul 21.
Inflammation stands as a pivotal factor in the pathogenesis of glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH). However, the vital role played by M1 macrophages, the principal constituents of the inflammatory process, remains largely underexplored. In this study, we employed reverse transcription-quantitative polymerase chain Reaction (RT-PCR), western blot, and flow cytometry to assess the impact of M1-conditioned medium on cultures of mouse bone marrow-derived mesenchymal stem cells (BMSCs) and Murine Long bone Osteocyte-Y4 (MLO-Y4) in vitro. Moreover, we quantified the levels of inflammatory cytokines in the M1-conditioned medium through the employment of an enzyme-linked immunosorbent assay (ELISA). For in vivo analysis, we examined M1 macrophages and investigated the NF-kB signaling pathway in specimens obtained from the femoral heads of animals and humans. We found that the number of M1 macrophages in the femoral head of GA-ONFH patients grew significantly, and in the mice remarkably increase, maintaining high levels in the intramedullary. In vitro, the M1 macrophage-conditioned medium elicited apoptosis in BMSCs and MLO-Y4 cells, shedding light on the intricate interplay between macrophages and these cell types. The presence of TNF-α within the M1-conditioned medium activated the NF-κB pathway, providing mechanistic insight into the apoptotic induction. Moreover, employing a robust rat macrophage clearance model and GA-ONFH model, we demonstrated a remarkable attenuation in TNF-α expression and NF-kB signaling subsequent to macrophage clearance. This pronounced reduction engenders diminished cellular apoptosis and engenders a decelerated trajectory of GA-ONFH progression. In conclusion, our study reveals the crucial involvement of M1 macrophages in the pathogenesis of GA-ONFH, highlighting their indispensable role in disease progression. Furthermore, early clearance emerges as a promising strategy for impeding the development of GA-ONFH.
炎症是糖皮质激素相关股骨头坏死（GA-ONFH）发病机制中的一个关键因素。然而，M1 巨噬细胞在炎症过程中的重要作用在很大程度上仍未得到充分探索。在这项研究中，我们采用逆转录定量聚合酶链反应（RT-PCR）、Western blot 和流式细胞术来评估 M1 条件培养基对体外培养的小鼠骨髓间充质干细胞（BMSCs）和 Murine Long bone Osteocyte-Y4（MLO-Y4）的影响。此外，我们通过酶联免疫吸附试验（ELISA）来定量 M1 条件培养基中的炎症细胞因子水平。对于体内分析，我们检查了股骨头中的 M1 巨噬细胞，并研究了动物和人类股骨头标本中的 NF-kB 信号通路。我们发现，GA-ONFH 患者股骨头中的 M1 巨噬细胞数量显著增加，而在小鼠中则显著增加，并在骨髓内保持高水平。在体外，M1 巨噬细胞条件培养基诱导 BMSCs 和 MLO-Y4 细胞凋亡，揭示了巨噬细胞与这些细胞类型之间的复杂相互作用。M1 条件培养基中的 TNF-α 激活了 NF-κB 通路，为凋亡诱导提供了机制上的见解。此外，我们利用一个强大的大鼠巨噬细胞清除模型和 GA-ONFH 模型，证明了在巨噬细胞清除后，TNF-α 表达和 NF-kB 信号显著减弱。这种明显的减少导致细胞凋亡减少，并减缓 GA-ONFH 的进展。总之，我们的研究揭示了 M1 巨噬细胞在 GA-ONFH 发病机制中的关键作用，强调了它们在疾病进展中的不可或缺的作用。此外，早期清除似乎是阻止 GA-ONFH 发展的一种很有前途的策略。
