Yang Shijie, Yu Beibei, Zhang Qing, Zhang Yongfeng, Fu Longhui, Zhou Bisheng, Wu Haining, Li Jianzhong, Gong Shouping
Department of Neurosurgery, The Second Affiliated Hospital of Xi'an Jiao Tong University, Xi'an, China.
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, 710004, China.
J Transl Med. 2025 Jan 13;23(1):60. doi: 10.1186/s12967-024-05916-y.
Spinal cord injury (SCI) triggers a complex inflammatory response that impedes neural repair and functional recovery. The modulation of macrophage phenotypes is thus considered a promising therapeutic strategy to mitigate inflammation and promote regeneration.
We employed microarray and single-cell RNA sequencing (scRNA-seq) to investigate gene expression changes and immune cell dynamics in mice following crush injury at 3 and 7 days post-injury (dpi). High-dimensional gene co-expression network analysis (hdWGCNA) and slingshot trajectory analysis were employed to identify key gene modules and macrophage differentiation pathways. Subsequently, immunofluorescence staining, flow cytometry, and western blotting were performed to validate the identified effects of amantadine on macrophage differentiation and inflammation.
To elucidate the molecular mechanisms underlying the injury response at the transcriptional level, we performed a microarray analysis followed by gene set enrichment analysis (GSEA). The results revealed that pathways related to phagocytosis and macrophage activation are significantly involved post-injury, shedding light on the regulatory role of macrophages in SCI repair. To further investigate macrophage dynamics within the injured spinal cord, we conducted scRNA-Seq, identifying three distinct macrophage subtypes: border-associated macrophages (BAMs), inflammatory macrophages (IMs), and chemotaxis-inducing macrophages (CIMs). Trajectory analysis suggested a differentiation pathway from Il-1b IMs to Mrc1 BAMs, and subsequently to Arg1 CIMs, indicating a potential maturation process. Given the importance of these pathways in the injury response, we utilized molecular docking to hypothesize that amantadine might modulate this process. Subsequent in vitro and in vivo experiments demonstrated that amantadine reduces Il-1b IMs and facilitates the transition to Mrc1 BAMs and Arg1 CIMs, likely through modulation of the HIF-1α and NF-κB pathways. This modulation promotes neural regeneration and enhances functional recovery following SCI.
Amantadine modulates macrophage phenotypes following SCI, reduces early inflammatory responses, and enhances neural function recovery. These findings highlight the therapeutic potential of amantadine as a treatment for SCI, and provide a foundation for future translational research into its clinical applications.
脊髓损伤（SCI）引发复杂的炎症反应，阻碍神经修复和功能恢复。因此，调节巨噬细胞表型被认为是减轻炎症和促进再生的一种有前景的治疗策略。
我们采用微阵列和单细胞RNA测序（scRNA-seq）来研究小鼠在损伤后3天和7天（dpi）遭受挤压损伤后的基因表达变化和免疫细胞动态。采用高维基因共表达网络分析（hdWGCNA）和弹弓轨迹分析来识别关键基因模块和巨噬细胞分化途径。随后，进行免疫荧光染色、流式细胞术和蛋白质印迹，以验证金刚烷胺对巨噬细胞分化和炎症的已确定作用。
为了在转录水平阐明损伤反应的分子机制，我们进行了微阵列分析，随后进行基因集富集分析（GSEA）。结果显示，与吞噬作用和巨噬细胞激活相关的途径在损伤后显著参与，揭示了巨噬细胞在脊髓损伤修复中的调节作用。为了进一步研究损伤脊髓内的巨噬细胞动态，我们进行了scRNA-Seq，识别出三种不同的巨噬细胞亚型：边界相关巨噬细胞（BAMs）、炎性巨噬细胞（IMs）和趋化诱导巨噬细胞（CIMs）。轨迹分析表明从Il-1b炎性巨噬细胞到Mrc1边界相关巨噬细胞，随后到Arg1趋化诱导巨噬细胞的分化途径，表明存在潜在的成熟过程。鉴于这些途径在损伤反应中的重要性，我们利用分子对接推测金刚烷胺可能调节这一过程。随后的体外和体内实验表明，金刚烷胺减少Il-1b炎性巨噬细胞，并促进向Mrc1边界相关巨噬细胞和Arg1趋化诱导巨噬细胞的转变，可能是通过调节HIF-1α和NF-κB途径。这种调节促进神经再生并增强脊髓损伤后的功能恢复。
金刚烷胺在脊髓损伤后调节巨噬细胞表型，减少早期炎症反应，并增强神经功能恢复。这些发现突出了金刚烷胺作为脊髓损伤治疗方法的治疗潜力，并为其临床应用的未来转化研究提供了基础。
