单细胞和批量 RNA 测序的组合揭示了脊髓损伤中的免疫浸润景观和靶向治疗药物。

BACKGROUND: In secondary spinal cord injury (SCI), the immune microenvironment of the injured spinal cord plays an important role in spinal regeneration. Among the immune microenvironment components, macrophages/microglia play a dual role of pro-inflammation and anti-inflammation in the subacute stage of SCI. Therefore, discovering the immune hub genes and targeted therapeutic drugs of macrophages/microglia after SCI has crucial implications in neuroregeneration. This study aimed to identify immune hub genes and targeted therapeutic drugs for the subacute phase of SCI. METHODS: Bulk RNA sequencing (bulk-RNA seq) datasets (GSE5296 and GSE47681) and single-cell RNA sequencing (scRNA-seq) dataset (GSE189070) were obtained from the Gene Expression Omnibus database. In the bulk RNA-seq, the R package 'limma,' 'WGCNA,' and 'CIBERSORT' were used to jointly screen key immune genes. Subsequently, the R package 'Seurat' and the R package 'celldex' were used to divide and annotate the cell clusters, respectively. After using the Autodock software to dock immune hub genes and drugs that may be combined, the effectiveness of the drug was verified using an experiment with the T9 SCI mouse model. RESULTS: In the bulk-RNA seq, , , and were identified as immune hub genes. Ten cell clusters were identified in scRNA-seq, and were mainly located in the microglia, while was mainly located in macrophages. Molecular docking results showed that the proteins corresponding to these immune genes could accurately bind to decitabine. In decitabine-treated mice, the pro-inflammatory factor (TNF-α, IL-1β) levels were decreased while anti-inflammatory factor (IL-4, IL-10) levels were increased at 2 weeks post-SCI, and macrophages/microglia transformed from M1 to M2. At 6 weeks post-SCI, the neurological function score and electromyography of the decitabine treatment group were also improved. CONCLUSION: In the subacute phase of SCI, in macrophages/microglia may be key therapeutic targets to promote nerve regeneration. In addition, low-dose decitabine may promote spinal cord regeneration by regulating the polarization state of macrophages/microglia.

背景：在继发性脊髓损伤（SCI）中，损伤脊髓的免疫微环境在脊髓再生中起着重要作用。在 SCI 的亚急性期，免疫微环境成分中的巨噬细胞/小胶质细胞在炎症反应中发挥着双重作用。因此，发现 SCI 后巨噬细胞/小胶质细胞的免疫枢纽基因和靶向治疗药物对神经再生具有重要意义。本研究旨在鉴定 SCI 亚急性期的免疫枢纽基因和靶向治疗药物。

方法：从基因表达综合数据库中获取批量 RNA 测序（bulk-RNA seq）数据集（GSE5296 和 GSE47681）和单细胞 RNA 测序（scRNA-seq）数据集（GSE189070）。在批量 RNA-seq 中，使用 R 包“limma”、“WGCNA”和“CIBERSORT”联合筛选关键免疫基因。随后，使用 R 包“Seurat”和 R 包“celldex”分别对细胞簇进行划分和注释。使用 Autodock 软件对接免疫枢纽基因和可能结合的药物后，使用 T9 SCI 小鼠模型验证药物的有效性。

结果：在批量 RNA-seq 中，、、和被鉴定为免疫枢纽基因。在 scRNA-seq 中鉴定出 10 个细胞簇，主要位于小胶质细胞中，主要位于巨噬细胞中。分子对接结果表明，这些免疫基因对应的蛋白可以准确地与地西他滨结合。在地西他滨处理的小鼠中，SCI 后 2 周，促炎因子（TNF-α、IL-1β）水平降低，抗炎因子（IL-4、IL-10）水平升高，巨噬细胞/小胶质细胞从 M1 向 M2 转化。SCI 后 6 周，地西他滨治疗组的神经功能评分和肌电图也得到改善。

结论：在 SCI 的亚急性期，巨噬细胞/小胶质细胞中的可能是促进神经再生的关键治疗靶点。此外，低剂量地西他滨可能通过调节巨噬细胞/小胶质细胞的极化状态促进脊髓再生。