Unveiling the Temporal Dynamics and Molecular Regulation Profiles of Neutrophil Extracellular Traps Following Spinal Cord Injury

Spinal cord injury (SCI) initiates secondary inflammatory processes that exacerbate tissue damage, with neutrophil extracellular traps (NETs) playing a significant role in amplifying these cascades. This study aimed to explore the temporal dynamics and key regulatory genes of NET formation in SCI to identify therapeutic targets. We integrated two transcriptomic datasets from the GEO database to identify differentially expressed NETs-related genes (NRGs) in SCI. WGCNA identified SCI-related modules, while GSVA assessed NET formation dynamics. Publicly available single-cell RNA sequencing data from the GEO database determined cell-specific expression patterns of key NRGs. Findings were validated through immunofluorescence, Western blot, and qPCR in a mouse SCI model. Regulatory networks were constructed, and potential therapeutic compounds were predicted using DSigDB and molecular docking. We identified seven key NRGs (Casp1, Ccl3, Fcgr2b, Itgam, Itgb2, Tlr2, Tlr4) in SCI. GSVA revealed peak NET score at day 1 post-injury, with attenuation at days 3 and 7. Single-cell transcriptome analysis demonstrated predominant expression of these key genes in neutrophils during the acute phase, most prominently at 1 day post-injury, which coincides with the most pronounced neutrophil infiltration. Immunofluorescence and Western blot analyses confirmed significantly elevated NET formation at 1 day post-SCI. qPCR verified the expression of all key NRGs. Regulatory network analysis identified CHD1 as an important transcription factor governing NET formation, while miRNA-mRNA network construction revealed sophisticated post-transcriptional regulation mechanisms. Drug prediction analysis identified atorvastatin as a promising therapeutic candidate with strong binding affinity to multiple key NET-related proteins. Our study provides insights into the temporal dynamics and molecular mechanisms of NET formation after SCI, identifying potential therapeutic targets to mitigate neutrophil-mediated secondary injury and improve functional outcomes.