Multiomics Profiling Identifies Tlr4 as a Therapeutic Target of Necroptosis in Spinal Cord Injury

Spinal cord injury (SCI) leads to a complex cascade of cellular events, among which necroptosis plays a critical role in exacerbating neuronal injury and inflammation. In this study, we aimed to identify and validate key genes associated with necroptosis in SCI using bulk RNA-seq data, followed by differential analysis and weighted gene coexpression network analysis (WGCNA). We identified several candidate necroptosis-related genes, and further least absolute shrinkage and selection operator (LASSO) regression highlighted five SCI-necroptosis differentially expressed genes (DEGs): toll-like receptor 4 (Tlr4), Nlrp3, Il1b, Tnfaip3, and Stat4. These genes were validated using RT-qPCR and western blot experiments. Our analysis revealed that necroptosis scores were significantly elevated following SCI. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) analysis revealed that Tlr4 was upregulated in myeloid cells (microglia and macrophages) and played a pivotal role in triggering downstream necroptosis, which was confirmed by protein levels. In vitro and in vivo experiments confirmed that Tlr4 inhibition attenuated necroptosis and inflammation. This study is the first to establish Tlr4 as a direct upstream regulator of the pRIPK1/pRIPK3/pMLKL necroptotic axis in SCI, distinct from its role as a general inflammatory mediator, suggesting Tlr4 as a promising therapeutic target for functional recovery.