C-C motif chemokine ligand 3 mediates inflammatory response via NLRP3 inflammasome and neuron damage after traumatic brain injury

OBJECTIVE: Traumatic brain injury (TBI) constitutes a major inducer of neuroinflammation within the initial 24 h, peak microglial reactivity by day 3 post-injury, and cytokine production through day 7. However, the precise underlying pathophysiology driving immune cell infiltration, inflammatory cascades, and subsequent neuronal damage remains incompletely defined. This study aims to investigate whether C-C motif chemokine ligand 3 (CCL3) plays a functional role in TBI-induced neuroinflammation and associated neuronal injury. METHODS: Datasets of RNA-seq analysis used were: GSE58485 is mice neocortex at 3 days post-TBI, GSE92363 is rat hippocampus at day 1 post-TBI, and GSE59645 is rat hippocampus at day 14 post-TBI. GEO datasets, GSE92363 and GSE59645, sequencing analysis of the normal rat hippocampus were merged to provide a baseline to identify key differentially expressed genes (DEGs). DEGs were used to perform functional annotations through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, execute gene set enrichment analysis (GSEA), and construct protein-protein interaction (PPI) networks. Microglia HAPI cells and H19-7 cells co-culture system was used to functionally validate microglia-derived neuroinflammatory damage mechanisms on neuronal cells. RESULTS: For screening of DEGs related to TBI, we first performed an integrated analysis of the GSE92363 and GSE59645 datasets. Initial-phase TBI exhibited characteristic immune landscape alterations, marked by significant macrophage and neutrophil infiltration with concurrent M1 macrophage polarization. GSEA further revealed microglial phagocytic pathway dysregulation as the most profoundly perturbed signaling cascade in TBI pathogenesis. Subsequently, we identified CCL3 as a master regulatory hub orchestrating TBI progression with PPI analysis. In HAPI cells, short hairpin RNA-mediated CCL3 knockdown effectively attenuated lipopolysaccharide-induced pro-inflammatory cytokine elevation and suppressed nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome pathway activation. In the HAPI-H19-7 (neuron) co-culture system, CCL3 silencing demonstrated significant neuroprotection by mitigating H19-7 neuronal apoptosis. CONCLUSION: CCL3 functions as a key regulatory chemokine orchestrating NLRP3 inflammasome-mediated microglial activation in post-TBI neuroinflammation. Inhibition of CCL3 may be a promising therapeutic target for ameliorating secondary neuronal injury following TBI.