Identification of Pyroptosis-Related Genes in Male Rats with Spared Nerve Injury-Induced Neuropathic Pain

Pyroptosis, a programmed inflammatory cell death mechanism, plays a significant role in neuropathic pain (NP) pathogenesis. However, the specific pyroptosis-related genes (PRGs) driving NP development remain poorly understood. This study employs systematic approaches to identify and validate PRGs, aiming to delineate their mechanistic contributions to NP progression.

To elucidate pyroptosis-related genes (PRGs) in neuropathic pain pathogenesis, we first performed integrated bioinformatics analysis of the GSE236754 dataset, revealing differentially expressed PRGs in the spinal cord dorsal horn of spared nerve injury (SNI) rats. Subsequent functional enrichment analyses coupled with protein-protein interaction network construction delineated pathway convergences among identified PRGs. Experimental validation utilizing SNI rat model, Western blot and immunofluorescence quantification confirmed protein expression patterns, and immunofluorescence mapping determined cellular localization collectively. Statistical analyses via ANOVA method.

Bioinformatics screening identified 11 candidate PRGs in the SNI model, particularly highlighting Nlrc4 and Nlrp3 as the most upregulated targets. Gene Ontology (GO) analysis demonstrated significant enrichment in three domains, pyroptosis regulation, inflammasome complex assembly, and cysteine-type endopeptidase activity associated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis specifically identified the "NOD-like receptor signaling pathway" as significantly enriched. Gene Set Enrichment Analysis (GSEA) further corroborated these findings. Behavioral quantification showed progressive mechanical hypersensitivity, with mechanical pain and cold pain reaching maximal sensitivity at day 7 post-injury (p<0.001). Western blot detected synchronized elevation of NLRP3 and NLRC4 inflammasome components and downstream effectors across the observation window (all p<0.05). Immunofluorescence analysis demonstrated a time-dependent increase in the expression of GSDMD-N, the pyroptotic-executing protein, a trend consistent with the findings from behavioral tests and Western blot analysis (p<0.05). And cellular localization analysis revealed neuron-predominant accumulation of GSDMD-N.

We identified 11 potential biomarkers for NP. Our data conclusively show that SNI induced NLRP3/NLRC4 inflammasome activation and neuronal pyroptosis in the rat spinal cord.