Nasal Exposure to Environmental PM2.5 Drives Testicular Pyroptosis and Tissue Damage: Evidence from Inhibitor...

Objective: Focusing on the process by which PM2.5 induces NLRP3/Caspase-1 axis activation and drives GSDMD-mediated pyroptosis, this study aimed to construct an integrated framework combining evidence from a PM2.5 animal model, intervention with a pyroptosis inhibitor, and reproducible screening of public transcriptomic databases. Methods: A total of 24 male Sprague-Dawley rats were randomly divided into four groups (n=6 per group): control (normal saline), Caspase-1 inhibitor Z-YVAD-FMK (5 µg/mg), PM2.5 (10 mg/kg), and PM2.5 + Caspase-1 inhibitor (PM2.5 exposure + Z-YVAD-FMK). After 4 weeks of exposure, mating tests were conducted followed by euthanasia. Testicular and epididymal tissues were collected for hematoxylin-eosin (HE) staining and sperm quality analysis. Key proteins associated with the pyroptotic response in testicular tissue were detected by Western Blot (WB). Concurrently, the public GEO dataset GSE189187 (GC-2spd cells, PM2.5-treated vs. control, 3 vs. 3) was analyzed for differential expression, followed by enrichment and Protein-Protein Interaction (PPI) network analyses. Hub genes were identified using the MCODE and CytoHubba plugins in Cytoscape software, and these network hubs were cross-validated with the evidence chain from the animal study. Results: Exposure to PM2.5 led to decreased sperm count and motility, along with a significant increase in sperm abnormality rate and a reduction in spermatogenic cell number. Western Blot analysis showed significantly elevated expression of the apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, and Gasdermin D protein in the PM2.5 group. These changes were reversed by intervention with the Caspase-1 pyroptosis inhibitor. Transcriptomic differential analysis identified 217 significant differentially expressed genes (DEGs: 91 up-regulated, 126 down-regulated). Separate GO and KEGG enrichment analyses (Over-Representation Analysis, ORA) for up- and down-regulated genes revealed that DEGs were primarily enriched in antiviral/interferon-related immune processes. Pathways associated with tissue injury were also significantly enriched. Two pyroptosis-related DEGs were identified (Hmox1 up-regulated, Stat1 down-regulated). A PPI network constructed from these candidate genes was subjected to topological analysis in Cytoscape, and further screening with CytoHubba yielded 10 hub genes, suggesting that the PM2.5-related transcriptional response involves an interferon-STAT axis-driven inflammatory interaction network. Conclusion: PM2.5 from vehicle exhaust impairs male reproductive function. The pyroptosis inhibitor protects reproductive function by suppressing the NLRP3/Caspase-1 signaling pathway. Furthermore, PM2.5 can activate the differential expression of pyroptosis-related genes at the transcriptional level, thereby promoting an inflammatory interaction network that contributes to systemic inflammation.