Integrative bioinformatic and experimental analyses identify the SPI1-NFE2L2/HMGB1 axis linking pyroptosis and immune...

Pyroptosis, a form of inflammatory programmed cell death, has been implicated in the pathogenesis of non-obstructive azoospermia (NOA). However, pyroptosis-related gene function in NOA pathogenesis has yet to be fully characterized. Publicly available datasets were interrogated to pinpoint differentially expressed genes in patients with NOA relative to healthy controls. These genes were cross-referenced with curated pyroptosis-related gene sets to define pyroptosis-related differentially expressed genes (PRDEGs). Functional enrichment, Protein-protein interaction mapping and diagnostic evaluation were carried out. Immune infiltration was quantified by single-sample gene set enrichment analysis, with subsequent correlation assessment. A busulfan-induced azoospermia mouse model was established, and hub genes were validated by RT-qPCR, Western blotting, and immunofluorescence. Fifteen PRDEGs were identified and were significantly enriched in pathways related to proliferation, differentiation, and apoptosis. NFE2L2 and HMGB1 were identified as top hub genes, with strong diagnostic performance (AUC > 0.9) and markedly increased expression. Both genes were strongly correlated with most immune cell types (r > 0.5, p < 0.05). In the mouse model, Nfe2l2 and Hmgb1 mRNA levels and NFE2L2 and HMGB1 protein levels were increased in testicular tissues, with extensive extracellular localization of HMGB1. Our findings substantiate the involvement of NFE2L2 and HMGB1 in NOA pathogenesis and reveal strong associations between their expression and the organization of testicular immune infiltrates. Collectively, the results implicate NFE2L2 and HMGB1 in NOA pathogenesis, highlighting their dual utility as both diagnostic indicators and druggable targets.