Astragaloside IV protects against PM2.5-induced spermatocyte injury via the cGAS-STING/Nrf2/HMOX1 axis.

Whether PM2.5-induced spermatocyte injury engages the cGAS-STING innate immune pathway remains unknown. This study integrated network pharmacology, GEO transcriptomic profiling (GSE189187), gene set enrichment analysis, molecular docking with positive-control benchmarking, single-cell RNA sequencing, and in vitro validation in GC-2 spermatocyte cells to address this question. Among 35 overlapping candidates between 447 AS-IV putative targets and 102 cGAS-STING/inflammation genes, Hmox1 was the sole gene significantly upregulated by PM2.5 (log₂FC = 2.15, AUC = 1.000). KEGG analysis identified six enriched innate immunity pathways, with the Cytosolic DNA-sensing pathway at adjusted P = 8.1 × 10⁻⁶. Molecular docking demonstrated that AS-IV bound cGAS (-10.2 kcal/mol) and STING (-11.3 kcal/mol) with affinities exceeding known inhibitors G150 (-8.8) and H-151 (-8.6). In GC-2 cells, PM2.5 (200 μg/mL) increased ROS 5.35-fold, elevated apoptosis to 42.83%, and activated cGAS-STING signaling. AS-IV co-treatment restored viability to 83.3%, enhanced proliferation to 37.96%, significantly suppressed cGAS/p-TBK1/p-IRF3, and activated Nrf2/HMOX1 (HMOX1 protein 1.99 ± 0.05-fold; mRNA 6.44-fold), reducing apoptosis to 18.27%, ROS, and LDH release, and normalizing the Bax/Bcl2 ratio from 5.0 to 2.1. These findings establish that AS-IV protects spermatocytes through coordinated cGAS-STING suppression and Nrf2-dependent HMOX1 activation. Causal necessity of cGAS-STING activation was confirmed by RU.521-mediated pathway blockade, and direct AS-IV binding to cGAS (KD = 0.619 μM) and STING (KD = 0.097 μM) was validated by surface plasmon resonance.