2,8-Dihydroxyquinoline ameliorates oligoasthenozoospermia by inhibiting MAPK14-mediated spermatogonial ferroptosis.

BACKGROUND: Oligoasthenozoospermia (OAS) is a major cause of male infertility. 2,8-Dihydroxyquinoline (2,8-DHQ), an endogenous antioxidant metabolite, remains unexplored in OAS. This study identified 2,8-DHQ as a bioactive XHBG metabolite and elucidated its mechanisms in improving OAS. METHODS AND RESULTS: Male BALB/c mice received glycosides of Tripterygium wilfordii (GTW) (140 mg/kg/day, oral gavage) for 4 weeks to induce OAS, confirmed by marked reductions in testicular/epididymal weights, sperm concentration, serum testosterone, and severe seminiferous tubule disruption. Then, mice were treated with XHBG (10.4, 20.8, 41.6 g/kg/day) or 2,8-DHQ (50 mg/kg/day) for another 4 weeks. Integrative network pharmacology and serum metabolomics identified 2,8-DHQ as a key bioactive metabolite of XHBG, with 137 overlapping targets with OAS. In H2O2-challenged GC-1 spermatogonia, the DARTS assay confirmed direct binding of 2,8-DHQ to Mitogen-Activated Protein Kinase 14 (MAPK14), and Western blot revealed its downregulation. 2,8-DHQ enhanced GC-1 cell viability and, in OAS mice, restored testicular weight, sperm count, testosterone, and histological architecture. Mechanistically, 2,8-DHQ lowered ROS and MDA, elevated GSH/GSSG ratio, reduced Fe2+ levels, and upregulated SLC7A11 and GPX4, indicating suppression of oxidative stress and ferroptosis. Importantly, MAPK14 overexpression abolished these protective effects both in vitro and in vivo, confirming that 2,8-DHQ acts via targeting MAPK14 to inhibit ferroptosis. CONCLUSION: 2,8-DHQ, an active metabolite of XHBG, ameliorates OAS by targeting MAPK14 to inhibit spermatogonial ferroptosis. These findings provide a scientific basis for the clinical use of XHBG and highlight 2,8-DHQ as a potential lead compound for male infertility.