Oridonin exposure induces testicular impairment through regulating ferroptosis via HIF-1α/HO-1 activation and STUB1-driven GPX4 ubiquitination-dependent degradation

Oridonin (ORI), a bioactive diterpenoid isolated from Rabdosia rubescens, possesses anti-inflammatory, Antibacterial and anti-tumor medicinal properties. However, its clinical application has been limited due to significant male reproductive toxicity, the underlying mechanisms of which remain elusive. In this study, we explore the potential mechanism of ORI-induced testicular injury and identify potential therapeutic targets for mitigating its reproductive toxicity. GC-MS metabolomics was employed to investigate differential metabolites in the serum of mice subjected to ORI-induced testicular injury. we demonstrated for the first time in vivo and in vitro that ORI-induced testicular Ferroptosis, accompanied by cysteine, GSH and NADPH depletion, ROS accumulation, intracellular ferrous enrichment, and changes in the expression of ferroptosis-related proteins. Molecular docking and co-immunoprecipitation (CO-IP) were performed to validate the STUB1-GPX4 interaction and GPX4 ubiquitination. Network toxicology identified potential targets, followed by experimental validation of HIF-1α/HO-1 pathway activation via Western blot, RT-qPCR, and immunohistochemistry (IHC). Mechanistically, ORI induced testicular Ferroptosis through two distinct pathways: (1) STUB1-mediated ubiquitination and degradation of GPX4, and (2) activation of the HIF-1α/HO-1 signaling axis, as predicted by network toxicology and confirmed experimentally. Collectively, this study provides the first evidence that ORI exposure induces testicular impairment via dual mechanisms: HIF-1α/HO-1 pathway activation and STUB1-dependent GPX4 ubiquitination, both of which collectively facilitate Ferroptosis. These findings not only elucidate the molecular basis of ORI induced-reproductive toxicity but also identify STUB1 and HIF-1α/HO-1 as potential therapeutic targets for prevention and treatment.

Ferroptosis; GPX4; HIF-1α/HO-1; Oridonin; Testicular injury; Ubiquitination.