Wulingzhi-Associated Ursolic Acid Alleviates Busulfan-Induced Oxidative Injury in Porcine Spermatogonial Stem Cells: Potential Involvement of AKT-Associated Signaling

Wulingzhi (WLZ), a traditional Chinese medicine with diverse chemical constituents, has shown antioxidant and cytoprotective activities; however, its potential to protect spermatogonial stem cells against chemotherapy-induced oxidative injury remains unclear. In this study, a busulfan-induced oxidative injury model was established in porcine spermatogonial stem cells (PSSCs) to investigate the protective effects of WLZ and explore potential bioactive compounds and associated molecular mechanisms by integrating ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS)-based chemical profiling, network pharmacology, molecular docking, and in vitro experiments. Chemical profiling of WLZ by UHPLC–MS/MS, followed by integration with constituents retrieved from BATMAN-TCM and HERB, established an integrated library of 89 candidate compounds. Network pharmacology and molecular docking computationally prioritized ursolic acid (UA) as a candidate WLZ-associated constituent and AKT1 as a candidate regulatory target for subsequent experimental evaluation. Experimental evaluation showed that both WLZ and UA attenuated busulfan-induced oxidative injury, as evidenced by improved cell viability, reduced ROS accumulation, and restored antioxidant capacity. The protective effects of UA were associated with changes in AKT1 transcript expression and were attenuated by the AKT inhibitor Miransertib. In contrast, the PI3K–AKT pathway agonist Recilisib independently improved antioxidant-related parameters in busulfan-exposed PSSCs in the absence of UA. UA treatment was also accompanied by increased transcript levels of Nrf2 and the antioxidant genes SOD, CAT, and GPX1. These findings indicate that UA, prioritized from the integrated WLZ candidate compound library, independently attenuates busulfan-induced oxidative injury in PSSCs and suggest a possible involvement of AKT-related signaling in the observed antioxidant responses. This study provides preliminary in vitro evidence that WLZ and UA attenuate busulfan-induced oxidative injury in PSSCs and supports further investigation of their potential protective effects against chemotherapy-associated male reproductive toxicity.