Andrographolide induces testicular damage by triggering ferroptosis in Sertoli cells via disruption of β-TrCP-mediated ATF4 ubiquitination and degradation

Andrographolide (Andro), the primary bioactive compound of Andrographis paniculata , has known bioactivities but also male reproductive toxicity with unclear mechanisms. This study investigated its testicular injury effects and mechanism, focusing on Sertoli cells (SCs) ferroptosis. Toxicity was evaluated in mice and SCs. Mechanisms were probed using RT-qPCR, Western blot, co-immunoprecipitation, and ubiquitination assays. LC-MS/MS assessed tissue distribution. Molecular docking and dynamics simulations characterized the Andro-β-TrCP interaction. The results demonstrated that Andro exposure caused significant testicular atrophy, blood-testis barrier (BTB) disruption, and spermatogenic impairment in mice. Meanwhile, pharmacokinetic analysis revealed SCs as the primary target of Andro-induced testicular damage, showing rapid Andro accumulation that was subsequently detected in sperm and the epididymis. Furthermore, Andro triggered ferroptosis in testicular injury and SCs, marked by increased ROS, MDA, iron overload, and lipid peroxidation. These effects were rescued by the ferroptosis inhibitor deferoxamine. Mechanistically, Andro directly bound to β-TrCP, downregulating its expression and subsequently stabilizing ATF4 by impairing its ubiquitin-mediated degradation. Silencing ATF4 inhibited ferroptosis and restored cell viability upon Andro challenge. The direct Andro-β-TrCP interaction was consistently confirmed by molecular docking, dynamics simulations, and CETSA. This study reveals that Andro triggers SCs ferroptosis by inhibiting β-TrCP-mediated ATF4 ubiquitination and degradation, leading to BTB disruption and spermatogenic impairment. These findings provide a theoretical basis for optimizing clinical application of Andro in the future.