Pristimerin disrupts blood-testis barrier (BTB) integrity via ferroptosis and apoptosis by the NUR77/ATF3/CHOP pathway.
Pristimerin (Pri), a natural triterpenoid from the Celastraceae family, exhibits potent anti-tumor and anti-inflammatory activities, yet its further development as a drug candidate is hampered by severe male reproductive toxicity and precise target cells and underlying mechanisms remain unclear. Therefore, this study aimed to identify the target cells and elucidate these mechanisms. In vivo, male mice were administered Pri to evaluate testicular injury, sperm quality, and BTB integrity, while LC-MS/MS was used to detect Pri distribution in testis, epididymis, and sperm, as well as its cellular uptake. In vitro, CCK-8 assessed cell viability, and inhibitor interventions identified contributions of distinct cell death pathways. Annexin V/PI, C11-BODIPY, and DCFH-DA/DHE were used to assess apoptosis and ferroptosis-related oxidative damage. Transcriptomics, RT-qPCR, Western blotting, and immunohistochemistry were performed to examine activation of the NUR77-ATF3-CHOP axis and molecular docking, MD simulations, CETSA, and siRNA-mediated knockdown validated direct NUR77 binding by Pri. Pri exposure induced testicular atrophy, BTB disruption, and sperm impairment, with accumulation in reproductive tissues. In vitro, Pri inhibited TM4 cell viability. Cell death was partially rescued by Z-VAD-FMK and Fer-1, indicating that Pri triggers apoptosis and ferroptosis. Mechanistically, Pri activated the NUR77-ATF3-CHOP axis and directly bound to NUR77, as confirmed by docking, MD simulations, and CETSA. Knockdown of NUR77 improved cell viability and suppressed Pri-induced ATF3 upregulation. Collectively, this study demonstrates that Pri directly targets NUR77, activating the NUR77-ATF3-CHOP axis and inducing both apoptosis and ferroptosis in Sertoli cells, leading to spermatogenesis dysfunction, thereby providing critical insights into Pri-induced testicular injury.