<i>Lycium barbarum</i> Polysaccharide Improves Iron Homeostasis in Spermatocytes and Sertoli Cells via NRF2 to Alleviate DEHP-Induced Male Reproductive Toxicity in Mice.

Male infertility, as a globally significant reproductive health issue, remains idiopathic in over 40% of cases. Reproductive disorders in males induced by environmental pollutants, such as di(2-ethylhexyl) phthalate (DEHP), have garnered considerable attention in recent years. DEHP induces testicular oxidative stress and ferroptosis via its active metabolite MEHP, thereby leading to spermatogenic dysfunction. <i>Lycium barbarum</i> polysaccharide (LBP), a traditional food and medicine homologous substance, exhibits potential antioxidant and reproductive protective properties. However, the underlying mechanism by which LBP intervenes in the toxicity induced by DEHP remains to be elucidated. This study explored the protective effect and molecular mechanism of LBP on DEHP-induced testicular injury through in vivo and in vitro experiments. The result showed that DEHP exposure (150 mg/L in free drinking water for 6 weeks) significantly decreased testicular weight, sperm concentration, and sperm motility in mice, while DEHP exposure induced pathological damage to testicular tissue, as evidenced by cavitation of seminiferous tubules, reduced numbers of spermatocytes, and vacuolar degeneration of Sertoli cells. However, LBP (450 mg/L) treatment significantly reversed testicular damage and sperm parameters. In vitro, MEHP reduced the viability of GC2 cells (spermatocyte cell line) and TM4 cells (Sertoli cell line), and LBP significantly restored cell activity. Mechanistically, exposure to DEHP/MEHP results in iron overload (elevated levels of free Fe²⁺), lipid peroxidation (increased MDA and reduced GSH), and dysregulated expression of key proteins involved in ferroptosis and iron homeostasis within the testis and cells. Furthermore, it was demonstrated that when NRF2 was specifically inhibited by ML385 or silenced via siRNA, the protective effects of LBP were abrogated, thereby validating the critical role of NRF2 in the regulation of iron homeostasis by LBP. In conclusion, LBP mitigates DEHP-induced testicular injury by activating NRF2 to regulate iron homeostasis in Sertoli cells and spermatocytes cells. This study not only offers a potential strategy for the prevention and treatment of male reproductive disorders caused by DEHP exposure, but also underscores the reproductive protective effects and application prospects of LBP in this context.