Netrin-1 protects against doxorubicin-induced cardiotoxicity by suppressing ferroptosis via the UNC5B-LKB1/CaMKKβ-AMPK-NRF2 axis.

Doxorubicin (DOX)-induced cardiotoxicity is strongly associated with ferroptosis and oxidative stress, yet the endogenous protective mechanisms remain incompletely understood. This study aimed to determine whether Netrin-1 protects against DOX-induced cardiomyocyte and myocardial injury and to elucidate the underlying signaling pathway. H9C2 cardiomyocytes and DOX-treated mice were used to evaluate the effects of Netrin-1 on ferroptosis, oxidative stress, and cardiac function. Ferroptosis markers, antioxidant proteins, and signaling molecules were assessed by biochemical assays, immunofluorescence, and Western blotting. Loss-of-function experiments were conducted using si-UNC5B, si-LKB1, si-CaMKKβ, Compound C, and AAV-shUNC5B to dissect pathway dependence. Netrin-1 significantly restored DOX-impaired cell viability, reduced membrane damage, and suppressed ferroptosis by decreasing Fe²⁺, MDA, and ROS while replenishing GSH. It normalized GPX4, ACSL4, ferritin, and TFR1 expression, uniquely activated AMPK, suppressed BACH1, enhanced HO-1, and promoted robust NRF2 nuclear translocation. UNC5B knockdown abolished these effects, indicating its essential role. Mechanistically, Netrin-1 activated AMPK through both LKB1 and CaMKKβ pathways, with dual knockdown producing additive suppression. In DOX-treated mice, Netrin-1 markedly improved cardiac function, lowered serum injury markers, alleviated histological damage, reduced apoptosis, and inhibited ferroptosis; these benefits were negated by AMPK inhibition or UNC5B depletion. Netrin-1 mitigated DOX-induced cardiotoxicity by suppressing ferroptosis and restoring antioxidant defenses through the UNC5B-LKB1/CaMKKβ-AMPK-NRF2 axis, highlighting Netrin-1 as a promising therapeutic candidate for preventing chemotherapy-induced cardiac injury.