Zearalenone Induces MLKL-Dependent Necroptosis in Goat Endometrial Stromal Cells via the Calcium Overload/ROS Pathway

Zearalenone (ZEA) is a fungal mycotoxin known to exert strong reproductive toxicity in animals. As a newly identified type of programmed cell death, necroptosis is regulated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like pseudokinase (MLKL). However, the role and mechanism of necroptosis in ZEA toxicity remain unclear. In this study, we confirmed the involvement of necroptosis in ZEA-induced cell death in goat endometrial stromal cells (gESCs). The release of lactate dehydrogenase (LDH) and the production of PI-positive cells markedly increased. At the same time, the expression of RIPK1 and RIPK3 mRNAs and P-RIPK3 and P-MLKL proteins were significantly upregulated in ZEA-treated gESCs. Importantly, the MLKL inhibitor necrosulfonamide (NSA) dramatically attenuated gESCs necroptosis and powerfully blocked ZEA-induced reactive oxygen species (ROS) generation and mitochondrial dysfunction. The reactive oxygen species (ROS) scavengers and N-acetylcysteine (NAC) inhibited ZEA-induced cell death. In addition, the inhibition of MLKL alleviated the intracellular Ca²⁺ overload caused by ZEA. The calcium chelator BAPTA-AM markedly suppressed ROS production and mitochondrial damage, thus inhibiting ZEA-induced necroptosis. Therefore, our results revealed the mechanism by which ZEA triggers gESCs necroptosis, which may provide a new therapeutic strategy for ZEA poisoning.