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.

Endometrial cell death is induced by bacterial infection, resulting in damage to the physical barriers and immune function. An in-depth understanding of the mechanisms of endometrial epithelial cell necroptosis might provide new insights into the treatment of uterine diseases. In the present study, we investigated the effect of Staphylococcus aureus on goat endometrial epithelial cell (gEEC) necroptosis, and the underlying molecular mechanism. We found that S. aureus induced significant necroptosis in gEECs by increasing the expression of key proteins of the RIPK1/RIPK3/MLKL axis; importantly, this effect was alleviated by inhibitors of RIPK1, RIPK3, and MLKL. Moreover, we found that the main triggers of gEEC necroptosis induced by S. aureus were not the toll-like receptors (TLRs) and tumor necrosis factor receptor (TNFR), but membrane disruption and ion imbalance. Moreover, we observed a significant decrease in the mitochondrial membrane potential, indicating mitochondrial damage, in addition to increased cytochrome c levels and reactive oxygen species (ROS) generation in S. aureus-infected gEECs; these, effects were also suppressed by the inhibitors of RIPK1, RIPK3, and MLKL. Taken together, these data revealed the molecular mechanism of S. aureus-induced gEEC necroptosis and provided potential new targeted therapies for clinical intervention in bacterial infections.

Osthole (Ost) is an active constituent of Cnidium monnieri (L.) Cusson which possesses anti-inflammatory and anti-oxidative properties. It also has estrogen-like activity and can stimulate corticosterone secretion. The present study was aimed to check the role of Ost on progesterone (P4) secretion in cultured granulosa cells obtained from hen preovulatory follicles. Different concentrations (5, 2.5, and 1.25 µg/mL) of Ost was added to granulosa cells for 6, 12, 18, and 24 h to investigate the level of progesterone secretions using enzyme linked immunosorbent assay (ELISA). The results showed that progesterone secretion was significantly increased in cells treated with Ost at 2.5 μg/mL. Also, qRT-PCR showed that mRNA expression of steroidogenic acute regulatory protein (StAR) was significantly up-regulated by Ost at 2.5 μg/mL concentration. Cytochrome P450 side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly up-regulated by Ost. However, no significant differences were observed for the expression of proliferating cell nuclear antigen (PCNA). The protein expression of StAR, P450scc and 3β-HSD were significantly up-regulated by Ost treatment. The concentration of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in cell lysates showed no change with Ost treatment at 2.5 μg/mL by ELISA. An ROS kit showed non-significant difference in the level of reactive oxygen species (ROS). In conclusion, Ost treatment at a concentration of 2.5 μg/mL for 24 h had significantly up-regulated P4 secretion by elevating P450scc, 3β-HSD and StAR at both gene and protein level in granulosa cells obtained from hen preovulatory follicles.

Increasing evidence indicates that autophagy and endoplasmic reticulum (ER) stress are involved in the regulation of cell death; however, the role of autophagy and ER stress in Staphylococcus aureus-induced endometrial epithelial cell damage is still unelucidated. In the present study, our results showed that infection with S. aureus increased the cytotoxicity and the protein expression of Bax, caspase-3, and cleaved-PARP-1 in goat endometrial epithelial cells (gEECs). Moreover, after infection, the expression of LC3II and autophagosomes were markedly increased. The autophagosome inhibitor 3-methyladenine (3-MA) significantly decreased the cytotoxicity and the expression of caspase-3, and cleaved-PARP-1; however, the autophagosome–lysosome fusion inhibitor chloroquine (CQ) increased their expression. Additionally, the protein expression of GRP78, EIF2α, and ATF4 were also markedly increased after infection. The ER stress inhibitor 4-PBA decreased the cytotoxicity and the expression of LC3II and apoptosis-related proteins in S. aureus-infected gEECs. Collectively, our findings prove that the accumulation of autophagosomes exacerbated S. aureus-induced gEECs apoptosis, and that ER stress was involved in the regulation of the autophagy and apoptosis.

Zearalenone is a common mycotoxin that impairs reproductive function, particularly by damaging ovarian granulosa cells. This study investigated the protective effect and underlying mechanism of scutellarin against zearalenone-induced injury in porcine ovarian granulosa cells. Cells were isolated and identified by follicle-stimulating hormone receptor immunofluorescence. Cell viability, cell-cycle distribution, and related molecular changes were evaluated using the MTT assay, flow cytometry, qRT-PCR, and Western blot. Scutellarin showed no obvious cytotoxicity within the tested range and attenuated the zearalenone-induced reduction in cell viability. Flow cytometry analysis demonstrated that scutellarin alleviated zearalenone-induced cell-cycle disturbance. At the molecular level, scutellarin upregulated the expression of cell-cycle-related factors, including CDK1, CDK2, CDK4, and PCNA, and increased the expression of Wnt/β-catenin signaling-related proteins, including WNT5A, β-catenin, c-MYC, and CCND1. WNT5A knockdown further indicated that scutellarin-mediated regulation of PCNA, CDK1, and CDK4 is WNT5A-dependent, whereas its effect on CDK2 may involve a WNT5A-independent mechanism. These findings indicate that scutellarin alleviates zearalenone-induced granulosa cell injury partly through WNT5A-associated modulation of cell-cycle-related proteins, providing mechanistic insights for its protective effects.