OGD/R‐induced ferroptosis and pyroptosis in retinal pigment epithelium cells: Role of PLD1 and PLD2 modulation

This study investigated the role of phospholipase D (PLD) in retinal ischemia–reperfusion (I/R) injury using an oxygen‐glucose deprivation/reperfusion (OGD/R) model commonly used in retinal I/R injury research. To create an in vitro cellular I/R model, pharmacological inhibitors and small interfering RNA (siRNA) were used to target PLD1 and PLD2 in retinal pigment epithelial (RPE) cells. Treatment with PLD inhibitors and siRNA reduced reactive oxygen species (ROS) and malondialdehyde (MDA) induced by OGD/R in RPE cells and increased the levels of superoxide dismutase (SOD) and glutathione (GSH), indicating a reduction in oxidative damage and improvement in the antioxidant system. Next, we showed that inhibiting PLD1 or PLD2 reduced intracellular iron levels and lipid peroxidation, which are critical factors in ferroptosis. Additionally, PLD1 and PLD2 modulated the expression of proteins involved in the regulation of ferroptosis, including GPX4, SLC7A11, FTH1, and ACSL4. We also investigated the roles of PLD1 and PLD2 in preventing pyroptosis, another form of programmed cell death associated with inflammation. Our study found that OGD/R significantly increased the production of pro‐inflammatory cytokines and activated caspase‐1, NLRP3, ASC, cleaved‐caspase 1 (C‐caspase‐1), and GSDMD‐N in RPE cells, indicating pyroptosis induction. However, PLD1 and PLD2 inhibition or knockdown significantly inhibited the production of pro‐inflammatory cytokines and activation of the NLRP3 inflammasome, Taken together, our findings support the hypothesis that the PLD signaling pathway plays a key role in OGD/R‐induced ferroptosis and pyroptosis induction and may be a potential therapeutic target for preventing or treating retinal dysfunction and degeneration. Significance statement Inhibiting PLD1 and PLD2 presents a novel and promising strategy for treating retinal damage. This study sheds light on intricate mechanisms that not only mitigate oxidative stress but also suppress ferroptosis and alleviate pyroptosis in retinal pigment epithelium cells. These findings significantly advance retinal health and disease treatment by unraveling interactions between phospholipase D isoforms and cellular stress responses, suggesting potential therapies for conditions marked by cell death pathways.