Luciferase-Based\nReporter System for Investigating\nGPx4-Mediated Ferroptosis and Its Therapeutic Implications in Diabetes

Ferroptosis, a distinct form of regulated cell death,\nis characterized\nby iron-dependent lipid peroxide accumulation in cell membranes from\ndysregulated cellular iron homeostasis and compromised antioxidant\ndefense mechanisms. Glutathione peroxidase 4 (GPx4) is crucial in\nthe regulation of ferroptosis by controlling lipid peroxide accumulation.\nRecent research established the association of ferroptosis with several\ndiseases, prompting investigation toward ferroptosis-targeted therapeutic\napproaches. However, there is a lack of sensor systems designed to\nevaluate ferroptosis modulation in intact cells. In this study, we\ndeveloped a highly sensitive luciferase-based reporter system to study\nGPx4-mediated ferroptosis in cells. We constructed a novel vector\nflanking the GPx4 promoter driving luciferase gene expression, demonstrating\nferroptosis-specific luciferase activity in transfected HEK293T cells.\nWe established stable cells expressing the construct and optimized\nits suitability for high-throughput screening using well-established\nferroptosis modulators. We identified eugenol, a phenolic compound,\nas a potent ferroptosis inhibitor using the developed reporter system.\nEugenol demonstrated dose-dependent protection against ferroptosis-induced\ndamage in pancreatic beta cells, as assessed by the expression of\nthe key markers such as GPx4, SLC7A11, NRF2, and HO1. Further, we\nshowed the regulation of iron levels and total iron-binding capacity\nof beta cells by eugenol in streptozotocin (STZ) -induced diabetic\nmice. Additionally, the diabetes-induced downregulation of GPx4 and\nantioxidant Nrf2 in pancreatic tissue was significantly mitigated\nby eugenol, as evidenced by both immunohistochemistry and gene expression\nanalysis. This research validates the functionality of the ferroptosis\nsensor and offers an approach to develop antidiabetic therapy by targeting\nferroptosis to protect beta-cell viability and function.