Luciferase-Based Reporter System for Investigating GPx4-Mediated Ferroptosis and Its Therapeutic Implications in Diabetes

Abstract

Ferroptosis, a distinct form of regulated cell death, is characterized by iron-dependent lipid peroxide accumulation in cell membranes from dysregulated cellular iron homeostasis and compromised antioxidant defense mechanisms. Glutathione peroxidase 4 (GPx4) is crucial in the regulation of ferroptosis by controlling lipid peroxide accumulation. Recent research established the association of ferroptosis with several diseases, prompting investigation toward ferroptosis-targeted therapeutic approaches. However, there is a lack of sensor systems designed to evaluate ferroptosis modulation in intact cells. In this study, we developed a highly sensitive luciferase-based reporter system to study GPx4-mediated ferroptosis in cells. We constructed a novel vector flanking the GPx4 promoter driving luciferase gene expression, demonstrating ferroptosis-specific luciferase activity in transfected HEK293T cells. We established stable cells expressing the construct and optimized its suitability for high-throughput screening using well-established ferroptosis modulators. We identified eugenol, a phenolic compound, as a potent ferroptosis inhibitor using the developed reporter system. Eugenol demonstrated dose-dependent protection against ferroptosis-induced damage in pancreatic beta cells, as assessed by the expression of the key markers such as GPx4, SLC7A11, NRF2, and HO1. Further, we showed the regulation of iron levels and total iron-binding capacity of beta cells by eugenol in streptozotocin (STZ) -induced diabetic mice. Additionally, the diabetes-induced downregulation of GPx4 and antioxidant Nrf2 in pancreatic tissue was significantly mitigated by eugenol, as evidenced by both immunohistochemistry and gene expression analysis. This research validates the functionality of the ferroptosis sensor and offers an approach to develop antidiabetic therapy by targeting ferroptosis to protect beta-cell viability and function.