Melatonin Protects BEAS-2B Cells Against Hypoxia/Reoxygenation-Induced Injury By Reducing MLKL Levels, Suppressing...

Necroptosis has been demonstrated to play a role in the process of lung ischemia-reperfusion injury (LIRI) in a variety of clinical conditions, including cardiopulmonary bypass and pulmonary embolism. Melatonin has been reported to exert a protective role by reducing oxidative stress and acute inflammatory reactions in LIRI. However, the effect of melatonin on necroptosis in LIRI remains unclear. The aim of this study was to investigate whether the protective effects of melatonin against H/R-induced injury are associated with modulation of necroptosis and related changes in mitochondrial function and oxidative stress. The cells were exposed to H/R (16/4 h) or normoxia, in the absence or presence of 2.5 µM melatonin. Cell viability was determined by the MTT method, while morphological changes in BEAS-2B cells resulting from H/R exposure were evaluated by fluorescence microscopy using acridine orange/ethidium bromide (AO/EtBr) staining. Mechanistic analyses, including flow cytometry-based cell cycle assessment, apoptosis detection (Annexin V-FITC), mitochondrial membrane potential evaluation (JC-1), and reactive oxygen species (ROS) measurements, were performed on BEAS-2B cells. The level of the mixed lineage kinase domain-like pseudokinase (MLKL), a key component of the necroptosis complex, was also quantified using an enzyme-linked immunosorbent assay. In addition, the ATP, pH, and lactate levels were determined. The findings indicated that melatonin treatment reduced the H/R-induced increase in the percentage of necrotic cells and increased the percentage of viable cells (p < 0.05). In addition, melatonin treatment reduced the increase in MLKL, ROS, and lactate levels, preserved mitochondrial membrane integrity, and increased intracellular ATP levels in BEAS-2B cells exposed to H/R (p < 0.05). In conclusion, this study suggests that melatonin protects BEAS-2B cells against H/R-induced injury, and that this protective effect may be associated with reduced oxidative stress, preservation of mitochondrial function, and suppression of necroptosis-related signaling.