Immunofluorescence staining showed that both MT and MT expressed in HT cells subjected to mechanical scratch injury or not. Most of the studies evaluated the effects of melatonin on cellular, histopathological and behavioral outcomes following TBI, including but are not limited to: markers of oxidative stress, inflammatory cytokines, edema, lesion size, and cognitive impairment.Although melatonin exerts multiple protective effects on treating TBI, including antiapoptotic, antioxidative, and antiinflammatory properties, there is no information regarding the effects of melatonin on ferroptosis after TBI.As a novel form of cell death, ferroptosis plays an important role in various pathological conditions, including TBI.Similarly, baicalein attenuated ferroptosis signaling and cognitive outcome after TBI.Together, the previous reports indicated that ferroptosis is involved in the pathogenesis of TBI.However, the underlying mechanisms of ferroptosis following TBI, and the antiferroptosis actions of melatonin have still not been clarified.Considering the halflife of melatonin is short, the animals received melatonin hour after TBI and once a day until sacrifice in this study.The effects of melatonin are observable both after a single administration. Numerous studies have suggested that endogenous glutathione exerts a neuroprotective effect against TBI.Cortical GSH content is significantly reduced following TBI in rats and mice.Inhibition of xc depletes GSH levels and impairs glutathione peroxidase activity, thereby increasing lipid peroxidation.Certain membrane lipids have been found to be oxidized during ferroptosis.In addition, we found the expression of MT and MT reduced in a timedependent fashion after TBI, while melatonin inhibited the MT and MT deficiency following TBI.However, there is no report about how melatonin treatment prevents a decrease in MT and MT expression after insults.More research work needs to address this in the future.We speculate that the dosage of luzindole matters or heterodimerization of melatonin receptors may affect the agonist response of luzindole.Moreover, inhibition of MAPKERK activation is correlated with antiferroptotic mechanisms in a renal ischemiareperfusion injury model.To our knowledge, this study is the first study directly investigating the effects of melatonin on ferroptosis after TBI.These findings indicate that a strategy using melatonin aimed at attenuating lipid peroxidation may be a viable approach to mitigate ferroptosis and the subsequent long term physical, cognitive, and emotional deficiencies caused by TBI.Another characteristic of ferroptosis is iron accumulation.Iron homeostasis appears to play an important role in the pathobiology of TBI.As a crucial micromineral required for all living beings, iron serves as a cofactor in heme and the ironsulfur cluster. However, abnormal iron deposition in specific regions of the brain is directly involved in the poor cognitive outcome, since nonheme bound iron accumulates in deep gray matter areas of the brain and may contribute to secondary pathological injury following TBI.These findings underscore the importance of maintaining iron homeostasis in the brain.In mammals, the uptake, transport, and storage of iron are tightly coordinated by various proteins and pathways to maintain iron homeostasis at both the cellular and systemic levels.In the central nervous system, cells including neurons, have evolved highly regulated mechanisms for controlling cellular iron levels, including the iron storage protein ferritin, iron importer transferrin receptor. Ferritin has regarded as the endpoint protein to store and remove excess iron to reduce cellular damage and stress.