Melatonin Suppresses Microglial Necroptosis By Regulating Deubiquitinating Enzyme A20 After Intracerebral Hemorrhage

Aims: Cell death is deeply involved in pathophysiology of brain injury after intracerebral hemorrhage(ICH).Necroptosis,one of the recently discovered forms of cell death,plays an important role in various diseases,including ICH.Previous studies have suggested that a considerable number of neurons undergoes necroptosis after ICH.However,necroptosis of microglia after ICH has not been reported to date.Melatonin is a hormone predominantly synthesized in and secreted from the pineal gland.It has been shown to have neuroprotective effects in various central nervous system diseases.In addition,melatonin has also been reported to have the ability to inhibit necroptosis.The purpose of this study was to explore the occurrence and mechanisms of necroptosis of microglia after ICH and to verify whether melatonin can suppress this process to alleviate secondary brain damage in ICH.Method: This study is divided into two parts: in vivo experiments and in vitro experiments.We used C57 mice to construct experimental ICH model,and evaluated the neuroprotective effects of melatonin at different time points after ICH.Immunofluorescence and western blotting were used to detect microglial necroptosis and the mechanism by which melatonin inhibits this process.In vitro experiments,OxyHb was used to treat BV2 microglia.Cell viability test,cytotoxicity test,ROS test,and flow cytometry were used for further verification.JC-1 was used to evaluate alteration of mitochondrial membrane potential in vitro.Results: Melatonin reduced brain edema and improved neurological function in mice after ICH.After ICH,the expression of necroptosis related proteins including RIP1,RIP3 and MLKL were significantly increased.Melatonin reduced the expression of these proteins and suppressed microglial necroptosis.Melatonin significantly alleviated the decrease of cell viability and mitochondrial damage caused by OxyHb in vitro.In addition,after ICH,the expression of deubiquitinase A20(also known as TNFAIP3)of microglia remarkedly increased,melatonin can inhibit the microglial necroptosis by regulating the expression of A20,thus exerting protective effect.Conclusion: In summary,we have demonstrated the role of microglial necroptosis in the pathogenesis of ICH.More importantly,A20 was identified as a novel target of melatonin,which opens perspectives for future research.