The Anti-necroptosis And Anti-neuroinflammation Mechanism Of Melatonin In Early Brain Injury After Subarachnoid Hemorrhage

Background:Subarachnoid Hemorrhage(SAH)is an acute cerebrovascular disease with a high rate of disability and mortality.85%of SAH is aneurysmal subarachnoid hemorrhage which is the most common type.Among the past decades,delayed cerebral vasospasm has been described as the main reason responding to unfavorable outcomes after SAH,while the pool anti-vasospastic treatment of SAH patients led to new concentrations on other pathogenesis.Recently,leading experts has focused on early brain injury(EBI)after SAH,eonsidering that EBI might play an important role on determining prognosis of SAH patients.EBI stands for the immediate brain injury of SAH patients within 72 hours after SAH,a very complicated pathophysiological process,including:release of inflammatory factors,occurrence of oxidative stress,toxicity of glutamic acid,lipid peroxidation,activation of calcium channel,apoptosis and necrocytosis.A lot of animal experiments have elucidated that neuronal cell death and neuro-inflammation are the two major consequences of EBI.Melatonin,a kind of neuroendocrine hormone,secreted by pineal body,can easily permeate the Blood Brain Barrier(BBB)with good tolerance.In recent years,researches demonstrated that melatonin plays a protective role on neural function after SAH through decreasing oxidative stress,inhibiting inflammatory reaction,activating autophagy and reducing apoptosis.Nevertheless,further investigations are still required to discover the details behind the mechanism.Hence,this research will focus on two main aspects of the neural protection of melatonin on SAH patients:anti-necroptosis and anti-inflammation.The first aspect is to explore the relationship between necroptosis and pathogenic process of SAH and to further investigate the function of melatonin treatment on necroptosis.The second aspect is to study the regulation of melatonin on NLRP3 inflammasome induced inflammation.Methods:Part ⅠExperiment Ⅰ:The SAH model was performed using the endovascular perforation technique.Adult male Sprague-Dawley(SD)rats were randomly assigned to the sham group and SAH groups.And SAH rats were further divided into SAH 6h group,SAH 12h group,SAH 24h group,SAH 48h group and SAH 72h group according to the duration between SAH and execution.Western blot was used to detect the RIPK3 levels in ipsilateral cortex.The cellular location of RIPK3 was detected by double immunofluorescence staining.Propidium iodide(PI)staining was performed to identify the necrotic neural cells and their relationship with RIPK3.The ultrastructure changes of necrotic neural cells were observed by transmission electron microscope(TEM).Experiment Ⅱ:Adult male SD rats were randomly assigned to the sham group,the SAH+vehicle group and the SAH + GSK872 group.According to the grouping rule,GSK872.the inhibitor of RIPK3 or vehicle was administered by intraventricular injection 30 minutes after SAH.Immunofluorescence and other methods were applied to evaluate cell death,brain edema and neurological function at 72 hours after SAH.Experiment Ⅲ:Adult male SD rats were randomly assigned to the sham group,the SAH + vehicle group and the SAH + Melatonin(Mel)group.According to the grouping rule,melatonin or vehicle was injected intraperitoneally 2 hours,12 hours and 24 hours after SAH.Western blot,immunofluorescence and other methods were applied to evaluate cell death,brain edema and neurological function at 72 hours after SAH.Experiment IV:Adult male SD rats were randomly assigned to the SAH + vehicle group,the SAH + Mel group and SAH + 3-methyladenine(3-MA)group.According to the grouping rule,GSK872 or vehicle was administered by intraventricular injection 30 minutes after SAH and melatonin or vehicle was injected intraperitoneally 2 hours,12 hours and 24 hours after SAH.Western blot,immunofluorescence and other methods were applied to evaluate cell death,brain edema and neurological function at 72 hours after SAH.Part ⅡThe SAH model was performed using the endovascular perforation technique.Adult male SD rats were randomly assigned to four groups:the sham group,the SAH +vehicle group,the SAH + melatonin(Mel)group,and the SAH + Mel +3-methyladenine(3-MA)group.According to the group,they were pretreated with 3-MA or vehicle 20 minutes before operation,and treated with melatonin or vehicle 2 hours after SAH,Brain samples were used to test mitophage and NLRP3 inflammasome related indicators by performing water content analysis,ROS assay,Western blot,immunohistochemistry and transmission electron microscopy.Results:PartⅠ(1)Expression of RIPK 3 increased at 6 hours,decreased at 12 hours,then increased again and peaked at 72 hours after SAH.(2)RIPK3 was primarily located in neurons.PI staining and transmission electron microscope indicated that necrotic neurons were observed at 72 hours after SAH.(3)Melatonin reduced expression of RIPK3 and MLKL,and the number of necrotic neural cells as well as the release of HMGB1.Melatonin decreased BBB disruption and subsequent brain edema and improved neurological function at 72 hours after SAH.(4)Autophagy inhibitor 3-MA abolished the effects of melatonin on RIPK3-mediated necrotic cell death,aggravated BBB disruption and brain edema,and deteriorated the neurological function.Part Ⅱ(1)Melatonin upregulated mitophagy-associated proteins(PINK1/Parkin)and reduced mitochondrial damage and ROS generation after SAH;(2)Melatonin inhibited NLRP3 inflammasome activation after SAH and attenuated the inflammatory response;(3)Melatonin reduced neuronal cell death,brain edema and neurological dysfunction following SAH;(4)Pretreatment with 3-MA reversed the beneficial effect of melatonin on the inhibition of NLRP3 inflammasome activation.Conclusions:1.This study demonstrated that RIPK3-dependent necroptosis was involved in early brain injury after SAH.Melatonin attenuated RIPK3-dependent necroptosis,decreased cytosolic translocation of HMGB1,BBB disruption and brain edema,and improved neurological function after SAH.The underlying mechanisms of these effects were closely related with autophagy activation.2.This study demonstrated that melatonin-upregulated mitophagy proteins played a protective role in post-SAH early brain injury.This neuroprotective effect was related to the inhibition of NLRP3 inflammasome activation and reduced inflammatory responses.