The Experimental Study On The Role Of RIP1/RIP3 Pathway On Secondary Brain Injury After Intracerebral Hemorrhage In Mice

Intracerebral hemorrhage(ICH)is a devastating neurological injury associated with significant morbility and mortality.After ICH,a complex performance of pathophysiologic processes occur in brain parenchyma,including hematoma formation,brain edema,and release of cytotoxic blood components,oxidative stress and inflammation.The synergistic actions of these pathological events lead to neural cell death and subsequent neurological dysfunction.Necroptosis is a newly identified type of programmed necrosis initiated by the activation of tumor necrosis factor alpha.Necroptosis is regulated by the kinase activity of receptor interacting protein kinase 1(RIP1)and its interaction with RIP3 after TNF-a or Fas stimulation.Necrostatin-1(Nec-1),a specific small molecule inhibitor of necroptosis specifically inhibits RIP1 kinase activity and RIP1-RIP3 interaction.Evidences have demonstrated the importance of necroptosis in neuronal cell death.Glutamate induced exicitoxicity causes cell death in ischaemia stroke,and evidence has shown that glutamate also participate in brain injury after ICH.Glutamate exerts its toxic effects through receptor-mediated excitotoxicity and non-receptor mediated oxidative toxicity.Hemin,oxidized heme,is a lipophilic cytotoxic oxidant and kills brain cells adjacent to the hematoma.Cell culture experiments have shown that hemin is sufficient to kill neurons and astrocytes within 24 hours.However,the mechanisms by which hemin or glutamate causes toxicity in cortical astrocytes and HT22 cells are not fully understood.The present study explored whether RIP1 and RIP3 pathways participate in hemin or glutamate induced necrotic cell death in HT22 cells,and the potential therapeutic strategies that can be employed to prevent brain damage.In the first part of our study,we analyzed the mechanisms involved in hemin and glutamate induced cell death in HT22 cells.We demonstrated that RIP1 inhibitor necrostaticn-1 blocked hemin or glutamate induced cell death.And reactive oxygen species(ROS)accumulation was responsible for necrotic cell death induced by hemin or glutamate in HT22 cells.Furthermore,we found that silencing of receptor interacting protein 3(RIP3)using siRNA attenuated hemin or glutamate induced necrotic cell death in HT22 cells.Taken together,our data suggested that regulation of neroptosis pathways including RIP1 and RIP3 might represent novel therapeutic targets after ICH.However,the role of necroptosis in intracerebral hemorrhage has not been studied.Another aim of this study was to investigate the potential therapeutics of RIP 1 inhibitor,necrostatin-1,through reducing necroptosis after ICH.In the second part of our study,collagenase was used to induce ICH in mice.Expression of RIP1 and RIP3 proteins were detected by western blot.Nec-1’s protective effect was evaluated in primary neurons subjected to hemoglobin toxicity.Nec-1 was injected into the right cerebralventricle 15 mins before ICH.Neurological function,brain edema,hemoglobin content was assessed after ICH.To explore the specific mechanism of Nec-1 after ICH,immnunoprecipitation,western blot,propidium iodide labeling,microglia immunofluorescence and proinflommatory cytokines mRNA were performed.Results showed that RIP 1 and RIP3 proteins were abundantly expressed and increased after ICH.Nec-1 treatment protected neurons from hemoglobin-induced cell death in primary neurons.Nec-1 pretreatment improved neurological function and attenuated brain edema in mice after ICH,but did not reduce brain bleeding.Moreover,Nec-1 reduced RIP1-RIP3 interaction and necrotic cell death,and also inhibited microglia activation and inflammatory response after ICH.In conclusion,our work suggests that necroptosis,regulated by RIP1 and RIP3,is an essential pathway for glutamate or hemin induced necrotic cell death in vitro and contributes to neurologic dysfunction after ICH in mice.Through inhibiting necroptosis,necrostatin-1 treatment exerts multifaceted neuroprotection in vitro and in vivo.These results highlight the potential therapeutic value of Nec-1 in protection against ICH,which could be translated to a novel neuroprotective strategy for ICH patients in the future and deserves further investigation.