Functions And Potential Molecular Mechanisms Of Resolvin D1 In Early Brain Injury After Experimental Subarachnoid Hemorrhage

Background: Subarachnoid hemorrhage(SAH)is a kind of severe hemorrhage disease in central nervous system,which causes high motality and disability as well as attracts widespread attention albeit only accounts 5% for stroke.Traditionally,cerebral vasospasm develops in large arteries is considered as the most important determinant of brain injury and outcome after SAH.However,recent studies show that prevention of vasospasm does not improve outcome in SAH patients.Finally,the influence of early brain injury(EBI)on SAH was brought in focus.Thus,finding effective drugs or reagents targeting on EBI is a prospective treatment for SAH,which is now still under primary investigation stage.Resolvin D1(Rv D1)is a kind of production of Docosahexaenoic acid(DHA)metabolism,which is occurring in the resolution phase of inflammation and engaging in regulation of inflammation process,i.e.,inhibiting neutrophils recruitment and tissue infiltration,attenuating inflammatory factors production,promoting neutrophil apoptosis and efferocytosis by macrophages.Rv D1 has been confirmed exerting good inflammation inhibition effect in many kind of diseases,such as acute kidney,pneumonia,gastrointestinal tract inflammation,peritonitis and inflammation induced pain.In the central nervous system,the study focus was mainly on analgesic effect of Rv D1 and the role of its receptor formyl peptide receptor 2(FPR2)in Aizheimer’s disease,meanwhile,several studies also investigated the effect of other FPR2 agonist on brain hemorrhage,ischemia and SAH.Nevertheless,there is almost no study focusing on the effect of Rv D1 on brain inflammation or other aspects.Therefore,this study was performed to observe whether Rv D1 could attenuate EBI after SAH to exert a protection effect on brain and furtherly,trying to investigate potential mechanisms.Methods: Our study investigated the effect of Rv D1 both in vivo and in vitro SAH model.Internal carotid artery endovascular perforation model was adopted for in vivo model;and hemoglobin stimulation model was adopted for in vitro model.Firstly,the expression location and time course change of Rv D1 receptor FPR2 after SAH were investigated by double immunofluorescence and western blotting in vivo and in vitro.Secondly,different dose of Rv D1 was administrated to observe its effect on brain inflammation especially neutrophils infiltration and glial activation after SAH as well as exploring its influence on nuclear factor-?B(NF-κB)and mitogen-activated protein kinase(MAPK)signaling activities using immunofluorescence,western blotting and real-time quantificative PCR.Then,furtherly investigating the effect of Rv D1 on hemoglobin(Hb)-induced microglial inflammatory response and underlying mechanisms.Finally,combining in vivo experiments and in vitro primary neuron culture,the effect of Rv D1 on neuronal damage and apoptosis was determined by western blotting,immunofluorescence,chemical quantification techniques and other methods to optimize our study.Results: The results indicated that the expression pattern of FPR2 after SAH was firstly elevated and then decreased,peaked at 2 days.FPR2 was mainly expressed on neurons according to the results of in vivo immunofluorescence while the western blotting results of primary cells showed that it not only expressed on neurons but also on microglia,together,FPR2 was not expressed on astrocytes.Both in in vivo or in vitro experiment,Rv D1 exerted good anti-inflammatory effect,evidenced as inhibiting neutrophils recruitment and infiltration,attenuating pro-inflammatory factors secretion and reduced glial activation.Furthermore,Rv D1 possibly could regulate microglial polarization.In regard of the mechanisms,Rv D1 might function via regulating interleukin-1 receptor-associated kinase 1(IRAK1)and TNF receptor associated factor6(TRAF6)to influence the activation of NF-κB and MAPK to exert its antiinflammation effect.Notably,the in vitro experiment indicated that in the late phase after Rv D1 addition,the pro-inflammatory factors rebounded to some extent,which might be a guidance for drug application interval.Finally,Rv D1 also have some immediate effects on neurons,presented as reducing neuronal damage and apoptosis.All of above indicate that Rv D1 could play multiple roles after SAH,which possibly involve many cell types and different signaling pathways.Conclusion: This study confirmed that FPR2 was expressed both on microglia and neurons,whose agonist(Rv D1)could exert good anti-inflammatory effect and simultaneously,has immediate effect on neuronal injury and apoptosis to some extent.The above indicate that FPR2 could be a potential therapy target and Rv D1 could be a potential good neuro-protection drug.