The Effect And Mechanism Of TLR4-Peli1 Axis Mediated Neuroinflammation In Early Brain Injury In Mice Following Subarachnoid Hemorrhage

Background:Subarachnoid hemorrhage(SAH)is a devastating clinical cerebrovascular disease that accounts for 5% of all strokes.Due to its high rate of mortality and morbidity,the management of SAH remains a tough clinical challenge faced by physicians.Early brain injury(EBI),which starts in the first 72 h following subarachnoid hemorrhage(SAH),has recently been regarded as a primary cause of mortality and disability.The pathophysiological events of EBI involve reduction in cerebral perfusion pressure,blood-brain barrier(BBB)disruption,brain edema and neuronal cell apoptosis,etc.It has been demonstrated that EBI following SAH is closely associated with neuroinflammation.The pathogenesis of SAH critically involves microglia,which are the innate immune cells in the central nervous system(CNS),and upon activation,cause production of pro-inflammatory cytokines.Therefore,preventing microglial activation and reducing inflammation might be a promising therapeutic strategy forSAH.Toll-like receptor 4(TLR4),one of the earliest found toll-like receptors(TLRs)subtype,plays an important role in inflammatory signal transduction of EBI following SAH.Importantly,the up-regulation of TLR4 expression in microglia mediates the microglial activation and activation of the inflammatory signaling pathway after SAH,which promotes neuronal apoptosis.Once activated,TLR4 leads to activation of mitogen-activated protein kinase(MAPK)signaling via MyD88-dependent pathway,subsequently mediating induction of pro-inflammatory cytokines.These studies indicate that TLR4 is a valid target for therapy of SAH.Peli1 is an E3 ubiquitin ligase that is abundantly expressed in microglia in CNS.Once activated,Peli1 mediates TLR4-induced pro-inflammatory cytokines in microglia.Peli1 functions as a critical mediator for MAPK activation in the MyD88-dependent pathway through K63 ubiquitination of cellular inhibitor of apoptosis proteins(cIAP).These indicate the potential role of Peli1 in regulation of neuroinflammation.Objective:This study was designed to investigate the role of Peli1 in EBI after experimental SAH.Furthermore,this study intend to explore the mechanism of Peli1 and its interaction with TLR4 on microglial activation and neuroinflammation in EBI after SAH.Methods:Part 11.The SAH model was induced by endovascular perforation in C57 mice.The Peli1 protein expression in mouse cerebral tissues and microglia cell line of BV2 was evaluated by western blot and immunofluorescence.2.The levels of TLR4,cIAP1/2 and MAPK family proteins(p-JNK and p-ERK)were detected by western blot at 6,24,48 and 72 h after SAH.Part 21.A lentiviral vector expressing shRNA targeting Peli1 was constructed and then transfected into BV2 cells.In order to establish the efficiency of Peli1-shRNA lentiviral vector,the expression of Peli1 mRNA in BV2 cells was detected by qRT-PCR.2.C57 mice were pretreated with intracerebral ventricle injection of lentivirus solution expressing Peli1-shRNA or scrambled sequence before the onset of SAH.C57 mice were randomly assigned into Sham group,SAH+Peli1-NC group and SAH+Peli1-KD group.Neurological deficit was evaluated by Garcia scores.The brain edema was detected by MRI measurement among three groups 48 h after SAH.The levels of TLR4,cIAP1/2,MyD88,apoptosis-related proteins(Bcl2 and Bax),MAPK family proteins(p-JNK and p-ERK)and iNOS were detected by western blot among three groups 48 h after SAH.The expression of IL-6 was evaluated by ELISA analysis.Part 31.The expression of TLR4 and microglia marker IBA1 were detected by immunofluorescence.2.C57 mice were pretreated with tail vein injection of TLR4 inhibitor of TAK242.C57 mice were randomly assigned into Sham group,SAH+vehicle group and SAH+TAK242 group.Neurological deficit was evaluated by Garcia scores 48 h after SAH.The protein levels of TLR4,MyD88,iNOS,Peli1,cIAP1/2,apoptosis-related proteins(Bcl2 and Bax)and MAPK family proteins(p-JNK and p-ERK)were detected by western blot among three groups 48 h after SAH.ResultsPart 11.We observed a time-dependent increase in Peli1 protein expression in EBI after experimental SAH.2.Peli1 was predominantly expressed in microglia,whereas it was lowly expressed in neurons,astrocytes and oligodendrocyte precursor cells.3.The expression of cIAP1/2,TLR4,p-ERK and p-JNK proteins were increased after SAH.The expression trend of Peli1 was associated with the expression trend of inflammatory regulation related proteins.4.The iNOS protein expression was increased after SAH.Peli1 and CD16/32 proteins were co-expressed in microglia after SAH.Part 21.The qRT-PCR analysis showed that the lentiviral vector ofPeli1-KD2,showing the highest interference efficiency,after transfection of BV2 cells with Peli1-shRNA lentiviral vector.2.Injection into the lateral ventricles of mice with Peli1-shRNA lentiviral vector repressed the protein levels of Peli1 48 h after SAH.3.Knockdown of Peli1 improved Garcia scores and reduced cerebral edema after SAH.4.Western blot analysis showed that down-regulation of Peli1 decreased the protein levels of Bax and increased the protein levels of Bcl248 h after SAH.Down-regulation of Peli1 reduced p-JNK and p-ERK proteins 48 h after SAH.ELISA analysis showed that down-regulation of Peli1 significantly suppressed the production of IL-6 48 h after SAH.5.Western blot analysis showed that down-regulation of Peli1 decreased the protein levels of i NOS.6.Western blot analysis showed that down-regulation of Peli1 decreased the protein levels of cIAP1/2 and K48,but it had no effect on MyD88 and TLR4 proteins after SAH.Part 31.Immunofluorescence analysis showed that TLR4 was co-expressed with IBA1 in microglia 48 h after SAH.2.TLR4 inhibitor of TAK242 treatment improved Garcia scores 48 h after SAH.3.Western blot analysis showed that TAK242 treatment suppressedTLR4 and MyD88 proteins 48 h after SAH.Meanwhile,TAK242 treatment decreased p-JNK and p-ERK proteins 48 h after SAH.4.Western blot analysis showed that TAK242 treatment decreased Bax protein,and increased Bcl2 protein.5.Western blot analysis showed that TAK242 treatment decreased i NOS protein,but it had no effect on Peli1 and cIAP1/2.ConclusionPart 11.As Peli1 expression was increased,and was associated with the expression of inflammatory regulation related proteins(cIAP1/2,TLR4,p-ERK and p-JNK),Peli1 may be associated with inflammatory regulation after SAH.2.Peli1 is predominantly expressed in microglia after SAH,indicating that Peli1 is associated with activation of M1 microglia.Part 21.Down-regulation of Peli1 significantly reduces the production of pro-inflammatory cytokines,and suppresses activation of M1-type microglia and apoptosis signaling after SAH.These combined effects reduces cerebral edema and improves neurological outcomes after SAH.Therefore,targeting Peli1 exerting neuroprotective effects during EBI after SAH.2.Peli1 contributes to production of pro-inflammatory cytokines bymediating cIAP1/2 activation,thus promoting the activation of MyD88-dependent MAPK pathway after experimental SAHPart 31.TLR4 mediates activation of M1-type microglia via MyD88-dependent MAPK signaling after SAH.2.TAK242 suppresses MAPK signaling via targeting TLR4 pathway,thus inhibiting activation of M1-type microglia and apoptosis signaling after SAH.Therefore,TLR4 exerting neuroprotective effects during EBI after SAH.