Effect Of IL-33-preconditioned Nsc Transplantation On Hypoxic-Ischemic Brain Injury In Neonatal Mice

Hypoxic-ischemic encephalopathy(HIE)is caused by perinatal asphyxia,which can lead to long-term neurological sequelae.Hypoxicischemia results in oligodendrocyte and neuronal loss due to post-injury excitotoxicity,inflammation and oxidative stress.As HIE involves different pathological factors and cell types,these determine the complexity of HIE treatment.Therefore,it has become a pressing issue to find a safe and effective treatment method.Neural stem cell(NSC)is multipotent cell that self-renew and differentiate into different types of neural cells.In recent years,NSC-based transplantation has emerged as a new potential therapeutic approach for the treatment of HIE.Experimental studies have shown that NSC transplantation can exert a protective effect after HIE through neuronal replacement or trophic effects including neuroprotection,promotion of vascularization,immunomodulation and axonal plasticity.It is well known that the ability of NSC proliferation and differentiation depends largely on the neural microenvironment.However,the poor neural microenvironment of the neonatal brain and the lack of nutritional support after HI,which have greatly affected the survival of NSCs after transplantation.IL-33,as a multifunctional cytokine,is highly expressed in the central nervous system(CNS).IL-33 is of vital importance for innate and adaptive immunity.IL-33 is able to polarize M2-type microglia at the site of injury to maintain brain tissue homeostasis.Studies have shown that IL-33 expression is increased after cerebral hypoxia-ischemia and alleviates HIinduced brain injury.In this study,we aim to investigate the regulatory effect of IL-33 on the functional activity of NSCs and the protective effect of IL-33 pretreatment NSC transplantation on neonatal mice after HIE,which will provide new approaches for the treatment of neonatal HIE by transplanting NSCs.Objectives:To investigate the effect of IL-33 on the functional activity of NSCs and the beneficial effect of IL-33-pretreated NSC transplantation on the neurological function of neonatal mouse after HI injury.Methods:We isolated and cultured NSCs from neonatal mouse brain,established NSCs glycotrophic deprivation model,and detected the protective effect of IL-33 on the functional activity of neural stem cells using immunofluorescence,RT-PCR,and flow cytometry.In vivo,we established a neonatal mouse HIE model and performed NSC transplantation using brain stereotactic technique to assess the extent of brain injury by brain appearance and neurobehavior examination.Next,the inflammatory response,neurovascular unit and white matter in the brain after NSC transplantation were examined with the methods of histopathology,molecular biology and immunology.Results:1、Effect of IL-33 on the functional activity of neural stem cellsUnder normoxia conditions,IL-33 promoted NSCs proliferation,neuronal differentiation,and upregulated neurotrophic factor expression.Under OGD conditions,IL-33 obviously inhibited oxidative stress in NSCs compared with the PBS group,further promoting neuronal differentiation and neurotrophic factor(BDNF,GDNF,NGF)expression in NSCs.These data suggest that IL-33 has a protective effect on the functional activity of neural stem cells.2、Regulatory mechanism of IL-33 on the functional activity of neural stem cellsWestern blot assay revealed that compared to the untreated group,IL-33 upregulated the expression of P-AKT and P-β-catenin in neural stem cells under normoxia and hypoxia.The expression of P-AKT and P-β-catenin was significantly decreased in IL-33 group when treatment with PI3 K inhibitor Ly294002.The expression of P-AKT was not significantly changed in IL-33 group when treatment with β-catenin inhibitor XAV939.These findings indicate that β-catenin is the downstream of PI3K/AKT signaling.Meanwhile,flow cytometry results showed that the proliferation of neural stem cells induced by IL-33 was inhibited following blockade ofβ-catenin.Immunofluorescence revealed that XAV939 restricted the neuronal differentiation of NSCs induced by IL-33.In short,these data show that IL-33 activates β-catenin through PI3K/AKT signaling and then regulates the functional activity of NSCs.3、IL-33-preconditioned NSC transplantation enhances neuroprotection of neonatal hypoxic-ischemic miceHIE model in neonatal mice was established and NSCs were transplanted by brain stereotaxic localization.Compared with the PBSpreconditioned NSC group,IL-33-preconditioned NSCs clearly reduced cerebral infarct size and improved neurobehavior in neonatal mice after HI injury.Meantime,IL-33-preconditioned NSCs inhibited brain inflammatory response by promoting M2-type microglia polarization,alleviated blood-brain barrier damage,as well as promoted white matter damage repair.Conclusion:IL-33 regulates the functional activity of NSCs via PI3K/AKT/β-catenin signaling pathway,which enhances the therapeutic efficiency of transplanted neural stem cells in the hypoxic-ischemic environment of the brain and thus improves hypoxic-ischemic brain injury in neonatal mice.