Mfat-1 Transgene Protects Adult Neural Stem Cells Against Cobat Chloride-Mediated Hypoxia Injury By Activating Nrf2/ARE Pathways

Background:Ischemic stroke is a devastating neurological disorder and one of the leading causes of death and serious disability in adults.Adult neural stem cells（NSCs）replacement therapy is a promising treatment for both structural and functional neurological recovery.Adult neural stem cells（NSCs）replacement therapy has become a promising treatment for ischemic stroke in recent years.The primary obstacle,however,is that hypoxic-ischemic damage inhibits the activity of adult NSCs and impairs their physiological function.In this study,mfat-1 transgene mouse model was used to investigate whether overproduction of n-3 PUFAs could protect adult NSCs against hypoxic damage induced by CoCl₂.Our results demonstrated for the first time that mfat-1 protects adult neural stem cells against CoCl₂-mediated hypoxia injury by activating Nrf2/ARE pathways.These results are important for innovating clinical intervention strategies for ischemic stroke.Objective:In the present study,we aimed to investigate the neuroprotective effects of the mfat-1 transgene on cobalt chloride（CoCl₂）-induced hypoxic-ischemic injury in cultured adult NSCs as well as its underlying mechanisms.Methods:In this study,the mfat-1 heterozygotes of C57/B6 and wild-type（WT）C57/B6 mice were interbred to produce mfat-1 transgene mice and WT littermates.The mfat-1 transgenic mice and WT littermates were identified by genotyping using polymerase chain reaction（PCR）amplification.The primary adult neural stem cells（NSCs）were isolated from the subventricular zone（SVZ）of adult mfat-1 transgenic mice and WT littermates.The primary neurospheres were passaged with Accutase solution（Sigma-Aldrich,USA）,and the passaged cells were called first passage（PI）.The third passage cells were used for all the subsequent experiments.Firstly,Lipids extraction from integrated mouse brain tissues and adult NSCs respectively was performed according to the general technique reported previously.And then CoCl₂ was used to simulate adult NSCs ischemia/hypoxia in vivo.With this CoCl₂-induced hypoxic-ischemic injury model,we investigated cell viability,cell apoptosis and cell proliferation were investigated to evaluate the protective effects of mfat-1 trangene on adult NSCs.In addition,Intracellular ROS levels were detected by ROS Fluorescent Probe-dihydroethidium（DHE）assay and intracellular GSH level assay was performed using a reduced glutathione assay kit（Njjcbio,China）.Nrf-2,HO-1,NQO-1,and glutamate cysteine ligase catalytic subunit（GCLC）mRNA expression were quantified by real-time PCR and western blot.Results:The results showed that in the CoCl₂-induced hypoxic-ischemic injury model,the mfat-1 transgene enhanced the viability of adult NSCs and suppressed CoCl₂-mediated apoptosis of adult NSCs;this process was related to the reduction of reactive oxygen species（ROS）.Additionally,the mfat-1 transgene promoted the proliferation of NSCs,as shown by increased bromodeoxyuridine labeling of adult NSCs.Quantitative RT-PCR and Western blot analysis showed that a much higher expression of nuclear factor erythroid 2-related factor 2（Nrf2）and its downstream genes（HO-1,NQO-1,GCLC）was found in the NSCsfat-I group after CoCl₂ exposure.Conclusion:Our results demonstrated for the first time that mfat-1 protects adult neural stem cells against CoCl₂-mediated hypoxia injury by activating Nrf2/ARE pathways.These results are important for innovating clinical intervention strategies for ischemic stroke.Taken together,our findings show that the mfat-1 transgene restores the CoCl₂-inhibited viability and proliferation of NSCs by activating the Nrf2/ARE pathways to inhibit oxidative stress injury.Further investigation of the function of the mfat-1 transgene in adult protective mechanisms may accelerate the development of adult NSC replacement therapy for ischemic stroke.