Study Of The Role And Mechanism Of Carbon Monoxide Preconditioned Neural Stem Cells In Hemorrhagic Stroke

Objective To study the cytotoxicity of iron overload on NSC and explore the neuroprotection and the mechanism of CO preconditioning on NSC challenged by iron overload as well as the effect of CO-preconditioned NSC on hemorrhagic stroke in vivo.Methods In vitro,ferrous chloride was applied to simulate iron overload environment after HS;CORM-2 was used to release carbon monoxide to precondition neural stem cells;CCK-8 assay was applied to assess the cytotoxicity of iron overload;TUNEL staining and flow cytometer analysis were used to investigate the apoptosis of NSCs;the protein expression was investigated by the immunofluorescence staining and Western blotting analysis;m RNA expressions of genes were tested by real time-PCR;ROS in NSCs was detected by the probe DCFH-DA;Nrf2Si RNA and inhibitor of NF-κB were used to verify the role of Nrf2 and NF-κB signaling pathways;in vivo,we used primary NSCs from red fluorescent protein transgenic(RFP Tg)fetal mice to detect the survival and differentiation of NSCs after transplanting;the neurological performance of each group was assessed.Results Iron overload induced significant apoptosis of NSCs.CO preconditioning inhibited the accumulation of ROS induced by iron overload in NSC.Moreover,CO preconditioning inhibited ROS dependant activation of NF-κB signaling induced by iron overload,and downregulated the expression of pro-apoptotic protein Bax and cleaved-caspase3,upregulated the expression of anti-apoptotic protein Bcl-2.Pretreatment with CO markedly protected NSCs against iron overload-induced apoptosis.CO preconditioning induced translocation and activation of Nrf2 and subsequent upregulation of NQO-1.The neuroprotection of CO is Nrf2 dependant.CO preconditioning upregulated the expression of GDNF、VEGF and BDNF.Compared with the control group,CO enhanced the survival of donor NSCs after HS in vivo.Moreover,transplantation of CO-preconditioned NSCs significantly improved neurological performance after HS.Conclusions CO reestablished the redox balance via activation of Nrf2 and subsequent inhibition of NF-κB to protect NSC against iron overload induced injury.Thus,preconditioning with CO,which reprograms NSCs to tolerate deleterious microenvironment after HS and to express higher levels of neurotrophic factors,is a promising approach to enhance the effectiveness of stem cell therapy in hemorrhagic stroke.