Repairing Effect Of Human Neural Stem Cell-derived Exosomes On Hypoxia-reperfusion Injured Neurons In Vitro

Stroke,especially cerebral ischemic stroke,is a highly lethal disease.Cerebral ischemic reperfusion(CIR)is the process of blood supply recovery in ischemic areas CIR can cause more serious damage than pure ischemia,known as CIR injury.At present,there is no effective therapy for CIR injury.Although stem cell therapy was once believed to become a viable option,no major breakthroughs have so far been achieved.Based on our pre-research and literature studies,we speculate that human neural stem cell-derived exosomes(hNSC-EXO)involve in the repair of post-CIR injured neurons and provide them with neuroprotective effects.The purpose of this study is to explore the therapeutic effect of hNSC-EXO on hypoxia-reperfusion(H/R)injured neurons in vitro.The research contents and results are as follows(1)Preparation and biological characterization of hNSC-EXOHuman neural stem cell(hNSC)culture supernatants were collected and centrafuged by ultra-high-speed centrifuge to separate and purify the exosomes.The structure,particle size and surface markers of hNSC-EXO were characterized,and the prepared exosomes were conformed to have the characteristics as reported in literatures.(2)The in vitro anti-oxidation and anti-apoptosis effect of hNSC-EXO on H/R injured neuronsAt the cellular level,a model of H/R injury was constructed for human neurons.The cell activity,apoptosis and reactive oxygen species(ROS)were detected using real-time cell analysis(RTCA),terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)and dichlorodihydrofluorescein diacetate(DCFH-DA)respectively.The expression of apoptosis and oxidative stress-related proteins in reperfusion damaged neurons and the nuclear transfer of Nrf2 after H/R were verified by Western blot analysis.It was found that hNSC-EXO improved the activity of neurons after H/R injury,inhibited neurons apoptosis,promoted Nrf2 nuclear transfer and alleviated oxidative stress.(3)Effect of hNSC-EXO on axonal elongation and angiogenesisThe effect of the exosomes on neuronal axon elongation was evaluated by opera PhenixTM screening system and ImageJ.Angiogenesis was evaluated by co-culture of human umbilical venous endothelial cells(HUVECs)with hNSC-EXO in Matrigel.The results showed that the exosomes promoted the elongation of neuronal axons and vascular regeneration,indicating the good regenerative repairing effect of hNSC-EXO.At present,there is no effective therapy for CIR injury.From the results of this study it can be inforred that the hNSC-EXO therapy may be considered a new strategy to repair injured nerve,so as to be used for the treatment of neurological diseases,especially CIR injury.The innovations of this paper include:(1)It is the first time to put forward that hNSC-EXO have reparing effect on H/R injured neurons.(2)The presence of hNSC-EXO was found for the first time to increase the expression levels of downstream antioxidant enzymes by promoting the nuclear transition of Nrf2,and to scavenge the reactive oxygen species under oxidative stress.(3)It was found that hNSC-EXO treatment could reduce neuronal apoptosis caused by oxidative stress.(4)It was discovered that hNSC-EXO could promote the elongation of neuronal axons and the angiogenesis of HUVECs,and promote nerve regeneration,which will be beneficial to facilitate the repair of neurons nerves.