Therapeutic mechanism of human neural stem cell-derived extracellular vesicles against hypoxia-reperfusion injury in vitro

This study aimed to explore that the human neural stem cell derived extracellular vesicles (hNSC-EVs) have therapeutic effect on neuronal hypoxia-reperfusion (H/R) injured neurons in vitro by mediating the nuclear translocation of NF-E2-related factor 2 (Nrf2) to regulate the expression of downstream oxidative kinases.The neuroprotective effects of hNSC-EVs were evaluated in an in vitro neuronal H/R model. Three parameters of hNSC-EVs, structure, phenotype and particle size, were characterized. At the cellular level, a human neuron cerebral ischemic reperfusion (CIR) injury model was constructed. Cell viability, apoptosis, and the amount of reactive oxygen species (ROS) were detected using real-time cell analysis (RTCA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and dichloro-dihydro-fluorescein diacetate (DCFH-DA), respectively. The neuronal axonal elongation was assessed by Opera Phenix™ screening system. The angiogenesis of human umbilical vein endothelial cells (HUVECs) was evaluated by co-culturing HUVECs with hNSC-EVs in Matrigel. The expression of apoptosis and oxidative stress-related proteins in cells and the nuclear transfer of Nrf2 following hypoxia-reperfusion (H/R) was verified by Western-blotting.We found that the hNSC-EVs can promote the survival of post-H/R injury neurons, inhibit neuronal apoptosis, and enhance nuclear transfer of Nrf2, in response to oxidative stress. We also found the hNSC-EVs can promote the elongation of neuronal axons and the angiogenesis of HUVECs.At present, there is no effective therapy for CIR injury. We suggest that the hNSC-EVs could be considered a new strategy to achieve nerve repair for the treatment of neurological diseases, especially stroke.