Combination of EPC-EXs and NPC-EXs with miR-126 and miR-210 overexpression produces better therapeutic effects on ischemic stroke by protecting neurons through the Nox2/ROS and BDNF/TrkB pathways

Neural progenitor cells (NPCs) and endothelial progenitor cell (EPCs) exhibit synergistical effects on protecting endothelial cell functions. MiR-126 and miR-210 can protect cell activities by regulating brain-derived neurotrophic factor (BDNF) and reactive oxygen species (ROS) production. Exosomes (EXs) mediate the beneficial effects of stem cells via delivering microRNAs (miRs). Here, we investigated the combination effects of EXs from EPCs (EPC-EXs) and NPCs (NPC-EXs), and determined whether these EXs with miR-126 (EPC-EXsmiR-126) and miR-210 overexpression (NPC-EXsmiR-210) had better effects on hypoxia/reoxygenation (H/R)-injured neurons and ischemic stroke (IS). Cultured neurons were subjected to hypoxia for 6 h and then co-cultured with culture medium, NPC-EXs, EPC-EXs, NPC-EXs + EPC-EXs or NPC-EXsmiR-210 + EPC-EXsmiR-126 under normoxia for 24 h. Cell apoptosis, ROS production, neurite outgrowth and BDNF level were analyzed. Permanent middle cerebral artery occlusion (MCAO) was performed on C57BL/6 mice to build IS model. The mice were injected with PBS or various EXs via tail vein 2 h after MCAO operation. After 24 h, infarct volume and neurological deficits score (NDS), neuronal apoptosis, ROS production and spine density of dendrites, and brain BDNF level were analyzed. For mechanism study, NADPH oxidase 2(Nox2) and BDNF receptor tyrosine kinase receptor B (TrkB) were determined, and TrkB inhibitor k-252a was used in in vitro and in vivo study. 1) The level of miR-210 or miR-126 was increased after NPC-EXs or EPC-EXs treatment respectively. 2) In H/R-injured neurons, NPC-EXs or EPC-EXs decreased cell apoptosis and ROS production and promoted neurite outgrowth, which were associated with the downregulation of Nox2 and the increase of BDNF and p-TrkB/TrkB level. 3) In MCAO mice, NPC-EXs or EPC-EXs decreased infarct volume and NDS, reduced neural apoptosis and ROS production, and promoted the spine density of dendrites. The levels of Nox2, BDNF and p-TrkB/TrkB in mouse brain tissues changed in similar patterns as seen in the in vitro study. 4) In both cell and mouse models, combination of NPC-EXs and EPC-EXs was more effective than NPC-EXs or EPC-EXs alone on all of these effects. 5) EPC-EXsmiR-126 + NPC-EXsmiR-210 had better effects compared to NPC-EXs + EPC-EXs, which were inhibited by k-252a. EPC-EXsmiR-126 combined NPC-EXsmiR-210 further orchestrate the combinative protective effects of EPC-EXs and NPC-EXs on IS, possibly by protecting H/R-injured neurons through the Nox2/ ROS and BDNF/TrkB pathways.