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
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
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EPC-EX和NPC-EX与miR-126和miR-210过表达相结合，通过Nox2/ROS和BDNF/TrkB途径保护神经元，对缺血性中风产生更好的治疗效果
DOI：
10.1016/j.expneurol.2022.114235
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发表时间：
2022-10-11
影响因子：
5.3
通讯作者：
Ma，Xiaotang
中科院分区：
文献类型：
--
作者：
Xu，Xiaobing;Zhang，Huiting;Ma，Xiaotang
背景/目的神经祖细胞（NPCs）和内皮祖细胞（EPCs）在保护内皮细胞功能方面具有协同作用。MiR-126和miR-210可以通过调节脑源性神经营养因子（BDNF）和活性氧（ROS）的产生来保护细胞活性。外泌体（EXs）通过传递microrna （miRs）介导干细胞的有益作用。在这里，我们研究了EPCs （EPC-EXs）和npc （NPC-EXs）的EXs的联合作用，并确定这些miR-126 （EPC-EXsmiR-126）和miR-210过表达（NPC-EXsmiR-210）的EXs是否对缺氧/再氧化（H/R）损伤的神经元和缺血性卒中（IS）有更好的作用。方法将培养好的神经元缺氧6 h后，与NPC-EXs、EPC-EXs、NPC-EXs + EPC-EXs或NPC-EXsmiR-210+ epc - exsmir -126共培养24 h，分析细胞凋亡、ROS生成、神经突生长和BDNF水平。采用永久性大脑中动脉闭塞法（MCAO）建立C57BL/6小鼠IS模型。MCAO术后2 h经尾静脉注射PBS或各种EXs。24 h后，分析梗死体积、神经功能缺损评分（NDS）、神经元凋亡、ROS生成、脊柱树突密度及脑BDNF水平。机制研究通过测定NADPH氧化酶2（Nox2）和BDNF受体酪氨酸激酶受体B (TrkB)，并采用TrkB抑制剂k-252a进行体外和体内研究。结果1)经NPC-EXs或EPC-EXs处理后，miR-210或miR-126水平分别升高。2)在H/ r损伤的神经元中，NPC-EXs或EPC-EXs可减少细胞凋亡和ROS的产生，促进神经突生长，这与Nox2的下调、BDNF和p-TrkB/TrkB水平的升高有关。3)在MCAO小鼠中，NPC-EXs或EPC-EXs可降低梗死体积和NDS，减少神经细胞凋亡和ROS的产生，提高脊柱树突密度。在体外研究中，小鼠脑组织中Nox2、BDNF和p-TrkB/TrkB水平的变化模式与体外研究相似。4)在细胞和小鼠模型中，NPC-EXs和EPC-EXs联合使用比NPC-EXs或EPC-EXs单独使用更有效。5) EPC-EXsmiR-126+ npc - exsmir -210的抑制效果优于NPC-EXs + EPC-EXs。结论epc - exsmir -126联合npc - exsmir -210进一步协调EPC-EXs和NPC-EXs对IS的联合保护作用，可能是通过Nox2/ ROS和BDNF/TrkB通路保护H/ r损伤的神经元。
Backgrounds/aimsNeural 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).MethodsCultured 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-126under 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.Results1) 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-210had better effects compared to NPC-EXs + EPC-EXs, which were inhibited by k-252a.ConclusionEPC-EXsmiR-126combined NPC-EXsmiR-210further 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.