Mesenchymal stromal cell-derived exosomes (MSC-EXs) could exert protective effects on recipient cells by transferring the contained microRNAs (miRs), and miR-132-3p is one of angiogenic miRs. However, whether the combination of MSC-EXs and miR-132-3p has better effects in ischemic cerebrovascular disease remains unknown. Mouse MSCs transfected with scrambler control or miR-132-3p mimics were used to generate MSC-EXs and miR-132-3p-overexpressed MSC-EXs (MSC-EXsmiR-132-3p). The effects of EXs on hypoxia/reoxygenation (H/R)-injured ECs in ROS generation, apoptosis, and barrier function were analyzed. The levels of RASA1, Ras, phosphorylations of PI3K, Akt and endothelial nitric oxide synthesis (eNOS), and tight junction proteins (Claudin-5 and ZO-1) were measured. Ras and PI3K inhibitors were used for pathway analysis. In transient middle cerebral artery occlusion (tMCAO) mouse model, the effects of MSC-EXs on the cerebral vascular ROS production and apoptosis, cerebral vascular density (cMVD), Evans blue extravasation, brain water content, neurological deficit score (NDS), and infarct volume were determined. MSC-EXs could deliver their carried miR-132-3p into target ECs, which functionally downregulated the target protein RASA1, while upregulated the expression of Ras and the downstream PI3K phosphorylation. Compared to MSC-EXs, MSC-EXsmiR-132-3p were more effective in decreasing ROS production, apoptosis, and tight junction disruption in H/R-injured ECs. These effects were associated with increased levels of phosphorylated Akt and eNOS, which could be abolished by PI3K inhibitor (LY294002) or Ras inhibitor (NSC 23766). In the tMCAO mouse model, the infusion of MSC-EXsmiR-132-3p was more effective than MSC-EXs in reducing cerebral vascular ROS production, BBB dysfunction, and brain injury. Our results suggest that miR-132-3p promotes the beneficial effects of MSC-EXs on brain ischemic injury through protecting cerebral EC functions.

中文翻译：

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miR-132-3p 引发增强间充质基质细胞衍生的外泌体对改善脑缺血性损伤的作用。

间充质基质细胞衍生的外泌体（MSC-EXs）可以通过转移所含的微小RNA（miRs）对受体细胞发挥保护作用，而miR-132-3p是血管生成miRs之一。然而，MSC-EXs和miR-132-3p的组合是否对缺血性脑血管疾病有更好的疗效尚不清楚。用加扰器对照或 miR-132-3p 模拟物转染的小鼠 MSCs 用于产生 MSC-EXs 和 miR-132-3p 过表达的 MSC-EXs (MSC-EXsmiR-132-3p)。分析了 EXs 对缺氧/复氧 (H/R) 损伤的 ECs 在 ROS 生成、细胞凋亡和屏障功能方面的影响。测量了 RASA1、Ras、PI3K、Akt 和内皮一氧化氮合成 (eNOS) 以及紧密连接蛋白 (Claudin-5 和 ZO-1) 的磷酸化水平。Ras 和 PI3K 抑制剂用于通路分析。在短暂的大脑中动脉闭塞 (tMCAO) 小鼠模型中，MSC-EXs 对脑血管 ROS 产生和凋亡、脑血管密度 (cMVD)、埃文斯蓝外渗、脑含水量、神经功能缺损评分 (NDS) 和确定梗死体积。MSC-EXs 可以将其携带的 miR-132-3p 递送到靶 ECs 中，从而在功能上下调靶蛋白 RASA1，同时上调 Ras 的表达和下游 PI3K 磷酸化。与 MSC-EXs 相比，MSC-EXsmiR-132-3p 在减少 H/R 损伤 ECs 中的 ROS 产生、细胞凋亡和紧密连接破坏方面更有效。这些影响与磷酸化 Akt 和 eNOS 水平升高有关，而这可以被 PI3K 抑制剂 (LY294002) 或 Ras 抑制剂 (NSC 23766) 消除。在 tMCAO 小鼠模型中，MSC-EXsmiR-132-3p 的输注在减少脑血管 ROS 产生、BBB 功能障碍和脑损伤方面比 MSC-EX 更有效。我们的研究结果表明，miR-132-3p 通过保护脑 EC 功能促进 MSC-EXs 对脑缺血性损伤的有益作用。