Cerebral ischemia-reperfusion (I/R) injury, the most common form of stroke, has high mortality and often brings persistent and serious brain dysfunction among survivors. Administration of adipose-derived mesenchymal stem cells (ASCs) has been suggested to alleviate the I/R brain injury, but the mechanism remains uncharacterized. Here, we aimed at investigating the mechanism of ASCs and their extracellular vesicles (EVs) in the repair of or protection from I/R injury. We established the middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation/reperfusion (OGD/RP) neuron model. ASCs or ASC-derived EVs (ASC-EVs) were co-cultured with neurons. RT-qPCR and Western blot analyses determined microRNA (miRNA)-22-3p, BMP2, BMF, and KDM6B expression in neurons upon treatment with ASC-EVs. Bioinformatics analysis predicted the binding between miR-22-3p and KDM6B. Using gain- and loss-of-function methods, we tested the impact of these molecules on I/R injury in vivo and in vitro. Treatment with ASCs and ASC-derived EVs significantly alleviated the I/R brain injury in vivo, elevated neuron viability in vitro, and decreased apoptosis. Interestingly, miR-22-3p was upregulated in ASC-EVs, and treatment with EV-miR-22-3p inhibitor led to increased apoptosis and decreased neuronal. Of note, miR-22-3p bound to and inhibited KDM6B, as demonstrated by dual-luciferase reporter gene assay and Western blot assay. Overexpression of KDM6B enhanced apoptosis of neurons in the OGD/RP model, and KDM6B bound to BMB2 and promoted its expression by binding to BMP2. Silencing of BMF reduced infarct volume and apoptosis in the stroke model. Results support a conclusion that ASC-EV-derived miR-22-3p could alleviate brain ischemic injury by inhibiting KDM6B-mediated effects on the BMP2/BMF axis. These findings compelling indicate a novel treatment strategy for cerebral ischemic injury.

中文翻译：

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外泌体microRNA-22-3p通过调节KDM6B / BMP2 / BMF轴减轻脑缺血性损伤

脑缺血-再灌注（I / R）损伤是中风的最常见形式，死亡率很高，并且常常在幸存者中引起持续且严重的脑功能障碍。已建议给予脂肪间充质干细胞（ASCs）来减轻I / R脑损伤，但该机制尚无定论。在这里，我们旨在研究ASC及其细胞外囊泡（EV）修复或保护I / R损伤的机制。我们建立了大脑中动脉闭塞（MCAO）模型和氧葡萄糖剥夺/再灌注（OGD / RP）神经元模型。ASC或ASC衍生的EV（ASC-EV）与神经元共培养。RT-qPCR和Western印迹分析确定了ASC-EV治疗后神经元中的microRNA（miRNA）-22-3p，BMP2，BMF和KDM6B表达。生物信息学分析预测了miR-22-3p和KDM6B之间的结合。使用功能获得和丧失功能的方法，我们在体内和体外测试了这些分子对I / R损伤的影响。用ASC和ASC衍生的EV进行治疗可显着减轻体内I / R脑损伤，体外神经元活力增强和凋亡减少。有趣的是，ASC-EVs中miR-22-3p上调，用EV-miR-22-3p抑制剂治疗导致凋亡增加和神经元减少。值得注意的是，miR-22-3p结合并抑制了KDM6B，如双萤光素酶报道基因检测和Western blot检测所证实。KDM6B的过表达增强了OGD / RP模型中神经元的凋亡，并且KDM6B与BMB2结合并通过与BMP2结合而促进其表达。BMF沉默可减少中风模型中的梗塞体积和细胞凋亡。结果支持一个结论，即ASC-EV衍生的miR-22-3p可通过抑制KDM6B介导的对BMP2 / BMF轴的作用来减轻脑缺血性损伤。这些令人信服的发现表明了一种针对脑缺血性损伤的新颖治疗策略。