Activation of pro-inflammatory microglia is an important mechanism of the cerebral ischemia-reperfusion (I/R)-induced neuronal injury and dysfunction. Mesenchymal stem cells (MSCs) together with their paracrine factors demonstrated curative potential in immune disorders and inflammatory diseases, as well as in ischemic diseases. However, it remains unclear whether conditioned medium from MSCs could effectively regulate the activation and polarization of microglia exposed to I/R stimulation. In this study, we investigated the effects of conditioned medium from bone marrow MSCs (BMSCs-CM) on I/R-stimulated microglia and the potential mechanism involved, as well as the way to obtain more effective BMSCs-CM. First, cell model of oxygen-glucose deprivation/reoxygenation (OGD/R) was established in microglia to mimic the I/R. BMSCs-CM from different culture conditions (normoxic: 21% O2; hypoxic: 1% O2; hypoxia preconditioning: preconditioning with 1% O2 for 24 h) was used to treat the microglia. Our results showed that BMSCs-CM effectively promoted the survival and alleviated the injury of microglia. Moreover, in microglia exposed to OGD/R, BMSCs-CM inhibited significantly the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), CD86 and inducible nitric oxide synthase, whereas upregulated the levels of anti-inflammatory cytokine (IL-10), CD206 and Arginase-1. These results suggested that BMSCs-CM promoted the polarization of anti-inflammatory microglia. In particular, BMSCs-CM from cultures with hypoxia preconditioning was more effective in alleviating cell injury and promoting anti-inflammatory microglia polarization than BMSCs-CM from normoxic cultures and from hypoxic cultures. Furthermore, inhibition of exosomes secretion could largely mitigate these effects of BMSCs-CM. In conclusion, our results suggested that hypoxia preconditioning of BMSCs could enhance the efficacy of BMSCs-CM in alleviating OGD/R-induced injury and in promoting the anti-inflammatory polarization of microglia, and these beneficial effects of BMSCs-CM owed substantially to exosomes.

中文翻译：

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缺氧预处理增强了间充质干细胞衍生的条件培养基在缺血/再灌注中将小胶质细胞向抗炎极化转变的功效。

促炎性小胶质细胞的激活是脑缺血再灌注 (I/R) 诱导的神经元损伤和功能障碍的重要机制。间充质干细胞 (MSC) 及其旁分泌因子在免疫疾病和炎症性疾病以及缺血性疾病中显示出治疗潜力。然而，尚不清楚来自 MSCs 的条件培养基是否能有效调节暴露于 I/R 刺激的小胶质细胞的激活和极化。在本研究中，我们研究了来自骨髓间充质干细胞 (BMSCs-CM) 的条件培养基对 I/R 刺激的小胶质细胞的影响及其潜在机制，以及获得更有效 BMSCs-CM 的方法。首先，在小胶质细胞中建立氧-葡萄糖剥夺/复氧（OGD/R）细胞模型以模拟 I/R。来自不同培养条件（常氧：21% O2；缺氧：1% O2；缺氧预处理：用 1% O2 预处理 24 小时）的 BMSCs-CM 用于治疗小胶质细胞。我们的结果表明，BMSCs-CM 有效地促进了小胶质细胞的存活并减轻了其损伤。此外，在暴露于 OGD/R 的小胶质细胞中，BMSCs-CM 显着抑制促炎细胞因子（TNF-α、IL-1β 和 IL-6）、CD86 和诱导型一氧化氮合酶的表达，而上调抗-炎性细胞因子 (IL-10)、CD206 和精氨酸酶-1。这些结果表明 BMSCs-CM 促进了抗炎小胶质细胞的极化。特别是，与来自常氧培养物和缺氧培养物的 BMSCs-CM 相比，来自缺氧预处理培养物的 BMSCs-CM 在减轻细胞损伤和促进抗炎小胶质细胞极化方面更有效。此外，抑制外泌体分泌可以在很大程度上减轻 BMSCs-CM 的这些影响。总之，我们的结果表明，BMSCs 的缺氧预处理可以增强 BMSCs-CM 在减轻 OGD/R 诱导的损伤和促进小胶质细胞的抗炎极化方面的功效，而 BMSCs-CM 的这些有益作用主要归功于外泌体.