间质干细胞衍生的细胞外囊泡通过 microRNA-140-5p 传递缓解蛛网膜下腔出血小鼠脑损伤中的 M1 小胶质细胞活化。

Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviate M1 Microglial Activation in Brain Injury of Mice With Subarachnoid Hemorrhage via microRNA-140-5p Delivery.

机构信息

Department of Neurosurgery, the Affiliated People's Hospital of Jiangsu University, Zhenjiang, P.R. China.

Department of Neurosurgery, Nanjing Medical University Affiliated Zhenjiang First People's Hospital, Zhenjiang, P.R. China.

出版信息

Int J Neuropsychopharmacol. 2022 Apr 19;25(4):328-338. doi: 10.1093/ijnp/pyab096.

BACKGROUND

It is documented that mesenchymal stem cells (MSCs) secrete extracellular vesicles (EVs) to modulate subarachnoid hemorrhage (SAH) development. miR-140-5p expression has been detected in MSC-derived EVs, while the mechanism of MSC-derived EVs containing miR-140-5p in SAH remains unknown. We aim to fill this void by establishing SAH mouse models and extracting MSCs and MSC-EVs.

METHODS

After ALK5 was silenced in SAH mice, neurological function was evaluated, neuron apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling with NeuN staining, and expression of serum inflammatory factors (interleukin-6, interleukin-1β, and tumor necrosis factor-α) was determined by enzyme-linked immunosorbent assay. The effect of ALK5 on NOX2 expression was assessed by western-blot analysis. Targeting the relationship between miR-140-5p and ALK5 was evaluated by dual luciferase assay. Following extraction of MSCs and MSC-EVs, EVs and miR-140-5p were labeled by PKH67 and Cy3, respectively, to identify the transferring of miR-140-5p by MSC-EVs. SAH mice were treated with EVs from miR-140-5p mimic/inhibitor-transfected MSCs to detect effects of MSC-EV-miR-140-5p on brain injury and microglial polarization.

RESULTS

ALK5 silencing increased the neurological score and reduced neuron apoptosis and neuroinflammation in SAH mice. ALK5 silencing inhibited M1 microglia activation by inactivating NOX2. ALK5 was a target gene of miR-140-5p. MSC-derived EVs contained miR-140-5p and transferred miR-140-5p into microglia. MSC-EV-delivered miR-140-3p reduced ALK5 expression to contribute to repression of brain injury and M1 microglia activation in SAH mice.

CONCLUSIONS

MSC-derived EVs transferred miR-140-5p into microglia to downregulate ALK5 and NOX2, thus inhibiting M1 microglia activation in SAH mice.

背景

有文献记载间充质干细胞（MSCs）通过分泌细胞外囊泡（EVs）来调节蛛网膜下腔出血（SAH）的发展。在 MSC 来源的 EVs 中检测到 miR-140-5p 的表达，但 MSC 来源的 EVs 中 miR-140-5p 对 SAH 的作用机制尚不清楚。我们旨在通过建立 SAH 小鼠模型并提取 MSCs 和 MSC-EVs 来填补这一空白。

方法

沉默 SAH 小鼠中的 ALK5 后，通过 TdT 介导的 dUTP-生物素缺口末端标记与 NeuN 染色检测神经元凋亡，并通过酶联免疫吸附试验测定血清炎性因子（白细胞介素-6、白细胞介素-1β 和肿瘤坏死因子-α）的表达。通过 Western-blot 分析评估 ALK5 对 NOX2 表达的影响。通过双荧光素酶报告基因实验评估 miR-140-5p 与 ALK5 之间的靶向关系。提取 MSCs 和 MSC-EVs 后，分别用 PKH67 和 Cy3 标记 EVs 和 miR-140-5p，以鉴定 MSC-EVs 转移 miR-140-5p 的情况。用 miR-140-5p 模拟物/抑制剂转染 MSC 的 EVs 处理 SAH 小鼠，以检测 MSC-EV-miR-140-5p 对脑损伤和小胶质细胞极化的影响。

结果

沉默 ALK5 可提高 SAH 小鼠的神经评分，减少神经元凋亡和神经炎症。沉默 ALK5 通过抑制 NOX2 抑制 M1 小胶质细胞的激活。ALK5 是 miR-140-5p 的靶基因。MSC 来源的 EVs 含有 miR-140-5p，并将 miR-140-5p 转移到小胶质细胞中。MSC-EV 传递的 miR-140-3p 降低了 ALK5 的表达，有助于抑制 SAH 小鼠的脑损伤和 M1 小胶质细胞的激活。