Department of Critical Care Medicine, The Affiliated Hospital of Qingdao University, Qingdao 266003, PR China.
Department of Critical Care Medicine, The Affiliated Hospital of Qingdao University, Qingdao 266003, PR China.
Int Immunopharmacol. 2021 Jun;95:107408. doi: 10.1016/j.intimp.2021.107408. Epub 2021 Apr 27.
Sepsis, as a disease affecting the microcirculation and tissue perfusion, results in tissue hypoxia and multiple organ dysfunctions. Bone mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) have been demonstrated to transfer trivial molecules (proteins/peptides, mRNA, microRNA and lipids) to alleviate sepsis. We sought to define the function of microRNA (miR)-17 carried in BMSC-EVs in sepsis.
The purity of the extracted BMSCs was identified and confirmed by detection of the surface markers by flow cytometry, followed by osteoblastic, adipogenic, and chondrocyte differentiation experiments. Subsequently, EVs were collected from the medium of BMSCs. The uptake of PKH-67-labeled BMSC-EVs or EVs carrying cy3-miR-17 by RAW264.7 cells was observed under laser confocal microscopy. Furthermore, a series of gain- and loss-of-function approaches were conducted to test the effects of LPS, miR-17 and BRD4 on the inflammatory factors (IL-1β, IL-6 and TNF-α), number of M1 macrophages and M2 macrophages, inflammatory-related signal pathway factors (EZH2, c-MYC and TRAIL), macrophage proliferation, and apoptosis in sepsis. The survival rates were measured in vivo.
BMSC-EVs was internalized by the RAW264.7 cells. BDR4 was verified as a target of miR-17, while the expression pattern of miR-17 was upregulated in BMSC-EVs. MiR-17 carried by BMSC-EVs inhibited LPS-induced inflammation and apoptosis of RAW264.7 cells, but improved the viability of RAW264.7 cells. Next, in vitro experiments supported that miR-17 inhibited LPS-induced inflammation in RAW264.7 cells through BRD4/EZH2/TRAIL axis. BRD4 overexpression reversed the effects of miR-17. Moreover, the therapeutic function of BMSC-EVs carried miR-17 was verified by in vivo experiments.
MiR-17 derived from BMSCs-EVs regulates BRD4-mediated EZH2/TRAIL axis to essentially inhibit LPS-induced macrophages inflammation.
脓毒症作为一种影响微循环和组织灌注的疾病，会导致组织缺氧和多器官功能障碍。骨髓间充质干细胞（BMSC）衍生的细胞外囊泡（EVs）已被证明可以传递微小分子（蛋白质/肽、mRNA、microRNA 和脂质）来缓解脓毒症。我们试图确定 BMSC-EVs 中携带的微小 RNA（miR）-17 的功能。
通过流式细胞术检测表面标志物来鉴定和确认提取的 BMSC 的纯度，然后进行成骨、成脂和成软骨分化实验。随后，从 BMSC 培养基中收集 EVs。使用激光共聚焦显微镜观察 PKH-67 标记的 BMSC-EVs 或携带 cy3-miR-17 的 EVs 被 RAW264.7 细胞摄取的情况。此外，通过一系列增益和缺失功能的方法来测试 LPS、miR-17 和 BRD4 对炎症因子（IL-1β、IL-6 和 TNF-α）、M1 巨噬细胞和 M2 巨噬细胞数量、炎症相关信号通路因子（EZH2、c-MYC 和 TRAIL）、巨噬细胞增殖和凋亡的影响在脓毒症中的作用。在体内测量存活率。
BMSC-EVs 被 RAW264.7 细胞内化。BDR4 被验证为 miR-17 的靶标，而 miR-17 的表达模式在 BMSC-EVs 中上调。BMSC-EVs 携带的 miR-17 抑制了 LPS 诱导的 RAW264.7 细胞炎症和凋亡，但提高了 RAW264.7 细胞的活力。接下来，体外实验支持 miR-17 通过 BRD4/EZH2/TRAIL 轴抑制 LPS 诱导的 RAW264.7 细胞炎症。BRD4 过表达逆转了 miR-17 的作用。此外，体内实验验证了 BMSC-EVs 携带 miR-17 的治疗功能。
BMSC-EVs 衍生的 miR-17 通过调节 BRD4 介导的 EZH2/TRAIL 轴来调节 LPS 诱导的巨噬细胞炎症。
