骨髓间充质干细胞来源的外泌体 miR-381 通过抑制 YTHDF1 表达激活 Treg 分化缓解肺缺血再灌注损伤。

Bone marrow mesenchymal stem cells-derived exosomal miR-381 alleviates lung ischemia-reperfusion injury by activating Treg differentiation through inhibiting YTHDF1 expression.

机构信息

Departments of Anesthesiology, The Third Affiliated Hospital of Soochow University, Changzhou 213003, Jiangsu, China.

Department of Thoracic Surgery, The Third Affiliated Hospital of Soochow University, Changzhou 213003, Jiangsu, China.

出版信息

Cell Signal. 2024 Dec;124:111440. doi: 10.1016/j.cellsig.2024.111440. Epub 2024 Sep 30.

DOI:10.1016/j.cellsig.2024.111440

PMID:39357613

Abstract

AIM

Our study aimed to investigate whether BMSCs-derived exosomal miR-381 promotes Treg cell differentiation in lung ischemia-reperfusion injury (LIRI), and the underlying mechanism.

METHODS

The in vitro and in vivo models of LIRI were established by hypoxia/reoxygenation (H/R) treatment and lung ischemia/reperfusion (I/R) surgery, respectively. BMSCs-derived exosomes were isolated and identified by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Cell viability, proliferation, and apoptosis were assessed by CCK-8, EdU, and flow cytometry assay, respectively. IL-18 secretion level in lung microvascular endothelial cells (LMECs) and lung tissue homogenate was examined by ELISA. Treg cell differentiation was determined using flow cytometry. The relationships between miR-381, YTHDF1, and IL-18 were investigated using dual-luciferase reporter gene, RIP, and/or RNA pull-down assays. MeRIP assay was employed to determine mA modification of IL-18 mRNA in LMECs. The ubiquitination level of Foxp3 protein in CD4 T cells was analyzed by Co-IP assay.

RESULTS

BMSCs-derived exosomes reduced LMECs injury and increased Treg cell differentiation in LIRI, whereas miR-381 inhibition in BMSCs weakened these impacts. Mechanistically, miR-381 inhibited IL-18 translation in LMECs by inhibiting YTHDF1 expression via binding to its 3'-UTR. As expected, YTHDF1 overexpression in LMECs abolished the effects of miR-381-overexpressed exosomes on LMECs injury and Treg cell differentiation. Moreover, LMECs-secreted IL-18 inhibited Treg cell differentiation by promoting the ubiquitination degradation of Foxp3 protein.

CONCLUSION

BMSCs-derived exosomal miR-381 suppressed IL-18 translation in LMECs through binding to YTHDF1 3'-UTR, thus suppressing the ubiquitination degradation of Foxp3 in CD4 T cells, which promoted Treg cell differentiation and mitigated LIRI development.

摘要

目的

本研究旨在探讨骨髓间充质干细胞衍生的外泌体 miR-381 是否促进肺缺血再灌注损伤（LIRI）中调节性 T 细胞（Treg）的分化，并探讨其潜在机制。

方法

通过缺氧/复氧（H/R）处理和肺缺血/再灌注（I/R）手术分别建立体外和体内 LIRI 模型。通过 Western blot、纳米颗粒跟踪分析和透射电子显微镜鉴定骨髓间充质干细胞衍生的外泌体。通过 CCK-8、EdU 和流式细胞术分别评估细胞活力、增殖和凋亡。通过 ELISA 检测肺微血管内皮细胞（LMECs）和肺组织匀浆中 IL-18 的分泌水平。通过流式细胞术测定 Treg 细胞分化。通过双荧光素酶报告基因、RIP 和/或 RNA 下拉测定研究 miR-381、YTHDF1 和 IL-18 之间的关系。采用 MeRIP 测定法确定 LMECs 中 IL-18 mRNA 的 mA 修饰。通过 Co-IP 测定分析 CD4 T 细胞中 Foxp3 蛋白的泛素化水平。

结果

骨髓间充质干细胞衍生的外泌体减轻了 LIRI 中的 LMECs 损伤并增加了 Treg 细胞分化，而骨髓间充质干细胞中 miR-381 的抑制作用削弱了这些影响。机制上，miR-381 通过结合其 3'-UTR 抑制 YTHDF1 的表达，从而抑制 LMECs 中 IL-18 的翻译。正如预期的那样，LMECs 中 YTHDF1 的过表达消除了 miR-381 过表达外泌体对 LMECs 损伤和 Treg 细胞分化的影响。此外，LMECs 分泌的 IL-18 通过促进 Foxp3 蛋白的泛素化降解抑制了 Treg 细胞的分化。