miR-146a-5p介导人脐带间充质干细胞外泌体改善芥子气肺损伤的作用与机制

Sulfur mustard (SM) is one of the major components in chemical weapons abandoned by Japan in China 70 years ago, which has been endangering the public health and the environment of the nation as well. It is also regarded as the most possible choice for potential chemical terrorism attack. Because of the complicated mechanism of toxicity, which has not yet been elucidated clearly, there are no specific antidotes for the treatment. In the preliminary experiments, we found that exosomes from human umbilical cord mesenchymal stem cells (uMSC-Exo) could significantly improve the survival rate on a mice model exposed to SM (P<0.01). In the same model, uMSC-Exo showed mesenchymal stem cells-like effects in ameliorating the pulmonary injuries induced by SM, as evidenced by attenuated histological alterations, decreased wet/dry weight ratio of the lungs, reduced total proteins of BALF, and down regulation of proinflammatory factors. According to high-throughput sequencing analysis of transcriptome, bioinformatics analysis, and the subsequent experimental verification in vitro, the microRNA miR-146a-5p seems to play an important role in the process. uMSC-Exo overexpressing miR-146a-5p could markedly improve the cell viability, inhibit apoptosis and increase the level of tight junction proteins as ZO-1 et al.; while the inhibition the expression of miR-146a-5p resulted in the disappearance of these effects. Therefore, we speculate that uMSC-Exo's mitigation effects on pulmonary injuries induced by SM are mediated by miR-146a-5p. We would continue to deeply investigate the efficacy of uMSC-Exo on SM-induced pulmonary injuries and explore the possible molecular mechanism, focusing on the mechanism of anti-inflammation, anti-apoptosis mechanism and regulation of cell junction. Target molecules for miR-146a-5p in the regulation process will be elucidated. These results will provide a strong foundation for the future miR-146a-5p application in SM-induced poisoning, which is of significance for national security and public health.

芥子气(sulfur mustard,SM)是日遗化武中的主要毒剂,也是最可能被用于化学恐怖袭击的毒剂。目前中毒机制不明,无特效药物。我们前期发现脐带间充质干细胞外泌体(uMSC-Exo)可显著提高SM中毒小鼠的生存率,减轻SM所致肺组织的炎性浸润、降低肺组织湿干重比等重要指标;组学数据与生物信息学分析发现miR-146a-5p在其中可能起关键作用,进一步体外验证表明,过表达miR-146a-5p的外泌体能明显提高细胞活力、抑制凋亡、促进紧密连接蛋白ZO-1等的表达,而抑制它后则失去这些效果。故我们推测,miR-146a-5p介导了uMSC-Exo对SM肺损伤的改善作用。围绕该假设,本项目将进一步从抗炎、抗凋亡及上皮屏障功能修复方面深入探讨uMSC-Exo通过miR-146a-5p改善SM肺损伤的作用与机制,明确可能的靶分子,为uMSC-Exo用于治疗SM中毒打下基础。

芥子气（sulfur mustard，SM）是外军装备的主要毒剂，中毒机制不明，无特效药物。干细胞及外泌体技术为SM损伤治疗带来了新方向。本项目通过体内外实验系统研究了人脐带间充质干细胞外泌体（hucMSC-EVs）在SM肺损伤中的治疗效果，筛选鉴定出其发挥作用的关键分子miR-146a-5p，解析了miR-146a-5p介导hucMSC-EVs通过抑制炎症与凋亡、促进肺上皮损伤修复的作用机制。主要研究结果如下：（1）hucMSC-EVs可有效改善SM诱发的肺损伤。其可显著提高SM中毒小鼠生存率，减轻SM所致肺组织的炎性浸润、降低肺组织湿干重比等重要肺损伤指标。（2）根据miRNA成分分析、转录组测序分析，结合生物信息学与细胞活力实验，筛选鉴定出miR-146a-5p是hucMSC-EVs发挥作用的关键分子。（3）miR-146a-5p介导hucMSC-EVs作用于TRAF6发挥抗炎作用。在SM损伤小鼠模型和小鼠巨噬细胞染毒模型中，miR-146a-5p过表达可显著降低TNF-α、IL-1β等促炎因子表达，升高抗炎因子IL-10，减轻炎症反应。作用靶点分析表明，miR-146a-5p可直接结合TRAF6，介导其下调，通过抑制TLR4/NF-κB信号通路减轻SM肺损伤中的炎症反应。（4）miR-146a-5p介导hucMSC-EVs作用于细胞凋亡增强核酸酶（AEN）发挥凋亡抑制作用。过表达miR-146a-5p的hucMSC-EVs可显著抑制SM肺上皮细胞凋亡。通过转录组学分析，结合双荧光素酶实验鉴定出miR-146a-5p直接作用的新靶点AEN。hucMSC-EVs通过miR-146a-5p靶向AEN，从而抑制caspase 3激活，降低线粒体膜电位，抑制SM诱导的细胞凋亡。（5）miR-146a-5p介导hucMSC-Evs促进肺上皮细胞屏障损伤修复。hucMSC-EVs中过表达miR-146a-5p可促进紧密连接蛋白claudin-1、ZO-1、occludin等表达量显著升高，促进紧密连接结构形成，从而提升肺上皮细胞屏障功能修复。.我们首次系统研究了hucMSC-Evs治疗SM致急性肺损伤的效应，探讨了机制，并鉴定出新靶点，为hucMSC-EVs未来在SM治疗领域的应用打下了坚实基础，为理解SM中毒机制提供了新思路，对国家公共安全和民众健康具有重要意义。