Alveolar macrophage exosomal tRNA-derived fragments tRF-22-8BWS7K092 promote ferroptosis to involve in acute lung injury

Macrophage exosomes play a demonstrative role in acute lung injury (ALI) by mediating cellu-lar communication. Transfer RNA-derived fragments (tRFs) possess potential functions in mul-tiple diseases through ferroptosis. The present study aims to reveal the role of macrophage exo-somal tRFs in ALI and to identify the relationship to ferroptosis. ALI mice model and cell model were established by lipopolysaccharide (LPS) induction. RNA sequencing was performed to identify the tRFs profile in exosomes of ALI mice. After interfering with the expression of can-didate tRFs in macrophages or lung epithelial cells (MLE-12), the effect of oxidative stress and expression of ferroptosis-related proteins were detected. Here, LPS induced lung injury was characterized by alveolar walls and interstitial spaces thickening, hemorrhage, and inflamma-tion. RNA sequencing identified 4 up- and 10 down-regulated differentially expressed tRFs (DEtRFs) in the exosomes of ALI mice. The GO function of exosomal DEtRFs enriched in gene regulation related terms such as transcription regulation and protein binding. Hippo signaling pathway was the most significantly enriched KEGG pathways for DEtRFs. LPS-induced macro-phage exosome (LPS-exo) inhibited the viability and the expression of GPX4 and FTH1, and en-hanced the oxidative stress in MLE-12 cells. Ferroptosis inhibitor reversed the inhibition of LPS-exo on cell viability. Also, tRF-22-8BWS7K092 inhibitor rescued the above effect of LPS-exo on MLE-12 cells. Besides, tRF-22-8BWS7K092 activates Hippo signaling pathway by binding Wnt5B, resulting in ferroptosis in MLE-12 cells. In conclusion, we obtained and characterized the exosomal tRFs expression profile in ALI mice. LPS-induced macrophage exosomal tRF-22-8BWS7K092 involved in the process of ALI by promoting ferroptosis. Our study provides a new molecular target for the treatment of ALI.

巨噬细胞外泌体通过介导细胞通讯，在急性肺损伤（ALI）中发挥关键调控作用。转运RNA衍生片段（tRFs）可通过铁死亡途径参与多种疾病的病理过程，具备潜在调控功能。本研究旨在揭示巨噬细胞外泌体tRFs在急性肺损伤中的作用，并明确其与铁死亡的关联。本研究采用脂多糖（LPS）诱导构建急性肺损伤小鼠模型与细胞模型。通过RNA测序鉴定急性肺损伤小鼠外泌体中的tRFs表达谱。在巨噬细胞或肺上皮细胞（MLE-12）中干扰候选tRFs的表达后，检测氧化应激水平及铁死亡相关蛋白的表达情况。结果显示，脂多糖诱导的肺损伤以肺泡壁及间质间隙增厚、出血与炎症浸润为主要特征。RNA测序共鉴定出急性肺损伤小鼠外泌体中4个上调、10个下调的差异表达tRFs（DEtRFs）。外泌体差异表达tRFs的基因本体（GO）功能富集于转录调控、蛋白质结合等基因调控相关条目。差异表达tRFs最显著富集的京都基因与基因组百科全书（KEGG）通路为Hippo信号通路。脂多糖诱导的巨噬细胞外泌体（LPS-exo）可抑制肺上皮细胞MLE-12的活性，下调谷胱甘肽过氧化物酶4（GPX4）与铁蛋白重链1（FTH1）的表达，并增强细胞氧化应激。铁死亡抑制剂可逆转脂多糖诱导的巨噬细胞外泌体对细胞活性的抑制作用。同时，tRF-22-8BWS7K092抑制剂可挽救脂多糖诱导的巨噬细胞外泌体对MLE-12细胞的上述效应。此外，tRF-22-8BWS7K092可通过结合Wnt5B激活Hippo信号通路，进而诱导MLE-12细胞发生铁死亡。综上，本研究获取并明确了急性肺损伤小鼠外泌体的tRFs表达谱。脂多糖诱导的巨噬细胞外泌体tRF-22-8BWS7K092通过促进铁死亡参与急性肺损伤的病理进程。本研究为急性肺损伤的临床治疗提供了全新的分子靶点。