Rosmarinic acid alleviates radiation-induced pulmonary fibrosis by downregulating tRNA N7-Methylguanosine modification-regulated fibroblast to myofibroblast transition through the exosomes pathway [tRNA-Seq]. Rosmarinic acid alleviates radiation-induced pulmonary fibrosis by downregulating tRNA N7-Methylguanosine modification-regulated fibroblast to myofibroblast transition through the exosomes pathway [tRNA-Seq]

Radiation-induced pulmonary fibrosis (RIPF) is a common complication after radiotherapy in thoracic cancer patients, and there is a lack of effective treatment methods. The aim of this study was to explore the protective effect of rosmarinic acid (RA) on RIPF in mice as well as the underlying mechanism.We found that RA exerted an antifibrotic effect on lung tissues of RIPF mouse models and inhibited the progression of FMT through exosomes derived from lung epithelial cells. Mechanistically, RA reduced the transcription and translation efficiency of SPHK1 in lung fibroblasts by decreasing the tRNA N7-methylguanosine modification and downregulating the expression of tRNAs in lung epithelial cell-derived exosomes after irradiation, as well as inhibiting the interaction of SPHK1 with the NAT10 protein in fibroblasts. Furthermore, exosomes derived from irradiated lung epithelial cells after RA intervention decreased the acetylation and cytoplasmic translocation of PFKFB3, suppressing the FMT process triggered by glycolysis, and ultimately decelerating the progression of RIPF. Overall design: In this study, m7G-tRNA-seq and tRNA-seq analysis were conducted to identify the m7G modified tRNAs. Western blotting, immunohistochemistry, northwestern blotting, northern blotting, immunofluorescence, wound-healing assays and Edu experiments were performed to explore the molecular mechanism by which RA regulated fibroblast to myofibroblast transformation (FMT) by affecting the exosomes of lung epithelial cells. Ribo-seq and mRNA-seq analysis were used to explore the underlying target mRNA. Seahorse assays and immunoprecipitation were carried out to elucidate the effects of RA on glycolysis and FMT processes via the regulation of PFKFB3 acetylation.

辐射诱导性肺纤维化（radiation-induced pulmonary fibrosis, RIPF）是胸癌患者放疗后常见并发症，目前尚无有效治疗手段。本研究旨在探讨迷迭香酸（rosmarinic acid, RA）对小鼠RIPF的保护作用及其潜在分子机制。研究发现，RA可对RIPF小鼠模型的肺组织发挥抗纤维化作用，并通过肺上皮细胞衍生的外泌体抑制成纤维细胞向肌成纤维细胞转化（fibroblast to myofibroblast transformation, FMT）的进程。机制层面，照射后RA可通过降低肺上皮细胞衍生外泌体中的tRNA N7-甲基鸟苷修饰、下调tRNA表达，降低肺成纤维细胞中SPHK1的转录与翻译效率，同时抑制成纤维细胞内SPHK1与NAT10蛋白的相互作用。此外，经RA干预后的照射肺上皮细胞衍生外泌体，可降低PFKFB3的乙酰化修饰与胞质转位，抑制糖酵解触发的FMT过程，最终延缓RIPF的疾病进展。整体实验设计：本研究通过m7G-tRNA测序（m7G-tRNA-seq）与tRNA测序（tRNA-seq）分析鉴定m7G修饰的tRNA；采用蛋白质印迹法（Western blotting）、免疫组织化学（immunohistochemistry）、西北印迹法（northwestern blotting）、Northern印迹法（northern blotting）、免疫荧光（immunofluorescence）、划痕愈合实验（wound-healing assays）与Edu实验（Edu experiments），探究RA通过调控肺上皮细胞外泌体进而影响FMT的分子机制；利用核糖体测序（Ribo-seq）与信使RNA测序（mRNA-seq）筛选潜在靶mRNA；采用海马细胞外通量分析仪实验（Seahorse assays）与免疫沉淀（immunoprecipitation），阐明RA通过调控PFKFB3乙酰化影响糖酵解与FMT过程的具体作用。