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

Rosmarinic acid alleviates radiation-induced pulmonary fibrosis by downregulating tRNA N7-Methylguanosine modification-regulated fibroblast to myofibroblast transition through the exosomes pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rosmarinic acid alleviates radiation-induced pulmonary fibrosis by downregulating tRNA N7-Methylguanosine modification-regulated fibroblast to myofibroblast transition through the exosomes pathway Tingting Zhang, Jinglin Mi, Zhechen Ouyang, Xinling Qin, Yiru Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3744363/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background 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. Results 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. Conclusions These findings suggest RA as a potential therapeutic agent for RIPF. Rosmarinic acid Exosomes tRNA N7-methylguanosine (m7G) Radiation-induced pulmonary fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Thoracic cancer is one of the most common malignancies. Patients with locally advanced chest tumors who are unable to undergo surgery or have high-risk factors after surgery often require radiation therapy. However, radiation-induced lung injury (RILI) and fibrosis are important factors that limit the dose and efficacy of radiotherapy. The early manifestation of RILI is radiation-induced pneumonia, and approximately 9%-30% of patients [ 1 ] gradually developed radiation-induced pulmonary fibrosis (RIPF) after 6 months. RIPF is characterized by the irreversible destruction of normal lung tissue structure and the deterioration of lung function. Although precision radiotherapy techniques, such as stereotactic body radiationtherapy and intensity modulated radiotherapy, have been widely applied in clinical practice nowadays, the incidence of RIPF remains high, and there is currently a lack of effective treatment plans. Therefore, finding effective drugs that are associated with low toxicity is crucial for the treatment of RIPF. In recent years, the use of traditional Chinese medicine in the prevention and treatment of pulmonary fibrosis has received considerable attention. Liu et al. [ 2 ] discovered that curcumin inhibited TGF-β2-induced lung fibroblast to myofibroblast differentiation and suppressed pulmonary fibrosis by reducing the activity of MMP-9. Emodin inhibited not only the expression of α-SMA, collagen IV, and fibronectin in human embryo lung fibroblasts exposed to TGF-β1 but also the activation of Smad2/3 and STAT3, thus blocking the differentiation of myofibroblasts and the deposition of extracellular matrix [ 3 ]. Rosmarinic acid (RA) is a polyphenolic compound that is isolated from the rosemary plant, which belongs to the Labiatae family [ 4 ]. RA exerts obvious anti-inflammatory and anti-tissue fibrosis effects on diseases such as enteritis, osteoarthritis, atopic dermatitis, and mastitis [ 5 ]. Hsieh et al. found that RA treatment reduced the levels of α-SMA, collagen I, and fibronectin in NRK-52E kidney cells and decreased renal interstitial fibrosis by inhibiting phosphorylated-AKT mediated epithelial-mesenchymal transition in vitro and in vivo [ 6 ]. Another study showed that RA regulated the AMPKα/Smad3 signaling axis to reduce the cardiac fibrosis that was caused by long-term arterial pressure overload and delay cardiac remodeling [ 7 ]. Similarly, two previous studies reported by our research group revealed that RA exerted a protective effect on the radiation-induced tissue inflammation and fibrosis. On the one hand, RA downregulated the expression of TNF-α and IL-6 by inhibiting the N-terminal kinase activity of p53/Jun, reduced the oxidative stress response and the apoptosis of parotid gland cells, and ultimately alleviated the radiation-induced parotid gland fibrosis [ 8 ]. On the other hand, RA reduced RhoA/ROCK and NF-κB phosphorylation levels by inhibiting MYPT1 expression, thereby suppressing the progression of RIPF in rats [ 9 ]. However, the mechanism of RA slowing radiation-induced tissue fibrosis still needs to be further explored. Fibroblast to myofibroblast transition (FMT) was an important characteristic of fibrosis after RILI [ 10 ]. Pulmonary fibroblasts can be transformed into myofibroblasts after radiation [ 11 ]. Myofibroblasts is the main source of extracellular matrix, which promotes the formation and development of RIPF [ 12 ]. Communication signals including chemokines, exosomes, and secretory proteins are important mechanisms for other types of cells in lung tissue to regulate the redifferentiation of lung fibroblasts [ 13 , 14 ]. In the present study, we found that RA alleviated the development of RIPF in mice by inhibiting the progression of FMT. Further exploration revealed that radiation-exposed epithelial cells can promote the occurrence of FMT through the exosomes pathway, while RA can counteract the effect of irradiated lung epithelial cells on lung fibroblasts. Mechanically, RA suppressed the METTL1/WDR4-mediated transfer RNA (tRNA) N7-methylguanosine (m7G) modification in exosomes of radiation-exposed lung epithelial cell, thereby lightening the translation efficiency and protein expression of the target gene SPHK1 regulated by tRNA m7G modification in lung fibroblasts through the exosomes pathway. Moreover, RA suppressed the acetylation of PFKFB3 in the nucleus and decreased the level of phosphorylated PFKFB3 in the cytoplasm, ultimately reducing FMT triggered by glycolysis in lung fibroblasts. Our findings provide the novel therapeutic targets for RIPF as well as insights into its molecular pathophysiology. Methods Animals and Treatments Eighteen male C57BL/6 mice (7 weeks of age) were obtained from the SipeiFu Biotechnology Co., Ltd (Beijing, China). To establish a RIPF model, mice were randomly divided into three groups: the Control (Ctrl) group, irradiation (IR) group and rosmarinic acid plus irradiation (RA + IR) group. The chest regions of mice in the IR group and RA + IR group received a single dose of 15 Gy (Varian, California, USA), and the remaining body parts were shielded. RA was purchased from MedchemExpress (New Jersey, USA) and orally administered from 7 days (1 mg/g/day) before irradiation to 24 weeks after irradiation, while mice in the Ctrl group and IR group were administrated with saline. The animal experiments were approved by the Animal Ethics Committee of GuangXi Medical University. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted by TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions, and then the concentration and quality of the RNA were measured. Total RNA was reverse transcribed into cDNA with the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan). qPCR was conducted to evaluate mRNA levels using the SYBR Green method. The expression level of genes was normalized to that of GAPDH and quantified by the 2 −△△CT method. Western blotting Cells were lysed with RIPA lysis buffer, the BCA Protein Assay Kit was used to measure the protein concentration (Beyotime, China), and proteins were separated by SDS‒PAGE and transferred to polyvinylidene difluoride membranes. Then, 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used to block the proteins for 1 h at room temperature, and the membranes were incubated with primary antibodies (1:1,000) overnight at 4°C. The, the membranes were incubated with secondary antibodies (1:5,000) for 1 h at room temperature. Enhanced chemiluminescence reagent was used to detect the protein signals. The following antibodies were used: METTL1 Polyclonal antibody (Proteintech, 14994-1-AP), Rabbit anti-WDR4 Polyclonal Antibody (absin, abs152662), SPHK1 Polyclonal antibody (Proteintech, 10670-1-AP), NAT10 Polyclonal antibody(Proteintech; 13365-1-AP), acetyl-lysine (Affinity, DF7729), PFKFB3 Antibody (Affinity, DF12016), Phospho-PFKFB3 (Ser461) Antibody (Affinity, AF3581), Phospho-AMPK alpha (Thr172) Antibody (Affinity, AF3423), AMPK alpha Antibody (Affinity, AF6423), Actin (Proteintech, 81115-1-RR) Cell lines, transfection and intervention The mouse lung epithelial cell lines TC-1 and MLE-12 were gifted from Guangxi Key Laboratory of Immunology and Metabolism for Liver Diseases, and mouse lung fibroblasts (MLFs) were purchased from Saios (Wuhan, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin streptomycin in a 37 °C incubator with 5% CO 2 . The cells were digested with 0.25% trypsin every 2–3 days. When the cells reached approximately 80% confluence, Lipofectamine 3000 (Invitrogen, USA) was utilized to transfect siRNAs and plasmids. METTL1 and WDR4 overexpression plasmids and METTL1 and WDR4 siRNAs were generated by GenePharma (Shanghai, China). TC-1 cells and MLE-12 cells were divided into the Ctrl, IR and RA + IR group, respectively. TC-1 and MLE-12 cells were pretreated with RA (150 µM) 24h prior to irradiation in both RA groups. A single dose of 4 Gy x-rays was delivered at a rate of 400 cGy/min to TC-1 and MLE-12 cells in all the irradiation groups. Extraction of exosomes Cell supernatants were centrifuged at 10,000 ×g for 45 min at 4 °C to remove the larger vesicles. The supernatants were extracted and filtered through a 0.45 µm filter. Then, the samples were centrifuged again at 10,000 ×g and at 4 °C for 70 min and resuspended in PBS. After removing the supernatants, 100 µL of PBS was used to resuspend the samples. Some exosomes (20 µL) were used for electron microscopy analysis, some exosomes (10 µL) were used for particle size analysis, and the remaining exosomes were stored at -80 °C. Edu incorporation and staining Edu assay was performed using BeyoClick™ Edu Cell Proliferation Kit with Alexa Fluor 594 following the manufacturer’s instruction (beyotime, china). In biref, Edu working solution was added to the cells and incubated for 2 h. The cells were subsequently fixed and permeabilized, and click additive solution was added to the samples and incubated in the dark for 30 min. DAPI was utilized to label the nuclei for 3 min. Images were captured by fluorescence microscopy. Hematoxylin and eosin (HE) staining HE staining was performed using Hematoxylin-Eosin (HE) Stain Kit following the manufacturer’s instruction (solarbio, china). In biref, Paraffin sections were dewaxed with xylene and dehydrated with alcohol, hematoxylin was used to stain the nuclei, and eosin staining was used to stain the cytoplasm. After dehydration and sealing with neutral resin, the sections were examined under a microscope, and images were collected and analyzed. Masson staining Masson staining was performed using Modified Masson's Trichrome Stain Kit following the manufacturer' s instruction (solarbio, china). In biref, After paraffin sections were dewaxed, the sections were stained with Weigert's iron hematoxylin for 5 min, washed with tap water, and differentiated with 1% hydrochloric acid alcohol for several seconds. The sections were stained with Ponceau red acid fuchsin solution for 5 min and treated with phosphomolybdic acid aqueous solution for approximately 3 min. The sections were counterstained with aniline blue solution for 5 min and then treated with 1% glacial acetic acid for 1 min. Then, the sections were dehydrated and sealed for microscopic examination, and the images were collected and analyzed. Wound-healing assays When the cells had grown to 80%, they were cultured in serum-free medium. After starvation for 12 h, the medium was discarded, and the cell monolayers were scratched with 200 µl pipette tips. The left and right sides of each intersection were photographed at 0 h and 24 h with the crossed point as the mark. Immunohistochemical staining Sections were incubated in a 60 °C incubator for 60 min, followed by dewaxing and hydration. Antigen retrieval was performed using citrate buffer. The sections were blocked with 5% goat serum and incubated with a-SMA, Collagen I and Fibronectin antibodies overnight. Secondary antibody incubation and DAB color development were performed. After counterstaining with hematoxylin, the sections were dehydrated, cleared, and sealed. Images were collected on the HAMAMATSU NANO ZOOMER system. Immunofluorescence Cells were seeded into climbing pieces, fixed using formaldehyde, and permeabilized using 0.5% Triton X-100. The cells were blocked with the BSA blocking solution, incubated with the primary antibodies at room temperature for 1 h, and incubated with the secondary antibodies in the dark at room temperature for 1 h. A drop of sealing agent was added to the climbing film, and the samples were evaluated under a fluorescence microscope. MeRIP-m7G-tRNA sequencing (m7G-tRNA-seq) Small RNAs with lengths less than 200 nt were enriched from total RNA with a mirVana Isolation Kit (Thermo Fisher). The GenSeq ® M7G MeRIP Kit (GenSeq, Inc.) was used to conduct a MeRIP experiment on the enriched small RNA according to the instructions of the kit. Merip/input RNA samples were demethylated with the alkB enzyme at 37°C for 100 min. After demethylation, small RNAs were used for small RNA library construction with the GenSeq ® Small RNA library prep Kit (GenSeq, Inc.). Libraries within the tRNA length range were purified by fragment screening and then sequenced on an Illumina NovaSeq sequencer. tRNA data were downloaded from the GtRNAdb website. According to their anticodons and scores, representative tRNAs were selected from among these tRNAs. Three bases of CCA were added to the 3' end of the tRNA sequences. After sequencing, image analysis and base recognition, the raw reads after quality control were harvested. First, q30 was used for quality control, and then cutadapt software (v1.9.3) was used to splice the original reads and remove low-quality reads. Finally, reads with a length > = 15 nt were retained to identify the spliced reads (i.e., trimmed reads). Then, the trimmed reads of each sample were aligned to the preprepared tRNA database using Bowtie2 software (v2.2.4). SAMtools (V1.3.1) was used to count the number of reads that were aligned to each tRNA as the original expression of the tRNA. The results of IP and input were normalized using the TPM method, and IP/input was calculated and considered the tRNA methylation level according to the normalized results. Fold Change > 1.5 was considered as differentially difference. tRNA sequencing (tRNA-seq) The tRNA sequencing service was provided by Shanghai Yunxu Biological Company, and the process was performed as follows. Briefly, small RNAs with lengths less than 200 nt were enriched from total RNA with a mirVana Isolation Kit (Thermo Fisher). The enriched small RNAs were treated with alkB enzyme for 100 min at 37°C. According to the instructions of the GenSeq ® small RNA Library Prep Kit (GenSeq, Inc.), a small RNA library was constructed from the treated samples. Libraries within the tRNA length range were enriched by fragment screening and then sequenced on an Illumina NovaSeq sequencer. tRNA data were downloaded from the GtRNAdb website. According to the anticodons and scores, representative tRNAs were selected from among these tRNAs. Three bases of CCA were added to the 3' end of their sequences. After Illumina sequencer sequencing, image analysis and base recognition, the raw reads after quality control were harvested. First, q30 was used for quality control, and then cutadapt software (v1.9.3) was used to splice the original reads and remove low-quality reads. Finally, reads with length > = 15 nt were retained to identify the spliced reads (i.e., trimmed reads). Then, the trimmed reads of each sample were aligned to the preprepared tRNA database using Bowtie2 software (v2.2.4). SAMtools (V1.3.1) was used to count the number of reads that were aligned to each tRNA as the original expression of the tRNA, and edger software was used for data normalization and differential expression screening. Differences were analyzed by t test. Fold Change > 2.0 and p values < 0.05 were considered as differentially difference. mRNA sequencing (mRNA-seq) Ribosomal RNA (rRNA) of samples was removed by rRNA Removal Kit (genseq, Inc.) kit, sequencing library was constructed by genseq ® The low input RNA library prep Kit (genseq, Inc.) according to the instructions. After that, the constructed sequencing library was subjected to quality control and quantification by Bioanalyzer 2100 system (Agilent Technologies, USA), followed by 150bp paired end sequencing using Illumina novaseq 6000 instrument. After sequencing with Illumina novaseq 6000 sequencer, the original data were obtained. First, the q30 value is used for raw data quality control. We use cutadapt software (v1.9.3) to remove connectors, remove low-quality reads, and obtain high-quality clean reads. Hisat2 software was used to align the clean reads to the reference genome, and then htseq software (v0.9.1) was used to obtain the original count number. Edger was used to normalize and calculate the fold change and p-value between the two groups of samples to screen the differentially expressed genes, Fold Change > 2.0 was considered as differentially expressed genes. Go function analysis and KEGG pathway analysis were performed using differentially expressed mRNA. Polyribosome-bound mRNA sequencing (Ribo-seq) Cells were treated with cycloheximide and lysed using lysis buffer and then digested with nuclease. The digested samples were separated into single ribosomes with a size exclusion column. Fragment selection was performed on RNA fragments that were protected by ribosomes using polyacrylamide gel electrophoresis, and then rRNA was removed from the samples using rRNA removal reagents. After purification, the RNA ends were repaired, and a 3 'connector was added; then, the samples were transformed into cDNA through reverse transcription. cDNA was purified by polyacrylamide gel electrophoresis, cyclized, and amplified by PCR. The amplified library that was obtained was purified and sequenced on a NovaSeq sequencer (Illumina). After sequencing with an Illumina NovaSeq 6000 sequencer, the raw data were obtained. First, the Q30 value was used for raw data quality control. Cutadapt software (v1.9.3) was used to remove connectors, remove low-quality reads, and obtain high-quality clean reads. Bowtie was used to compare the disconnected data to rRNA sequences and to obtain clean reads that had not been aligned to rRNA. Tophat2 software was used to compare clean reads to the reference genome. Then, HTSeq software (v0.9.1) was used to obtain the original count number, edgeR or DESeq2 was used for standardization, and the multiple changes and p values between the two groups of samples were calculated to identify differentially expressed genes. By default, edgeR was used for differential analysis. Correlation analysis between the mRNA-seq and the Ribo-seq was perform. The ratio of FPKM of each gene in Ribo-seq to FPKM in mRNA-seq was consider as the translation efficiency (TE). Fold Change > 2.0 was considered as differentially difference. Northern blotting and northwestern blotting For northern blotting, 2µg total RNA samples were mixed with RNA loading buffer (2X) and denatured at 65 C for 15 min, then, the sample were added into 15% Urea-PAGE electrophoresis in 1X TBE buffer. The separated RNAs were then transferred into a positively charged nylon membrane, cross-linking was performed by Ultraviolet (UV) light. The tRNAs or U6 snoRNA were blotted with corresponding digoxigenin-labeled probes. For Northwestern blotting, the the RNA-containing nylon membranes was crosslinked with UV and blotted with an anti-m7G antibody (RN017M, Medical Biological Laboratories, Nagoya, Japan). After incubation of BeyoECL Moon buffer with nylon membrane, the signals were detected according to previous reports [ 15 – 17 ]. Coomassie Blue Staining PAGE gel was soaked by BeyoBlue™ Coomassie Blue Super Fast Staining Solution (Beyotime Biotechnology, China) and dyed at room temperature on a side sway shaker for 30 minutes, then discard the decolorization solution, add deionized water for decolorization on a shaker. Co-Immunoprecipitation (CoIP) Cells were lysed and incubated with indicated antibody overnight, then Protein A/G-MagBeads was added to the sample, eluting was conducted by Elution buffer, the indicated proteins was obtained. Seahorse assay The extracellular acidification rate (ECAR) of cells was measure using a Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Agilent). In brief, 1 × 10 4 Cells were seeded into Agilent Seahorse XFe96 plates and cultured for 12 h in a standard incubator. After that, the cells were treated with 10 mM glucose, 2µM oligo-mycin and 2-deoxy- D -glucose in different ports of the Seahorse cartridge. Statistical analysis All the continuous variables are presented as the mean ± standard deviation (SD). Differences between the two groups were analyzed by Student’s t test. The data were analyzed and visualized by SPSS 21.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 7 (GraphPad, San Diego, CA, USA). P < 0.05 was considered to indicate statistically significant differences. Results RA prevented RIPF and inhibited the progression of FMT Our previous study have shown that RA can alleviate RIPF in rats [ 9 ], and it is recognized that FMT plays a core role in the pathogenesis of organ fibrosis [ 18 ]. In the present study, we further evaluated the effect of RA on RIPF and FMT in C57BL/6 mice. It is observed that RA-treated mice had improved lung morphology, with less lung collapse and fibrous nodules (Fig. 1 A). Lung index that was referred to lung/body weight ratio decreased significantly in the RA + IR group comparing with that in the IR group (Fig. 1 B). The RA-treated mice showed attenuated fibrosis versus irradiated mice without RA treatment, as indicated by less thickened alveolar walls, fibrotic foci and collagen deposition, which was in line with decreases in the ashcroft score and collagen volume fraction (Fig. 1 C and 1 D). The expression of FMT markers α-SMA, collagen I and fibronectin in RA-treated mice was lower than that in irradiated mice (Fig. 1 E and 1 F). These results indicated that RA exerted an antifibrotic effect on lung tissues of RIPF mouse models and suppressed the progression of FMT in irradiated mouse lung tissues. RA decelerated the progression of FMT through exosomes pathway To explore the mechanism underlying RIPF, exosomes derived from the lung epithelial cells were identified and quantified by transmission electron microscopy. Nanoparticle tracking analysis demonstrated that the average sizes of exosomes isolated from TC-1 and MLE-12 cells were approximately 84.49 and 86.93 nm, respectively (Fig. 2 A and 2 B). Additionally, uptake of exosomes by MLFs were observed after 24h when coincubating MLFs with TC-1 cell-derived exosomes (TC-1-exo) or MLE-12 cell-derived exosomes (MLE-12-exo) that were labeled with PKH67 (red) (Fig. 2 C). Interestingly, immunofluorescence staining showed that incubating MLFs with exosomes that derived from irradiated epithelial cells with RA treatment (RA + IR-TC-1/MLE-12-exo) inhibited the induction of α-SMA expression in MLFs (Fig. 2 D). Besides, Edu staining and wound-healing assays showed that treating MLFs with RA + IR-TC-1/MLE-12-exo significantly decreased the effect of IR-TC-1/MLE-12-exo on inducing proliferation and migration (Fig. 2 E and 2 F). Together, these results indicated that RA suppressed FMT of MLFs induced by exosomes that were derived from irradiated lung epithelial cells. RA reversed the increase in expression and m7G modification level of tRNA that induced by irradiation in lung epithelial cells Many studies have shown that tRNA epigenetic modifications are associated with the pathological processes of numerous diseases including organ fibrosis [ 17 , 19 – 21 ]. To assess the correlation between RIPF and tRNA m7G modification that is one of the most common epigenetic modifications, m7G-tRNA-seq and tRNA-seq analysis were performed in TC-1 cells (Table S1 and S2). We found that the expression and m7G modification level of tRNA-ArgCCG and tRNA-CysGCA were significantly higher in the IR group than those in the Ctrl group (Fig. 3 A and 3 B). Northwestern and northern blotting analysis further confirmed the results, and found that the increase in expression and m7G modification level of tRNA-ArgCCG and tRNA-CysGCA caused by irradiation could be reversed by RA (Fig. 3 C and 3 D). We then investigated the effect of RA on the METTL1/WDR4 complex, which is the main catalytic complex for tRNA m7G modification in eukaryotic cells. Western blotting results revealed that RA reduced the upregulation of METTL1 and WDR4 protein expression induced by irradiation (Fig. 3 E). In addition, silencing METTL1 and WDR4 eliminated the promoting effect of irradiaton on the expression and m7G modification of the indicated tRNA (Fig. 3 F). Taken together, our results indicated that the tRNA level and m7G modification increased in the lung epithelial cells after irradiation, and RA reversed these changes. RA suppressed the process of FMT by downregulating METTL1/WDR4-mediated tRNAs m7G modification in exosomes of lung epithelial cells To further confirm whether tRNA level and m7G modification also increasing in exosomes, we performed northwestern, northern blotting and immunofluorescence staining. The results revealed that the levels of m7G modification and expression of tRNAs in exosomes of the irradiated lung epithelial cells were higher than controls, while RA treatment can partially reverse those effect of irradiation on exosomes of lung epithelial cells (Fig. 4 A). In addition, exosomes of the METTL1/WDR4 overexpressed TC-1 cells (oeMETTL1/MDR4-TC-1-exo) showed a high tRNAs expression and m7G modification level, but no similar results were observed in the RA intervention group (Fig. 4 B). m7G modification levels in MLFs that were incubated with exosomes of TC-1 cells in the Ctrl, IR, and RA + IR (Ctrl-TC-1-exo, IR-TC-1-exo and RA + IR-TC-1-exo) group were analyzed by northwestern and northern blotting, and the results confirmed that the expression of tRNAs m7G modification increased in the IR-TC-1-exo group and RA + IR-TC-1-exo group, especially the former showing a more significant increase (Fig. 4 C). Besides, immunofluorescence staining showed that incubating MLFs with oeMETTL1/MDR4-TC-1/MLE-12-exo plus RA diminished the expression of α-SMA induced by these exosomes (Fig. 4 D, Fig. 4 I and Fig. S1 A). Consistently, Edu staining and wound-healing assays indicated that MLFs treated with oeMETTL1/MDR4-TC-1-exo and RA had a lower proliferation and migration ability than those without RA intervention (Fig. 4 E- 4 H, Fig. S1 B, and Fig. S1 C). In conclusion, these results confirmed that RA had a negative regulatory effect on tRNAs m7G modification level in exosomes of lung epithelial cells. RA regulated the translation efficiency of SPHK1 and subsequently affected the interaction between SPHK1 and NAT10 Since tRNA is mainly involved in protein synthesis, it is plausible to speculate that the abnormal tRNA expression may affect mRNA translation. In our present study, Ribo-seq and mRNA-seq analysis was performed to explore the underlying target mRNA. We identified mRNA that had no significant difference in expression levels assesed by mRNA-Seq but had significant difference in translation efficiency assesed by Ribo-seq as the target genes for modifying tRNA [ 22 ]. Comparing with MLFs in the Ctrl-TC-1-exo group, MLFs in the IR-TC-1-exo group had 3101 mRNAs with decreased translation ratios (TRs) and 2086 mRNAs with increased TRs (Fig. 5 A, Table S3 ), in which we noticed sphingosine kinase type 1 isoform (SPHK1) had a significantly increased TR. SPHK1 has been reported to regulate pulmonary, hepatic, and renal fibrosis [ 23 – 25 ], yet the role of SPHK1 in RIPF has not been elucidated. KEGG enrichment analysis of these mRNAs with increased TRs showed significant enrichment in the Wnt signaling pathway, MAPK signaling pathway, and ECM-receptor interaction (Fig. 5 B). qRT-PCR and western blotting further revealed that IR-TC-1-exo and RA + IR-TC-1-exo had little effect on the mRNA expression of SPHK1 in MLFs, but had a significant impact on protein expression (Fig. 5 C and 5 D). Subsequently, polyribosome-qPCR assay confirmed that the translation efficiency of SPHK1 in the IR-TC-1-exo group increased compared to the Ctrl-TC-1-exo group, while the translation efficiency of SPHK1 was found to be decreasing in the RA + IR-TC-1-exo group compared to the IR-TC-1-exo group (Fig. 5 E). Recently, N-acetyltransferase 10 (NAT10) was reported to enhance pulmonary fibrosis [ 26 ]. Based on HDOCK database retrieval, NAT10 was found to be potentially bound to SPHK1 (Fig. 5 F). The coomassie blue staining assay further prompted the interaction between NAT10 and SPHK1 (Fig. 5 G). What’s more, CoIP assays verified that SPHK1 directly interacted with NAT10 (Fig. 5 H). Taken together, our results indicated that IR-TC-1-exo heightened the translation ratio of SPHK1 in MLFs and affected the binding between SPHK1 and NAT10 proteins, while RA can counteract the effect of IR-TC-1-exo on MLFs. RA diminished glycolysis by reducing acetylated PFKFB3 and cytoplasmic translocation Myofibroblasts may use aerobic glycolysis as an additional source of bioenergetics and biosynthesis to meet the demands related to fast growth and proliferation [ 27 ]. In this study, the Seahorse assay showed that compared with the control group, the extracellular acidification rate (ECAR) of the IR-TC-1-exo group significantly increased, while the RA + IR-TC-1-exo group only slightly increased (Fig. 6 A), indicating that RA may inhibit the glycolysis of MLFs via exosomes pathway derived from lung epithelial cells. Previous studies have proven that the acetylation of metabolic enzymes is a critical mechanism underlying metabolic modulation [ 28 ]. PFKFB3, as a key enzyme in glycolysis, has been proven to be a driving factor for various organ fibrosis diseases [ 29 – 31 ], which prompted us to investigate the mechanism underlying PFKFB3 regulation by exosomes. The results showed that IR-TC-1-exo treatment can improve the acetylation level of PFKFB3, and the addition of RA partially counteracts this effect (Fig. 6 B). NAT10 belongs to the Gcn5-related N-acetyltransferase family, which has been reported to acetylate RNAs and proteins [ 32 ]. Interestingly, we observed that Remodelin (inhibitor of NAT10) reduced the acetylation of PFKFB3 (Fig. 6 C). PFKFB3 is localized to the nucleus, which may occur due to the inclusion of a classical nuclear localization signal (KKPR, amino acids 472–475) [ 33 ]. Mass spectrometric analysis by Li et al. revealed six lysine (K) residues in PFKFB3 that undergo acetylation (K12, K284, K302, K451, K472 and K473) [ 33 ]. Consequently, we speculated that K472 and K473 were the major acetylation sites that interfered with the nuclear localization of PFKFB3. As expected, we found that mutation of the K472R and K473R sites greatly reduced the acetylation of PFKFB3 (Fig. 6 D). FISH assays indicated that wild type (WT) PFKFB3 was localized in the nucleus, while the K472Q and K473Q mutants localized in the cytoplasm (Fig. 6 E). In addition, cytoplasmic PFKFB3 has a stronger effect on promoting glycolysis [ 34 ]. AMPK activates PFKFB3 and promotes glycolysis through the phosphorylation of PFKFB3 at S461 [ 35 ]. To verify the cytoplasmic localization of PFKFB3 has an effect on its phosphorylation at the S461 site, western bloting was performed. The results showed that S461 phosphorylation increased in the IR-TC-1-exo group, and this effect was reversed in the RA + IR-TC-1-exo group (Fig. 6 F). Cells expressing the K472Q or K473Q mutants exhibited higher phosphorylation of S461 and activity of PFKFB3 (Fig. 6 G and 6 H) as well as a higher ECAR than those in cells expressing WT PFKFB3 (Fig. 6 I). Taken together, these results indicated that IR-TC-1-exo promoted PFKFB3 acetylation at K472 and K473 and induced the translocation of PFKFB3 from the nucleus to the cytoplasm in MLFs. Additionally, cytoplasmic PFKFB3 was phosphorylated by AMPK, and then the glycolytic process was enhanced. On the other hand, RA reduced these changes. Discussion RIPF is a serious complication of radiotherapy that is difficult to reverse. At present, there is a lack of effective treatment methods for it. In this study, we have uncovered several interesting findings: high-throughput sequencing results showed that the expression and m7G modification level of tRNA increased in the exosomes derived from irradiated lung epithelial cells. In addition, RA inhibited the translation efficiency and protein expression of SPHK1 that regulated by tRNA m7G modification, as well as suppressing the acetylation of PFKFB3 in the nucleus. Ulteriorly, it decreasd the level of phosphorylated PFKFB3 in the cytoplasm, and ultimately reducing FMT triggered by glycolysis in lung fibroblasts. Numerous studies have shown that cells can transmit important signaling molecules, such as RNA and proteins, and thus promote FMT through exosomes. Huang et al. found that exosomal SPP1 derived from silica-treated macrophages can trigger FMT and promote pulmonary fibrosis to form silicosis [ 36 ]. Li et al. reported that miR-192-5p in exosomes derived from adipose tissue mesenchymal stem cells reduced collagen deposition and FMT by targeting the IL-17RA/Smad signaling axis, thereby exerting an inhibitory effect on scar hyperplasia [ 37 ]. In this study, we extracted exosomes of irradiated lung epithelial cells. Transmission electron microscopy and nanoparticle tracing analysis confirmed that irradiated TC-1 cell-derived exosomes can be taken up by MLFs, which promoted the expression of the FMT marker protein α-SMA and enhanced the proliferation and migration of MLFs. After treatment with RA, exosomes of irradiated lung epithelial cells (RA + IR-TC-1-exo) significantly inhibited the FMT of MLFs compared to IR-TC-1-exo. These results indicated that RA can inhibit FMT via exosomes derived from lung epithelial cells. In recent years, increasing evidence has shown that epigenetic modifications of tRNAs are associated with the pathological processes of various diseases. For example, dysregulated tRNA m7G modification exerted a carcinogenic effect on the esophageal squamous cell carcinoma [ 17 ]. In patients with NSUN2 gene mutations, the lack of specific 5-cytosine methylation at the C47 and C48 sites of the tRNA Asp led to moderate to severe intellectual impairment, facial deformities, and distal myopathy [ 19 ]. Another studie have pointed out that the key pathogenesis that is caused by CDKAL1 deficiency in diabetes patients is the β-mistranslation of Lys codons in cells, which leads to a decrease in glucose-stimulated insulin synthesis; the underlying molecular mechanism may be related to the 2-methylthio-modification of N 6 -threonylcarbonyladenosine at position-37 in tRNA UUUU Lys3 [ 20 ]. However, there is currently no research on the epigenetic modification of tRNA in RIPF. m7G modification is one of the most common epigenetic modifications of RNA, playing an important role in maintaining the integrity and stability of tRNA. Our study conducted m7G-tRNA-seq and tRNA-seq analysis and found that the m7G modification and expression of tRNA-ArgCCG and tRNA-CysGCA were most significantly increased in the irradiated lung epithelial cells. Further validation showed that RA treatment could reverse these change and radiation-induced high expression of m7G methyltransferase complex components (METTL1 and WDR4). In addition, we also found that after upregulating METTL1 and WDR4 in lung epithelial cells and incubating MLFs with their exosomes, the expression level of α-SMA significantly increased, and the proliferation and migration of MLFs were enhanced. These results suggested that RA may downregulate the transmission of tRNA-ArgCCG and tRNA-CysGCA to MLFs by inhibiting these tRNA m7G modification in the lung epithelial cells, ultimately slowing down the FMT process in MLFs. Glycolysis is an important metabolic pathway that occurs in almost all living cells. Research has shown that glycolysis is enhanced in fibroblasts and myofibroblasts of the liver, lungs, and kidneys during chronic inflammation and fibrosis [ 38 ]. Besides, it is reported that during the process of fibrosis in organs such as the lungs, cell-dependent energy metabolism gradually shifted from oxidative phosphorylation to glycolysis, which is also known as the Warburg effect [ 39 ]. However, the role of glycolysis in RIPF is not yet clear. PFKFB3 catalyzes the synthesis and hydrolysis of the small molecule fructose-2,6-diphosphate, which is a potent activator of the glycolytic pathway. Among the four members of the PFKFB protein family, PFKFB3 is the only protein that is located in the nucleus [ 40 ]. Our study showed that after incubating MLFs with IR-TC-1-exo, the level of acetylated PFKFB3 at the K472 and K473 sites was enhanced, as well as the level of phosphorylation at the S461 site increased. On the other hand, RA inhibited these changes. Based on the FISH experiments, we observed that PFKFB3 protein was originally primarily expressed in the nucleus of MLFs, while PFKFB3 protein was translocated from the nucleus to the cytoplasm after incubation with IR-TC-1-exo in MLFs. However, treatment with RA could reverse the nuclear-cytoplasmic translocation of PFKFB3. Similar results were also observed by Li et al. [ 33 ], who found that the chemotherapy drug cisplatin accumulated PFKFB3 in the cytoplasm and promoted glycolysis. Specifically, the lysine residue at position 472 of the PFKFB3 protein was acetylated, which inactivated the nuclear localization signal of PFKFB3 and promoted its retention in the cytoplasm. PFKFB3, which was located in the cytoplasm, was more susceptible to phosphorylation by the kinase AMPK, leading to the activation of PFKFB3 and promoting glycolysis, thereby protecting cells from apoptosis. Together, our study reveals a novel mechanism for regulating the activity of metabolic regulatory enzyme PFKFB3 through acetylation in RIPF and suggests targeted inhibition of PFKFB3 by RA may be a new clinical strategy for treating RIPF. Conclusion In summary, these findings presented here suggested that RA alleviated RIPF by downregulating tRNA m7G modification and expression level-regulated FMT through the exosomes pathway. RA may be a potential therapeutic drug for slowing down the progression of RIPF. Declarations Supplementary Information The online version contains supplementary material available at GSE249534 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE249534). Acknowledgements We thank the support of Guangxi Key Laboratory of Immunology and Metabolism for Liver Diseases, Key Laboratory of Early Prevention and Treatment for Regional High-Frequency Tumors of Guangxi Medical University. Author contributions T.Z, J.M and Z.O conducted the cellular and animal experiments, X.Q, Y.W and Z.L helped to the animal experiments, S.H and K.H helped to data analysis and figure design, M.H and R.W participated in the design of the study and performed the statistical analysis, T.Z drafted the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the “Medical Excellence Award” Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University, the Basic Ability Enhancement Project of Young Teachers in Guangxi Zhuang Autonomous Region (No. 2023KY0120) and Nanning Qingxiu district key research and development plan for Science and Technology (No. 2020019). Data Availability All data generated or analyzed during this study are included in this published article. All the authors declare no conflicts of interest in this study. Ethics approval and consent to participate All procedures conducted in this work comply with the ethical standards of the institution and/or the National Research Council. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References He Y, Thummuri D, Zheng G, Okunieff P, Citrin DE, Vujaskovic Z, et al. Cellular senescence and radiation-induced pulmonary fibrosis. Translational research: the journal of laboratory and clinical medicine. 2019;209:14–21. Liu D, Gong L, Zhu H, Pu S, Wu Y, Zhang W, et al. Curcumin Inhibits Transforming Growth Factor β Induced Differentiation of Mouse Lung Fibroblasts to Myofibroblasts. Front Pharmacol. 2016;7:419. Guan R, Zhao X, Wang X, Song N, Guo Y, Yan X, et al. Emodin alleviates bleomycin-induced pulmonary fibrosis in rats. Toxicol Lett. 2016;262:161–72. Noor S, Mohammad T, Rub MA, Raza A, Azum N, Yadav DK, et al. Biomedical features and therapeutic potential of rosmarinic acid. 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Nucleic Acids Res. 2020;48:3638–56. Li FL, Liu JP, Bao RX, Yan G, Feng X, Xu YP, et al. Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis. Nat Commun. 2018;9:508. Yalcin A, Clem BF, Simmons A, Lane A, Nelson K, Clem AL, et al. Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. J Biol Chem. 2009;284:24223–32. Marsin AS, Bouzin C, Bertrand L, Hue L. The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase. J Biol Chem. 2002;277:30778–83. Huang R, Hao C, Wang D, Zhao Q, Li C, Wang C, et al. SPP1 derived from silica-exposed macrophage exosomes triggers fibroblast transdifferentiation. Toxicol Appl Pharmcol. 2021;422:115559. Li Y, Zhang J, Shi J, Liu K, Wang X, Jia Y, et al. Exosomes derived from human adipose mesenchymal stem cells attenuate hypertrophic scar fibrosis by miR-192-5p/IL-17RA/Smad axis. Stem Cell Res Ther. 2021;12:221. Xu S, Cheuk YC, Jia Y, Chen T, Chen J, Luo Y, et al. Bone marrow mesenchymal stem cell-derived exosomal miR-21a-5p alleviates renal fibrosis by attenuating glycolysis by targeting PFKM. Cell Death Dis. 2022;13:876. Ding H, Jiang L, Xu J, Bai F, Zhou Y, Yuan Q, et al. Inhibiting aerobic glycolysis suppresses renal interstitial fibroblast activation and renal fibrosis. Am J Physiol Ren Physiol. 2017;313:F561–f75. De Bock K, Georgiadou M, Schoors S, Kuchnio A, Wong BW, Cantelmo AR, et al. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell. 2013;154:651–63. Additional Declarations No competing interests reported. Supplementary Files figS1.tif Supplemental Fig. 1. RA suppressed the process of FMT in MLFs by downregulating METTL1/WDR4-mediated tRNAs m7G modification via exsomes of MLE-12 cells. (A ) The expression of α-SMA of MLFs was assessed by Immunofluorescence staining. (B) The cell proliferation viability of MLFs was tested by Edu staining. (C) The migration ability was detected by wound healing assay. The precise n value (number of biologically-independent replicates) is 3. *P < 0.05 **P < 0.01. tableS1.xlsx Supplemental table 1. Expression profile of the m7G-modified tRNAs based on m7G-tRNA-seq. tableS2.xlsx Supplemental table 2. Expression profile of the differentially expressed tRNAs. tableS3.xlsx Supplemental table 3. Expression profile of identified genes with increased or decreased translation ratios examined by Ribo-seq and mRNA-seq. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3744363","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":259420267,"identity":"871d2301-3fd9-41d4-8280-42714bdc2a20","order_by":0,"name":"Tingting Zhang","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Zhang","suffix":""},{"id":259420268,"identity":"6d62c4b4-2752-4c25-8a0d-0a7a5db68053","order_by":1,"name":"Jinglin Mi","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinglin","middleName":"","lastName":"Mi","suffix":""},{"id":259420269,"identity":"88a911c4-469a-4852-b3b0-dfe4d261e349","order_by":2,"name":"Zhechen Ouyang","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhechen","middleName":"","lastName":"Ouyang","suffix":""},{"id":259420270,"identity":"a0f56181-76ee-450d-9f37-45badbd17c43","order_by":3,"name":"Xinling Qin","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinling","middleName":"","lastName":"Qin","suffix":""},{"id":259420273,"identity":"0caf7e87-35b0-4517-b71f-876ebfce75ce","order_by":4,"name":"Yiru Wang","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiru","middleName":"","lastName":"Wang","suffix":""},{"id":259420275,"identity":"0224024a-c85a-4105-9586-f21580c8e773","order_by":5,"name":"Zhixun Li","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhixun","middleName":"","lastName":"Li","suffix":""},{"id":259420276,"identity":"ae47169f-4741-4d4d-8cd8-7143e0322eac","order_by":6,"name":"Siyi He","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siyi","middleName":"","lastName":"He","suffix":""},{"id":259420277,"identity":"c25a92cf-8576-4612-a694-f51e0e13f57c","order_by":7,"name":"Kai Hu","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Hu","suffix":""},{"id":259420278,"identity":"0b2930f2-45d5-4633-8c00-ee5082c27d3b","order_by":8,"name":"Rensheng Wang","email":"","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rensheng","middleName":"","lastName":"Wang","suffix":""},{"id":259420279,"identity":"286caf7b-36be-4e2a-a2b5-55ce96632988","order_by":9,"name":"Weimei Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBACAyjNw8DA2PgATZCwlmYDNEH8WkCATYIoLeYSyc8efm2zkdFtb26r/Nq2LbGBvXmbBEPNHZxaLGekmRvLtqXxmJ052HZb5sztxAaeY2USDMee4XbYjQQzaclth3nMbiS23ZaoAGqRyDGTYGw4jEdL+jeglv88ZvcfthVLGAC1yL8hpCXHTPLjtgNAWxjbGD+AbeEhoOXMmzJpxn/JQL8kNksznLlt3MaTVmyRcAyPluPp2yR/nLGzNzt+/OHHn223ZfvZD2+88aEGtxYQYOZBZrCBWAl4NQCj/Qc6YxSMglEwCkYBMgAAFzhZpCncxbkAAAAASUVORK5CYII=","orcid":"","institution":"the First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Weimei","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2023-12-12 14:29:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3744363/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3744363/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":48299779,"identity":"c1715b4b-06b9-48f8-839b-e31275865efb","added_by":"auto","created_at":"2023-12-15 21:42:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1442698,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of RA on pathological changes and collagen deposition in lung tissue of mice at 24 weeks after whole thoracic irradiation. (A) Representative images of lung tissues. (B) Lung index was shown which referred to lung/body weight ratio. (C-F) Representative pictures and statistical analysis of hematoxylin-eosin staining, masson staining and immunohistochemical staining of FMT markers. The precise n value (number of biologically-independent replicates) is 6. *P \u0026lt; 0.05 **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/c2569716f4d28b659aa315e9.png"},{"id":48299781,"identity":"06e8b19e-980c-450c-9c7e-41623a73625b","added_by":"auto","created_at":"2023-12-15 21:42:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1016269,"visible":true,"origin":"","legend":"\u003cp\u003eRA decelerated the progression of FMT in MLFs through exosomes pathway. (A) Exosomes derived from lung epithelial cells were characterized by electron microscopy. (B) Distribution of the size and concentration of extracted exosomes. (C) PKH67-labeled exosomes derived from TC-1 and MLE-12 cells were ingested by MLFs. (D) The expression levels and statistical analysis of α-SMA were detected by immunofluorescence staining. (E) The cell viability was tested by Edu staining. (F) The migration ability was detected by wound healing assay. The precise n value (number of biologically-independent replicates) is 3. *P \u0026lt; 0.05 **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/fe5fabf33a6d29fd1ee0829e.png"},{"id":48299972,"identity":"d5219755-8493-4110-aed2-9cd0cfbdd2a5","added_by":"auto","created_at":"2023-12-15 21:50:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":441730,"visible":true,"origin":"","legend":"\u003cp\u003eRA reversed the increase of tRNAs expression and m7G modification levels in irradiated lung epithelial cells. (A) Quantification of tRNAs m7G modification level. (B) Expression profile of tRNAs. (C and D) Northwestern and northern blot of indicated tRNAs were performed in TC-1 cells and mouse lung tissue. (E) The expression levels of METTL1/WDR4 complex were evaluated by western blot. (F) The effect of METTL1 and WDR4 on tRNA expression and m7G modification were detected using northwestern and northern blot. The precise n value (number of biologically-independent replicates) is 3. *P \u0026lt; 0.05 **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/09c9c6cde755860027d770fe.png"},{"id":48299589,"identity":"da52d813-7dd0-4435-afb2-7c7b41bba039","added_by":"auto","created_at":"2023-12-15 21:34:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":824182,"visible":true,"origin":"","legend":"\u003cp\u003eRA suppressed the process of FMT in MLFs by downregulating METTL1/WDR4-mediated tRNAs m7G modification via exsomes. (A and B) Expression levels of representative m7G-modified tRNAs in exosomes were examined by northwestern and northern blot using the indicated tRNA probes. (C) MLFs cells were incubated with exosomes derived fromTC-1 cells, the expression and m7G modification level of indicated tRNAs were detected by northwestern and northern blot assays. (D and I) The expression of α-SMA was assessed by Immunofluorescence staining. (E and G) The cell viability was tested by Edu staining. (F and H) The migration ability was detected by wound healing assay. The precise n value (number of biologically-independent replicates) is 3. *P \u0026lt; 0.05 **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/7e57eb9983ebcc7ac62e5336.png"},{"id":48299594,"identity":"2994aaaa-f264-43b0-b8b4-ca7137598507","added_by":"auto","created_at":"2023-12-15 21:34:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":689441,"visible":true,"origin":"","legend":"\u003cp\u003eSPHK1 is a target gene of m7G-modified tRNA and interacted with NAT10. (A) Ribo-seq and mRNA-seq analysis were performed to explore the target mRNA. (B) KEGG analysis of increased translated mRNAs. (C and D) qRT-PCR and Western blot analysis of SPHK1 in MLFs. (E) qRT-PCR based TE analysis of SPHK1 was performed using the polyribosome mRNAs. (F) The relationship between SPHK1 and NAT10 was predicted by HDOCK database. (G) SPHK1-interacted proteins were identified by coomassie blue staining. (H) The interaction of SPHK1 protein with NAT10 protein was evaluated by CoIP assay. The precise n value (number of biologically-independent replicates) is 3. *P \u0026lt; 0.05 **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/dee019a43da209f3cc0ba46b.png"},{"id":48299586,"identity":"77894231-b1c1-4501-9c56-ae7047e292a2","added_by":"auto","created_at":"2023-12-15 21:34:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":610913,"visible":true,"origin":"","legend":"\u003cp\u003eRA diminished glycolysis by reducing acetylated PFKFB3 and cytoplasmic translocation. (A) The ECARs were evaluated by Seahorse XFe96 Extracellular Flux analyser. (B) Acetylation levels of PFKFB3 were examined by western blot. (C) After treating with Remodelin (inhibitor of NAT10), acetylation levels of PFKFB3 in MLFs was examined. (D) Acetylation sites of PFKFB3 was examined by immunoprecipitation. (E) The effect of K472Q and K473Q mutation on distribution of PFKFB3 in MLFs was assessed by Immunofluorescence staining. (F) Phosphorylation levels of PFKFB3 and AMPK were examined. (G and H) The effect of K472Q and K473Q mutation on phosphorylation levels of PFKFB3 was examined. (I) The effect of K472Q and K473Q mutation on ECAR was evaluated by seahorse assay. The precise n value (number of biologically-independent replicates) is 3. *P \u0026lt; 0.05 **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/37776e6a3eb508df75adc7eb.png"},{"id":48299973,"identity":"40f7718d-7009-4a10-a8be-b519b338f98c","added_by":"auto","created_at":"2023-12-15 21:50:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":618782,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic model of RA protection against RIPF.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/bb3050b3ab6ceee45e8b4223.png"},{"id":48461503,"identity":"f8131ecb-295d-468a-96a0-49836d538272","added_by":"auto","created_at":"2023-12-19 13:37:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3320932,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/955f6678-dbfe-48f2-a021-343b25814ca1.pdf"},{"id":48299597,"identity":"f280a89c-57e3-40ee-a70e-77662fae5282","added_by":"auto","created_at":"2023-12-15 21:34:06","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14414544,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Fig. 1. RA suppressed the process of FMT in MLFs by downregulating METTL1/WDR4-mediated tRNAs m7G modification via exsomes of MLE-12 cells. (A ) The expression of α-SMA of MLFs was assessed by Immunofluorescence staining. (B) The cell proliferation viability of MLFs was tested by Edu staining. (C) The migration ability was detected by wound healing assay. The precise n value (number of biologically-independent replicates) is 3. *P \u0026lt; 0.05 **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/1473c45009e84a1119f89bd6.tif"},{"id":48299595,"identity":"54bedf40-9523-453e-a4b3-da7eb05dce0f","added_by":"auto","created_at":"2023-12-15 21:34:05","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30246,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental table 1. Expression profile of the m7G-modified tRNAs based on m7G-tRNA-seq.\u003c/p\u003e","description":"","filename":"tableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/c70f36e09229780f8804c74d.xlsx"},{"id":48299971,"identity":"cff56360-385d-400d-acb9-f738eade5410","added_by":"auto","created_at":"2023-12-15 21:50:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20435,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental table 2. Expression profile of the differentially expressed tRNAs.\u003c/p\u003e","description":"","filename":"tableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/707962a5d1d6429e605ac268.xlsx"},{"id":48299782,"identity":"8cdf7469-3836-4791-93d6-5e78f1fbfc73","added_by":"auto","created_at":"2023-12-15 21:42:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2491737,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental table 3. Expression profile of identified genes with increased or decreased translation ratios examined by Ribo-seq and mRNA-seq.\u003c/p\u003e","description":"","filename":"tableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3744363/v1/345dc170cb909665e6c397a8.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rosmarinic acid alleviates radiation-induced pulmonary fibrosis by downregulating tRNA N7-Methylguanosine modification-regulated fibroblast to myofibroblast transition through the exosomes pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThoracic cancer is one of the most common malignancies. Patients with locally advanced chest tumors who are unable to undergo surgery or have high-risk factors after surgery often require radiation therapy. However, radiation-induced lung injury (RILI) and fibrosis are important factors that limit the dose and efficacy of radiotherapy. The early manifestation of RILI is radiation-induced pneumonia, and approximately 9%-30% of patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] gradually developed radiation-induced pulmonary fibrosis (RIPF) after 6 months. RIPF is characterized by the irreversible destruction of normal lung tissue structure and the deterioration of lung function. Although precision radiotherapy techniques, such as stereotactic body radiationtherapy and intensity modulated radiotherapy, have been widely applied in clinical practice nowadays, the incidence of RIPF remains high, and there is currently a lack of effective treatment plans. Therefore, finding effective drugs that are associated with low toxicity is crucial for the treatment of RIPF.\u003c/p\u003e \u003cp\u003eIn recent years, the use of traditional Chinese medicine in the prevention and treatment of pulmonary fibrosis has received considerable attention. Liu et al. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] discovered that curcumin inhibited TGF-β2-induced lung fibroblast to myofibroblast differentiation and suppressed pulmonary fibrosis by reducing the activity of MMP-9. Emodin inhibited not only the expression of α-SMA, collagen IV, and fibronectin in human embryo lung fibroblasts exposed to TGF-β1 but also the activation of Smad2/3 and STAT3, thus blocking the differentiation of myofibroblasts and the deposition of extracellular matrix [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Rosmarinic acid (RA) is a polyphenolic compound that is isolated from the rosemary plant, which belongs to the Labiatae family [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. RA exerts obvious anti-inflammatory and anti-tissue fibrosis effects on diseases such as enteritis, osteoarthritis, atopic dermatitis, and mastitis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hsieh et al. found that RA treatment reduced the levels of α-SMA, collagen I, and fibronectin in NRK-52E kidney cells and decreased renal interstitial fibrosis by inhibiting phosphorylated-AKT mediated epithelial-mesenchymal transition in vitro and in vivo [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Another study showed that RA regulated the AMPKα/Smad3 signaling axis to reduce the cardiac fibrosis that was caused by long-term arterial pressure overload and delay cardiac remodeling [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similarly, two previous studies reported by our research group revealed that RA exerted a protective effect on the radiation-induced tissue inflammation and fibrosis. On the one hand, RA downregulated the expression of TNF-α and IL-6 by inhibiting the N-terminal kinase activity of p53/Jun, reduced the oxidative stress response and the apoptosis of parotid gland cells, and ultimately alleviated the radiation-induced parotid gland fibrosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. On the other hand, RA reduced RhoA/ROCK and NF-κB phosphorylation levels by inhibiting MYPT1 expression, thereby suppressing the progression of RIPF in rats [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the mechanism of RA slowing radiation-induced tissue fibrosis still needs to be further explored.\u003c/p\u003e \u003cp\u003eFibroblast to myofibroblast transition (FMT) was an important characteristic of fibrosis after RILI [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Pulmonary fibroblasts can be transformed into myofibroblasts after radiation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Myofibroblasts is the main source of extracellular matrix, which promotes the formation and development of RIPF [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Communication signals including chemokines, exosomes, and secretory proteins are important mechanisms for other types of cells in lung tissue to regulate the redifferentiation of lung fibroblasts [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the present study, we found that RA alleviated the development of RIPF in mice by inhibiting the progression of FMT. Further exploration revealed that radiation-exposed epithelial cells can promote the occurrence of FMT through the exosomes pathway, while RA can counteract the effect of irradiated lung epithelial cells on lung fibroblasts. Mechanically, RA suppressed the METTL1/WDR4-mediated transfer RNA (tRNA) N7-methylguanosine (m7G) modification in exosomes of radiation-exposed lung epithelial cell, thereby lightening the translation efficiency and protein expression of the target gene SPHK1 regulated by tRNA m7G modification in lung fibroblasts through the exosomes pathway. Moreover, RA suppressed the acetylation of PFKFB3 in the nucleus and decreased the level of phosphorylated PFKFB3 in the cytoplasm, ultimately reducing FMT triggered by glycolysis in lung fibroblasts. Our findings provide the novel therapeutic targets for RIPF as well as insights into its molecular pathophysiology.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eAnimals and Treatments\u003c/h2\u003e\nEighteen male C57BL/6 mice (7 weeks of age) were obtained from the SipeiFu Biotechnology Co., Ltd (Beijing, China). To establish a RIPF model, mice were randomly divided into three groups: the Control (Ctrl) group, irradiation (IR) group and rosmarinic acid plus irradiation (RA\u0026thinsp;+\u0026thinsp;IR) group. The chest regions of mice in the IR group and RA\u0026thinsp;+\u0026thinsp;IR group received a single dose of 15 Gy (Varian, California, USA), and the remaining body parts were shielded. RA was purchased from MedchemExpress (New Jersey, USA) and orally administered from 7 days (1 mg/g/day) before irradiation to 24 weeks after irradiation, while mice in the Ctrl group and IR group were administrated with saline. The animal experiments were approved by the Animal Ethics Committee of GuangXi Medical University.\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA Extraction and Quantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e\n\u003cp\u003eTotal RNA was extracted by TRIzol reagent (Invitrogen, USA) according to the manufacturer\u0026rsquo;s instructions, and then the concentration and quality of the RNA were measured. Total RNA was reverse transcribed into cDNA with the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan). qPCR was conducted to evaluate mRNA levels using the SYBR Green method. The expression level of genes was normalized to that of GAPDH and quantified by the 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eWestern blotting\u003c/h2\u003e\n\u003cp\u003eCells were lysed with RIPA lysis buffer, the BCA Protein Assay Kit was used to measure the protein concentration (Beyotime, China), and proteins were separated by SDS‒PAGE and transferred to polyvinylidene difluoride membranes. Then, 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used to block the proteins for 1 h at room temperature, and the membranes were incubated with primary antibodies (1:1,000) overnight at 4\u0026deg;C. The, the membranes were incubated with secondary antibodies (1:5,000) for 1 h at room temperature. Enhanced chemiluminescence reagent was used to detect the protein signals. The following antibodies were used: METTL1 Polyclonal antibody (Proteintech, 14994-1-AP), Rabbit anti-WDR4 Polyclonal Antibody (absin, abs152662), SPHK1 Polyclonal antibody (Proteintech, 10670-1-AP), NAT10 Polyclonal antibody(Proteintech; 13365-1-AP), acetyl-lysine (Affinity, DF7729), PFKFB3 Antibody (Affinity, DF12016), Phospho-PFKFB3 (Ser461) Antibody (Affinity, AF3581), Phospho-AMPK alpha (Thr172) Antibody (Affinity, AF3423), AMPK alpha Antibody (Affinity, AF6423), Actin (Proteintech, 81115-1-RR)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eCell lines, transfection and intervention\u003c/h2\u003e\n\u003cp\u003eThe mouse lung epithelial cell lines TC-1 and MLE-12 were gifted from Guangxi Key Laboratory of Immunology and Metabolism for Liver Diseases, and mouse lung fibroblasts (MLFs) were purchased from Saios (Wuhan, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin streptomycin in a 37 \u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were digested with 0.25% trypsin every 2\u0026ndash;3 days.\u003c/p\u003e\n\u003cp\u003eWhen the cells reached approximately 80% confluence, Lipofectamine 3000 (Invitrogen, USA) was utilized to transfect siRNAs and plasmids. METTL1 and WDR4 overexpression plasmids and METTL1 and WDR4 siRNAs were generated by GenePharma (Shanghai, China).\u003c/p\u003e\n\u003cp\u003eTC-1 cells and MLE-12 cells were divided into the Ctrl, IR and RA\u0026thinsp;+\u0026thinsp;IR group, respectively. TC-1 and MLE-12 cells were pretreated with RA (150 \u0026micro;M) 24h prior to irradiation in both RA groups. A single dose of 4 Gy x-rays was delivered at a rate of 400 cGy/min to TC-1 and MLE-12 cells in all the irradiation groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eExtraction of exosomes\u003c/h2\u003e\n\u003cp\u003eCell supernatants were centrifuged at 10,000 \u0026times;g for 45 min at 4 \u0026deg;C to remove the larger vesicles. The supernatants were extracted and filtered through a 0.45 \u0026micro;m filter. Then, the samples were centrifuged again at 10,000 \u0026times;g and at 4 \u0026deg;C for 70 min and resuspended in PBS. After removing the supernatants, 100 \u0026micro;L of PBS was used to resuspend the samples. Some exosomes (20 \u0026micro;L) were used for electron microscopy analysis, some exosomes (10 \u0026micro;L) were used for particle size analysis, and the remaining exosomes were stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eEdu incorporation and staining\u003c/h2\u003e\n\u003cp\u003eEdu assay was performed using BeyoClick\u0026trade; Edu Cell Proliferation Kit with Alexa Fluor 594 following the manufacturer\u0026rsquo;s instruction (beyotime, china). In biref, Edu working solution was added to the cells and incubated for 2 h. The cells were subsequently fixed and permeabilized, and click additive solution was added to the samples and incubated in the dark for 30 min. DAPI was utilized to label the nuclei for 3 min. Images were captured by fluorescence microscopy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eHematoxylin and eosin (HE) staining\u003c/h2\u003e\n\u003cp\u003eHE staining was performed using Hematoxylin-Eosin (HE) Stain Kit following the manufacturer\u0026rsquo;s instruction (solarbio, china). In biref, Paraffin sections were dewaxed with xylene and dehydrated with alcohol, hematoxylin was used to stain the nuclei, and eosin staining was used to stain the cytoplasm. After dehydration and sealing with neutral resin, the sections were examined under a microscope, and images were collected and analyzed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eMasson staining\u003c/h2\u003e\n\u003cp\u003eMasson staining was performed using Modified Masson's Trichrome Stain Kit following the manufacturer' s instruction (solarbio, china). In biref, After paraffin sections were dewaxed, the sections were stained with Weigert's iron hematoxylin for 5 min, washed with tap water, and differentiated with 1% hydrochloric acid alcohol for several seconds. The sections were stained with Ponceau red acid fuchsin solution for 5 min and treated with phosphomolybdic acid aqueous solution for approximately 3 min. The sections were counterstained with aniline blue solution for 5 min and then treated with 1% glacial acetic acid for 1 min. Then, the sections were dehydrated and sealed for microscopic examination, and the images were collected and analyzed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eWound-healing assays\u003c/h2\u003e\n\u003cp\u003eWhen the cells had grown to 80%, they were cultured in serum-free medium. After starvation for 12 h, the medium was discarded, and the cell monolayers were scratched with 200 \u0026micro;l pipette tips. The left and right sides of each intersection were photographed at 0 h and 24 h with the crossed point as the mark.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e\n\u003cp\u003eSections were incubated in a 60 \u0026deg;C incubator for 60 min, followed by dewaxing and hydration. Antigen retrieval was performed using citrate buffer. The sections were blocked with 5% goat serum and incubated with a-SMA, Collagen I and Fibronectin antibodies overnight. Secondary antibody incubation and DAB color development were performed. After counterstaining with hematoxylin, the sections were dehydrated, cleared, and sealed. Images were collected on the HAMAMATSU NANO ZOOMER system.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\n\u003cp\u003eCells were seeded into climbing pieces, fixed using formaldehyde, and permeabilized using 0.5% Triton X-100. The cells were blocked with the BSA blocking solution, incubated with the primary antibodies at room temperature for 1 h, and incubated with the secondary antibodies in the dark at room temperature for 1 h. A drop of sealing agent was added to the climbing film, and the samples were evaluated under a fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eMeRIP-m7G-tRNA sequencing (m7G-tRNA-seq)\u003c/h2\u003e\n\u003cp\u003eSmall RNAs with lengths less than 200 nt were enriched from total RNA with a mirVana Isolation Kit (Thermo Fisher). The GenSeq \u0026reg; M7G MeRIP Kit (GenSeq, Inc.) was used to conduct a MeRIP experiment on the enriched small RNA according to the instructions of the kit. Merip/input RNA samples were demethylated with the alkB enzyme at 37\u0026deg;C for 100 min. After demethylation, small RNAs were used for small RNA library construction with the GenSeq \u0026reg; Small RNA library prep Kit (GenSeq, Inc.). Libraries within the tRNA length range were purified by fragment screening and then sequenced on an Illumina NovaSeq sequencer.\u003c/p\u003e\n\u003cp\u003etRNA data were downloaded from the GtRNAdb website. According to their anticodons and scores, representative tRNAs were selected from among these tRNAs. Three bases of CCA were added to the 3' end of the tRNA sequences. After sequencing, image analysis and base recognition, the raw reads after quality control were harvested. First, q30 was used for quality control, and then cutadapt software (v1.9.3) was used to splice the original reads and remove low-quality reads. Finally, reads with a length\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;15 nt were retained to identify the spliced reads (i.e., trimmed reads). Then, the trimmed reads of each sample were aligned to the preprepared tRNA database using Bowtie2 software (v2.2.4). SAMtools (V1.3.1) was used to count the number of reads that were aligned to each tRNA as the original expression of the tRNA. The results of IP and input were normalized using the TPM method, and IP/input was calculated and considered the tRNA methylation level according to the normalized results. Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 was considered as differentially difference.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003etRNA sequencing (tRNA-seq)\u003c/h2\u003e\n\u003cp\u003eThe tRNA sequencing service was provided by Shanghai Yunxu Biological Company, and the process was performed as follows. Briefly, small RNAs with lengths less than 200 nt were enriched from total RNA with a mirVana Isolation Kit (Thermo Fisher). The enriched small RNAs were treated with alkB enzyme for 100 min at 37\u0026deg;C. According to the instructions of the GenSeq \u0026reg; small RNA Library Prep Kit (GenSeq, Inc.), a small RNA library was constructed from the treated samples. Libraries within the tRNA length range were enriched by fragment screening and then sequenced on an Illumina NovaSeq sequencer.\u003c/p\u003e\n\u003cp\u003etRNA data were downloaded from the GtRNAdb website. According to the anticodons and scores, representative tRNAs were selected from among these tRNAs. Three bases of CCA were added to the 3' end of their sequences. After Illumina sequencer sequencing, image analysis and base recognition, the raw reads after quality control were harvested. First, q30 was used for quality control, and then cutadapt software (v1.9.3) was used to splice the original reads and remove low-quality reads. Finally, reads with length\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;15 nt were retained to identify the spliced reads (i.e., trimmed reads). Then, the trimmed reads of each sample were aligned to the preprepared tRNA database using Bowtie2 software (v2.2.4). SAMtools (V1.3.1) was used to count the number of reads that were aligned to each tRNA as the original expression of the tRNA, and edger software was used for data normalization and differential expression screening. Differences were analyzed by t test. Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;2.0 and p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as differentially difference.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003emRNA sequencing (mRNA-seq)\u003c/h2\u003e\n\u003cp\u003eRibosomal RNA (rRNA) of samples was removed by rRNA Removal Kit (genseq, Inc.) kit, sequencing library was constructed by genseq \u0026reg; The low input RNA library prep Kit (genseq, Inc.) according to the instructions. After that, the constructed sequencing library was subjected to quality control and quantification by Bioanalyzer 2100 system (Agilent Technologies, USA), followed by 150bp paired end sequencing using Illumina novaseq 6000 instrument. After sequencing with Illumina novaseq 6000 sequencer, the original data were obtained. First, the q30 value is used for raw data quality control. We use cutadapt software (v1.9.3) to remove connectors, remove low-quality reads, and obtain high-quality clean reads. Hisat2 software was used to align the clean reads to the reference genome, and then htseq software (v0.9.1) was used to obtain the original count number. Edger was used to normalize and calculate the fold change and p-value between the two groups of samples to screen the differentially expressed genes, Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;2.0 was considered as differentially expressed genes. Go function analysis and KEGG pathway analysis were performed using differentially expressed mRNA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003ePolyribosome-bound mRNA sequencing (Ribo-seq)\u003c/h2\u003e\n\u003cp\u003eCells were treated with cycloheximide and lysed using lysis buffer and then digested with nuclease. The digested samples were separated into single ribosomes with a size exclusion column. Fragment selection was performed on RNA fragments that were protected by ribosomes using polyacrylamide gel electrophoresis, and then rRNA was removed from the samples using rRNA removal reagents. After purification, the RNA ends were repaired, and a 3 'connector was added; then, the samples were transformed into cDNA through reverse transcription. cDNA was purified by polyacrylamide gel electrophoresis, cyclized, and amplified by PCR. The amplified library that was obtained was purified and sequenced on a NovaSeq sequencer (Illumina). After sequencing with an Illumina NovaSeq 6000 sequencer, the raw data were obtained. First, the Q30 value was used for raw data quality control. Cutadapt software (v1.9.3) was used to remove connectors, remove low-quality reads, and obtain high-quality clean reads. Bowtie was used to compare the disconnected data to rRNA sequences and to obtain clean reads that had not been aligned to rRNA. Tophat2 software was used to compare clean reads to the reference genome. Then, HTSeq software (v0.9.1) was used to obtain the original count number, edgeR or DESeq2 was used for standardization, and the multiple changes and p values between the two groups of samples were calculated to identify differentially expressed genes. By default, edgeR was used for differential analysis. Correlation analysis between the mRNA-seq and the Ribo-seq was perform. The ratio of FPKM of each gene in Ribo-seq to FPKM in mRNA-seq was consider as the translation efficiency (TE). Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;2.0 was considered as differentially difference.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eNorthern blotting and northwestern blotting\u003c/h2\u003e\n\u003cp\u003eFor northern blotting, 2\u0026micro;g total RNA samples were mixed with RNA loading buffer (2X) and denatured at 65 C for 15 min, then, the sample were added into 15% Urea-PAGE electrophoresis in 1X TBE buffer. The separated RNAs were then transferred into a positively charged nylon membrane, cross-linking was performed by Ultraviolet (UV) light. The tRNAs or U6 snoRNA were blotted with corresponding digoxigenin-labeled probes. For Northwestern blotting, the the RNA-containing nylon membranes was crosslinked with UV and blotted with an anti-m7G antibody (RN017M, Medical Biological Laboratories, Nagoya, Japan). After incubation of BeyoECL Moon buffer with nylon membrane, the signals were detected according to previous reports [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eCoomassie Blue Staining\u003c/h2\u003e\n\u003cp\u003ePAGE gel was soaked by BeyoBlue\u0026trade; Coomassie Blue Super Fast Staining Solution (Beyotime Biotechnology, China) and dyed at room temperature on a side sway shaker for 30 minutes, then discard the decolorization solution, add deionized water for decolorization on a shaker.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eCo-Immunoprecipitation (CoIP)\u003c/h2\u003e\n\u003cp\u003eCells were lysed and incubated with indicated antibody overnight, then Protein A/G-MagBeads was added to the sample, eluting was conducted by Elution buffer, the indicated proteins was obtained.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eSeahorse assay\u003c/h2\u003e\n\u003cp\u003eThe extracellular acidification rate (ECAR) of cells was measure using a Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Agilent). In brief, 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e Cells were seeded into Agilent Seahorse XFe96 plates and cultured for 12 h in a standard incubator. After that, the cells were treated with 10 mM glucose, 2\u0026micro;M oligo-mycin and 2-deoxy- D -glucose in different ports of the Seahorse cartridge.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eAll the continuous variables are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between the two groups were analyzed by Student\u0026rsquo;s t test. The data were analyzed and visualized by SPSS 21.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 7 (GraphPad, San Diego, CA, USA). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistically significant differences.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003eRA prevented RIPF and inhibited the progression of FMT\u003c/h2\u003e\n\u003cp\u003eOur previous study have shown that RA can alleviate RIPF in rats [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], and it is recognized that FMT plays a core role in the pathogenesis of organ fibrosis [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the present study, we further evaluated the effect of RA on RIPF and FMT in C57BL/6 mice. It is observed that RA-treated mice had improved lung morphology, with less lung collapse and fibrous nodules (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Lung index that was referred to lung/body weight ratio decreased significantly in the RA\u0026thinsp;+\u0026thinsp;IR group comparing with that in the IR group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). The RA-treated mice showed attenuated fibrosis versus irradiated mice without RA treatment, as indicated by less thickened alveolar walls, fibrotic foci and collagen deposition, which was in line with decreases in the ashcroft score and collagen volume fraction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The expression of FMT markers \u0026alpha;-SMA, collagen I and fibronectin in RA-treated mice was lower than that in irradiated mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results indicated that RA exerted an antifibrotic effect on lung tissues of RIPF mouse models and suppressed the progression of FMT in irradiated mouse lung tissues.\u003c/p\u003e\n\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n\u003ch2\u003eRA decelerated the progression of FMT through exosomes pathway\u003c/h2\u003e\n\u003cp\u003eTo explore the mechanism underlying RIPF, exosomes derived from the lung epithelial cells were identified and quantified by transmission electron microscopy. Nanoparticle tracking analysis demonstrated that the average sizes of exosomes isolated from TC-1 and MLE-12 cells were approximately 84.49 and 86.93 nm, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, uptake of exosomes by MLFs were observed after 24h when coincubating MLFs with TC-1 cell-derived exosomes (TC-1-exo) or MLE-12 cell-derived exosomes (MLE-12-exo) that were labeled with PKH67 (red) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Interestingly, immunofluorescence staining showed that incubating MLFs with exosomes that derived from irradiated epithelial cells with RA treatment (RA\u0026thinsp;+\u0026thinsp;IR-TC-1/MLE-12-exo) inhibited the induction of \u0026alpha;-SMA expression \u003cspan class=\"Underline\"\u003ein\u003c/span\u003e MLFs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Besides, Edu staining and wound-healing assays showed that treating MLFs with RA\u0026thinsp;+\u0026thinsp;IR-TC-1/MLE-12-exo significantly decreased the effect of IR-TC-1/MLE-12-exo on inducing proliferation and migration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). Together, these results indicated that RA suppressed FMT of MLFs induced by exosomes that were derived from irradiated lung epithelial cells.\u003c/p\u003e\n\u003cstrong\u003eRA reversed the increase in expression and m7G modification level of tRNA that induced by irradiation in lung epithelial cells\u003c/strong\u003e\u003cbr /\u003e\n\u003cp\u003eMany studies have shown that tRNA epigenetic modifications are associated with the pathological processes of numerous diseases including organ fibrosis [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. To assess the correlation between RIPF and tRNA m7G modification that is one of the most common epigenetic modifications, m7G-tRNA-seq and tRNA-seq analysis were performed in TC-1 cells (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). We found that the expression and m7G modification level of tRNA-ArgCCG and tRNA-CysGCA were significantly higher in the IR group than those in the Ctrl group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Northwestern and northern blotting analysis further confirmed the results, and found that the increase in expression and m7G modification level of tRNA-ArgCCG and tRNA-CysGCA caused by irradiation could be reversed by RA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). We then investigated the effect of RA on the METTL1/WDR4 complex, which is the main catalytic complex for tRNA m7G modification in eukaryotic cells. Western blotting results revealed that RA reduced the upregulation of METTL1 and WDR4 protein expression induced by irradiation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). In addition, silencing METTL1 and WDR4 eliminated the promoting effect of irradiaton on the expression and m7G modification of the indicated tRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). Taken together, our results indicated that the tRNA level and m7G modification increased in the lung epithelial cells after irradiation, and RA reversed these changes.\u003c/p\u003e\n\u003cstrong\u003eRA suppressed the process of FMT by downregulating METTL1/WDR4-mediated tRNAs m7G modification in exosomes of lung epithelial cells\u003c/strong\u003e\u003cbr /\u003e\n\u003cp\u003eTo further confirm whether tRNA level and m7G modification also increasing in exosomes, we performed northwestern, northern blotting and immunofluorescence staining. The results revealed that the levels of m7G modification and expression of tRNAs in exosomes of the irradiated lung epithelial cells were higher than controls, while RA treatment can partially reverse those effect of irradiation on exosomes of lung epithelial cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, exosomes of the METTL1/WDR4 overexpressed TC-1 cells (oeMETTL1/MDR4-TC-1-exo) showed a high tRNAs expression and m7G modification level, but no similar results were observed in the RA intervention group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). m7G modification levels in MLFs that were incubated with exosomes of TC-1 cells in the Ctrl, IR, and RA\u0026thinsp;+\u0026thinsp;IR (Ctrl-TC-1-exo, IR-TC-1-exo and RA\u0026thinsp;+\u0026thinsp;IR-TC-1-exo) group were analyzed by northwestern and northern blotting, and the results confirmed that the expression of tRNAs m7G modification increased in the IR-TC-1-exo group and RA\u0026thinsp;+\u0026thinsp;IR-TC-1-exo group, especially the former showing a more significant increase (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Besides, immunofluorescence staining showed that incubating MLFs with oeMETTL1/MDR4-TC-1/MLE-12-exo plus RA diminished the expression of \u0026alpha;-SMA induced by these exosomes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Consistently, Edu staining and wound-healing assays indicated that MLFs treated with oeMETTL1/MDR4-TC-1-exo and RA had a lower proliferation and migration ability than those without RA intervention (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH, Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eB, and Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In conclusion, these results confirmed that RA had a negative regulatory effect on tRNAs m7G modification level in exosomes of lung epithelial cells.\u003c/p\u003e\n\u003cstrong\u003eRA regulated the translation efficiency of SPHK1 and subsequently affected the interaction between SPHK1 and NAT10\u003c/strong\u003e\u003cbr /\u003e\n\u003cp\u003eSince tRNA is mainly involved in protein synthesis, it is plausible to speculate that the abnormal tRNA expression may affect mRNA translation. In our present study, Ribo-seq and mRNA-seq analysis was performed to explore the underlying target mRNA. We identified mRNA that had no significant difference in expression levels assesed by mRNA-Seq but had significant difference in translation efficiency assesed by Ribo-seq as the target genes for modifying tRNA [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Comparing with MLFs in the Ctrl-TC-1-exo group, MLFs in the IR-TC-1-exo group had 3101 mRNAs with decreased translation ratios (TRs) and 2086 mRNAs with increased TRs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e), in which we noticed sphingosine kinase type 1 isoform (SPHK1) had a significantly increased TR. SPHK1 has been reported to regulate pulmonary, hepatic, and renal fibrosis [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], yet the role of SPHK1 in RIPF has not been elucidated. KEGG enrichment analysis of these mRNAs with increased TRs showed significant enrichment in the Wnt signaling pathway, MAPK signaling pathway, and ECM-receptor interaction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). qRT-PCR and western blotting further revealed that IR-TC-1-exo and RA\u0026thinsp;+\u0026thinsp;IR-TC-1-exo had little effect on the mRNA expression of SPHK1 in MLFs, but had a significant impact on protein expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Subsequently, polyribosome-qPCR assay confirmed that the translation efficiency of SPHK1 in the IR-TC-1-exo group increased compared to the Ctrl-TC-1-exo group, while the translation efficiency of SPHK1 was found to be decreasing in the RA\u0026thinsp;+\u0026thinsp;IR-TC-1-exo group compared to the IR-TC-1-exo group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). Recently, N-acetyltransferase 10 (NAT10) was reported to enhance pulmonary fibrosis [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Based on HDOCK database retrieval, NAT10 was found to be potentially bound to SPHK1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). The coomassie blue staining assay further prompted the interaction between NAT10 and SPHK1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). What\u0026rsquo;s more, CoIP assays verified that SPHK1 directly interacted with NAT10 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). Taken together, our results indicated that IR-TC-1-exo heightened the translation ratio of SPHK1 in MLFs and affected the binding between SPHK1 and NAT10 proteins, while RA can counteract the effect of IR-TC-1-exo on MLFs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n\u003ch2\u003eRA diminished glycolysis by reducing acetylated PFKFB3 and cytoplasmic translocation\u003c/h2\u003e\n\u003cp\u003eMyofibroblasts may use aerobic glycolysis as an additional source of bioenergetics and biosynthesis to meet the demands related to fast growth and proliferation [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, the Seahorse assay showed that compared with the control group, the extracellular acidification rate (ECAR) of the IR-TC-1-exo group significantly increased, while the RA\u0026thinsp;+\u0026thinsp;IR-TC-1-exo group only slightly increased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), indicating that RA may inhibit the glycolysis of MLFs via exosomes pathway derived from lung epithelial cells. Previous studies have proven that the acetylation of metabolic enzymes is a critical mechanism underlying metabolic modulation [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. PFKFB3, as a key enzyme in glycolysis, has been proven to be a driving factor for various organ fibrosis diseases [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e], which prompted us to investigate the mechanism underlying PFKFB3 regulation by exosomes. The results showed that IR-TC-1-exo treatment can improve the acetylation level of PFKFB3, and the addition of RA partially counteracts this effect (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). NAT10 belongs to the Gcn5-related N-acetyltransferase family, which has been reported to acetylate RNAs and proteins [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Interestingly, we observed that Remodelin (inhibitor of NAT10) reduced the acetylation of PFKFB3 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). PFKFB3 is localized to the nucleus, which may occur due to the inclusion of a classical nuclear localization signal (KKPR, amino acids 472\u0026ndash;475) [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Mass spectrometric analysis by Li et al. revealed six lysine (K) residues in PFKFB3 that undergo acetylation (K12, K284, K302, K451, K472 and K473) [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Consequently, we speculated that K472 and K473 were the major acetylation sites that interfered with the nuclear localization of PFKFB3. As expected, we found that mutation of the K472R and K473R sites greatly reduced the acetylation of PFKFB3 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). FISH assays indicated that wild type (WT) PFKFB3 was localized in the nucleus, while the K472Q and K473Q mutants localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). In addition, cytoplasmic PFKFB3 has a stronger effect on promoting glycolysis [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. AMPK activates PFKFB3 and promotes glycolysis through the phosphorylation of PFKFB3 at S461 [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. To verify the cytoplasmic localization of PFKFB3 has an effect on its phosphorylation at the S461 site, western bloting was performed. The results showed that S461 phosphorylation increased in the IR-TC-1-exo group, and this effect was reversed in the RA\u0026thinsp;+\u0026thinsp;IR-TC-1-exo group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Cells expressing the K472Q or K473Q mutants exhibited higher phosphorylation of S461 and activity of PFKFB3 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH) as well as a higher ECAR than those in cells expressing WT PFKFB3 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI). Taken together, these results indicated that IR-TC-1-exo promoted PFKFB3 acetylation at K472 and K473 and induced the translocation of PFKFB3 from the nucleus to the cytoplasm in MLFs. Additionally, cytoplasmic PFKFB3 was phosphorylated by AMPK, and then the glycolytic process was enhanced. On the other hand, RA reduced these changes.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRIPF is a serious complication of radiotherapy that is difficult to reverse. At present, there is a lack of effective treatment methods for it. In this study, we have uncovered several interesting findings: high-throughput sequencing results showed that the expression and m7G modification level of tRNA increased in the exosomes derived from irradiated lung epithelial cells. In addition, RA inhibited the translation efficiency and protein expression of SPHK1 that regulated by tRNA m7G modification, as well as suppressing the acetylation of PFKFB3 in the nucleus. Ulteriorly, it decreasd the level of phosphorylated PFKFB3 in the cytoplasm, and ultimately reducing FMT triggered by glycolysis in lung fibroblasts.\u003c/p\u003e \u003cp\u003eNumerous studies have shown that cells can transmit important signaling molecules, such as RNA and proteins, and thus promote FMT through exosomes. Huang et al. found that exosomal SPP1 derived from silica-treated macrophages can trigger FMT and promote pulmonary fibrosis to form silicosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Li et al. reported that miR-192-5p in exosomes derived from adipose tissue mesenchymal stem cells reduced collagen deposition and FMT by targeting the IL-17RA/Smad signaling axis, thereby exerting an inhibitory effect on scar hyperplasia [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, we extracted exosomes of irradiated lung epithelial cells. Transmission electron microscopy and nanoparticle tracing analysis confirmed that irradiated TC-1 cell-derived exosomes can be taken up by MLFs, which promoted the expression of the FMT marker protein α-SMA and enhanced the proliferation and migration of MLFs. After treatment with RA, exosomes of irradiated lung epithelial cells (RA\u0026thinsp;+\u0026thinsp;IR-TC-1-exo) significantly inhibited the FMT of MLFs compared to IR-TC-1-exo. These results indicated that RA can inhibit FMT via exosomes derived from lung epithelial cells.\u003c/p\u003e \u003cp\u003eIn recent years, increasing evidence has shown that epigenetic modifications of tRNAs are associated with the pathological processes of various diseases. For example, dysregulated tRNA m7G modification exerted a carcinogenic effect on the esophageal squamous cell carcinoma [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In patients with NSUN2 gene mutations, the lack of specific 5-cytosine methylation at the C47 and C48 sites of the tRNA\u003csup\u003eAsp\u003c/sup\u003e led to moderate to severe intellectual impairment, facial deformities, and distal myopathy [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Another studie have pointed out that the key pathogenesis that is caused by CDKAL1 deficiency in diabetes patients is the β-mistranslation of Lys codons in cells, which leads to a decrease in glucose-stimulated insulin synthesis; the underlying molecular mechanism may be related to the 2-methylthio-modification of N\u003csup\u003e6\u003c/sup\u003e-threonylcarbonyladenosine at position-37 in tRNA\u003csub\u003eUUUU\u003c/sub\u003e\u003csup\u003eLys3\u003c/sup\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, there is currently no research on the epigenetic modification of tRNA in RIPF.\u003c/p\u003e \u003cp\u003em7G modification is one of the most common epigenetic modifications of RNA, playing an important role in maintaining the integrity and stability of tRNA. Our study conducted m7G-tRNA-seq and tRNA-seq analysis and found that the m7G modification and expression of tRNA-ArgCCG and tRNA-CysGCA were most significantly increased in the irradiated lung epithelial cells. Further validation showed that RA treatment could reverse these change and radiation-induced high expression of m7G methyltransferase complex components (METTL1 and WDR4). In addition, we also found that after upregulating METTL1 and WDR4 in lung epithelial cells and incubating MLFs with their exosomes, the expression level of α-SMA significantly increased, and the proliferation and migration of MLFs were enhanced. These results suggested that RA may downregulate the transmission of tRNA-ArgCCG and tRNA-CysGCA to MLFs by inhibiting these tRNA m7G modification in the lung epithelial cells, ultimately slowing down the FMT process in MLFs.\u003c/p\u003e \u003cp\u003eGlycolysis is an important metabolic pathway that occurs in almost all living cells. Research has shown that glycolysis is enhanced in fibroblasts and myofibroblasts of the liver, lungs, and kidneys during chronic inflammation and fibrosis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Besides, it is reported that during the process of fibrosis in organs such as the lungs, cell-dependent energy metabolism gradually shifted from oxidative phosphorylation to glycolysis, which is also known as the Warburg effect [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, the role of glycolysis in RIPF is not yet clear. PFKFB3 catalyzes the synthesis and hydrolysis of the small molecule fructose-2,6-diphosphate, which is a potent activator of the glycolytic pathway. Among the four members of the PFKFB protein family, PFKFB3 is the only protein that is located in the nucleus [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our study showed that after incubating MLFs with IR-TC-1-exo, the level of acetylated PFKFB3 at the K472 and K473 sites was enhanced, as well as the level of phosphorylation at the S461 site increased. On the other hand, RA inhibited these changes. Based on the FISH experiments, we observed that PFKFB3 protein was originally primarily expressed in the nucleus of MLFs, while PFKFB3 protein was translocated from the nucleus to the cytoplasm after incubation with IR-TC-1-exo in MLFs. However, treatment with RA could reverse the nuclear-cytoplasmic translocation of PFKFB3. Similar results were also observed by Li et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], who found that the chemotherapy drug cisplatin accumulated PFKFB3 in the cytoplasm and promoted glycolysis. Specifically, the lysine residue at position 472 of the PFKFB3 protein was acetylated, which inactivated the nuclear localization signal of PFKFB3 and promoted its retention in the cytoplasm. PFKFB3, which was located in the cytoplasm, was more susceptible to phosphorylation by the kinase AMPK, leading to the activation of PFKFB3 and promoting glycolysis, thereby protecting cells from apoptosis. Together, our study reveals a novel mechanism for regulating the activity of metabolic regulatory enzyme PFKFB3 through acetylation in RIPF and suggests targeted inhibition of PFKFB3 by RA may be a new clinical strategy for treating RIPF.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, these findings presented here suggested that RA alleviated RIPF by downregulating tRNA m7G modification and expression level-regulated FMT through the exosomes pathway. RA may be a potential therapeutic drug for slowing down the progression of RIPF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at GSE249534 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE249534).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the support of Guangxi Key Laboratory of Immunology and Metabolism for Liver Diseases, Key Laboratory of Early Prevention and Treatment for Regional High-Frequency Tumors of Guangxi Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.Z, J.M and Z.O conducted the cellular and animal experiments, X.Q, Y.W and Z.L helped to the animal experiments, S.H and K.H helped to data analysis and figure design, M.H and R.W participated in the design of the study and performed the statistical analysis, T.Z drafted the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the \u0026ldquo;Medical Excellence Award\u0026rdquo; Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University, the Basic Ability Enhancement Project of Young Teachers in Guangxi Zhuang Autonomous Region (No. 2023KY0120) and Nanning Qingxiu district key research and development plan for Science and Technology (No. 2020019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003eAll the authors declare no conflicts of interest in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures conducted in this work comply with the ethical standards of the institution and/or the National Research Council.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHe Y, Thummuri D, Zheng G, Okunieff P, Citrin DE, Vujaskovic Z, et al. 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Cell. 2013;154:651\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rosmarinic acid, Exosomes, tRNA, N7-methylguanosine (m7G), Radiation-induced pulmonary fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-3744363/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3744363/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusions\u003c/b\u003e These findings suggest RA as a potential therapeutic agent for RIPF.\u003c/p\u003e","manuscriptTitle":"Rosmarinic acid alleviates radiation-induced pulmonary fibrosis by downregulating tRNA N7-Methylguanosine modification-regulated fibroblast to myofibroblast transition through the exosomes pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-15 21:33:59","doi":"10.21203/rs.3.rs-3744363/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7bd05004-f221-4df0-8ab9-20ef6042bff0","owner":[],"postedDate":"December 15th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-19T13:29:24+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-15 21:33:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3744363","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3744363","identity":"rs-3744363","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}