The Gene Expression Omnibus (GEO) database was searched for datasets on patients with IPF, and differential gene analysis was subsequently conducted on datasets GSE49072, GSE40839, GSE135065 and GSE150910 (14–17) using the ‘limma’ package (version 3.66.0) in R (4.5.2). The screening criterion for differentially expressed genes included |log₂fold-change|>1 and an adjusted P-value <0.05. Venn diagrams were generated using the R package ggvenn (version 0.1.19).

The GEO database was searched for datasets containing survival information regarding patients with IPF. Survival analysis was conducted using datasets GSE70866 and GSE27957 (18,19). The Kaplan-Meier method was used to analyze the overall survival period of patients with IPF, defining death as the endpoint event. Patients in each dataset were divided into high- and low-expression groups based on the optimal cutoff value of SLC7A5 mRNA expression, which was determined separately for each dataset: 6.78 for GSE70866 and 7.03 for GSE27957, and log-rank tests were performed to compare the survival of patients between groups. The survival curves were visualized using the R package ‘survminer’ (version 0.4.9) and a risk table was provided (Fig. 1).

RNA was extracted from HFL1 cells using the total RNA extraction kit [Seven Innovation (Beijing) Biotechnology Co., Ltd.; cat. no. SM132] and sent to Novogene for sequencing. The quality and integrity of the processed RNA samples were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Sequencing libraries were prepared using the Fast RNA-seq Lib Prep Kit V2 (cat. no. RK20306; ABclonal Biotech Co., Ltd.) according to the manufacturer's instructions. The library concentration was determined by quantitative PCR (qPCR), and the final loading concentration of the library was 1.5 ng/µl. Sequencing was performed on the NovaSeq X Plus platform (Illumina, Inc.) with paired-end 150 bp (PE150) sequencing reads. The ‘limma’ package (version 3.66.0) in R (4.5.2) was used to conduct differential analysis on the expression data from transcriptome sequencing.

Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were both conducted using the ‘clusterProfiler’ package in R. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA_4.3.2 software. The hallmark gene sets, C2 curated gene set collection and KEGG subset from the Molecular Signatures Database (MSigDB; http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp#C2) was used for GSEA. GSEA was performed on the transcriptomic data of datasets GSE101286 (20) and GSE135065. Furthermore, the differentially expressed genes identified via transcriptome sequencing were inputted into the Search Tool for the Retrieval of Interacting Genes/Proteins database platform (v12.0; cn.string-db.org) in order to conduct protein-protein interaction (PPI) analyses. P<0.05 and false discovery rate <0.25 were considered to indicate statistically significant differences.

In the present study, healthy wild-type male C57BL/6J mice aged 8–10 weeks were selected as the experimental animals. Mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Charles River Laboratories, Inc.) and each mouse weighed ~25 g. The mice used in the present study were raised in a specific pathogen-free environment. The environmental conditions were maintained at a constant indoor temperature of 20–26°C with a relative humidity of 40–70%, 12-h light/dark cycle, and noise controlled <60 dB to provide a quiet environment. The mice were given ad libitum access to sufficient drinking water and food, which had been sterilized. The mouse cages and bedding materials were sterilized by autoclaving (high-pressure steam) and regularly replaced each week. All animal experiments involved in the present study were approved by the Experimental Animal Protection and Ethics Committee of Harbin Medical University (Harbin, China; approval no. YJSDW2023-024).

A pulmonary fibrosis animal model was established in C57BL/6J mice via intratracheal instillation of bleomycin (BLM; cat. no. s1214; Selleck Chemicals). Mice were fasted and dehydrated for 1–2 h before anesthesia. Subsequently, mice were anesthetized by inhalation of isoflurane (induction concentration, 4%; maintenance concentration, 2%) and 3.5 mg/kg of BLM was injected into the trachea of model mice to establish a model of IPF; mice in the normal saline (NS) control group were injected with the same volume of NS.

JPH203 (cat. no. TQ0081; TargetMol Chemicals Inc.) was used to treat mice with pulmonary fibrosis induced by BLM. From days 1 to 13 following the establishment of the BLM model, mice were administered JPH203 by intraperitoneal injection daily, with mice divided into additional groups based on drug doses (12.5 and 25 mg/kg/d).

Immediately after the BLM infusion was completed, the anesthesia recovery status of mice was observed, which included monitoring the breathing, heart rate and body temperature of mice. After modeling, mice in the NS, BLM group and BLM + JPH203 groups were housed separately. The health status of mice was observed daily for 14 consecutive days without interruption after BLM induction, which involved monitoring for changes in body weight, appearance of the mice, clinical symptoms, behavioral responses and social behaviors. On the 14th day following BLM infusion, the mice were euthanized and lung, heart, liver, spleen and kidney tissues were collected. Mice were euthanized before reaching the experimental endpoint if the following humane endpoints were displayed: i) A decrease in body weight of 20–25%; ii) cachexia or wasting symptoms; iii) breathing difficulties, specifically regarding saliva flowing from the mouth and nose, cyanosis, arching of the back or lying down; iv) a complete loss of appetite for 24 h or a poor appetite (<50% of normal appetite) for 3 days; v) inability to eat or drink without anesthesia; or vi) mice were only able stand with great difficulty for 24 h. These humane endpoints were similar to those reported in previous studies (21,22). Euthanasia was performed by inducing deep anesthesia via inhalation of 4% isoflurane followed by rapid dislocation of the head and neck. After euthanasia, death was confirmed via the following observations: i) No respiratory movement in the chest or nostrils; ii) a lack of heartbeat observed through palpation of the femoral artery; iii) using a flashlight to illuminate the pupils, no contraction response was observed; and iv) no pain response or limb withdrawal after pressing the toes of euthanized mice.

A total of 42 mice were used in the present study (NS group, n=14; BLM group, n=14; BLM + 12.5 mg/kg group, n=7; BLM + 25 mg/kg group, n=7). A total of 2 mice were euthanized prematurely, and 3 mice died prior to reaching humane endpoint criteria; the deaths of these mice were observed between two daily welfare checks, predominantly occurring between days 1 to 7 following model establishment, which is referred to as the acute lung injury stage (23,24). Before these 3 mice died, they did not exhibit any of the typical symptoms of respiratory distress, such as saliva flowing from the mouth and nose, cyanosis, arching of the back or lying down, nor did these mice meet the other predetermined humane endpoint criteria of the present study. The cause of the death in these mice was speculated to be BLM-related lung injury and respiratory deficiency (21,25,26). The mortality rate of BLM-induced model mice in the present study was within the range of 10–80% as reported in previous literature (27–29). The remaining 37 mice all reached the 14-day set endpoint.

The human embryonic lung fibroblast cell lines HFL1 (iCell-h017) and MRC-5 (iCell-h146) were both purchased from iCell Bioscience Inc. The authenticity of these cell lines was confirmed through short tandem repeat analysis. MRC-5 cells were cultured in DMEM (cat. no. C3103-0500; Shanghai VivaCell Biosciences, Ltd.), whereas HFL1 cells were cultured in F12K medium (cat. no. iCell-0007; iCell Bioscience Inc.). Cells were cultured in an incubator at a constant temperature of 37°C with 5% CO₂. Before experiments using these cells, all cells were tested for mycoplasma and those showing negative results for mycoplasma contamination were used for subsequent formal experiments.

Recombinant TGF-β1 protein (cat. no. TMPY-02638; TargetMol Chemicals Inc.) was added to the culture medium of both cell lines at a concentration of 10 ng/ml. After incubation with recombinant TGF-β1 for 48–72 h, the cells were collected for subsequent experiments. For the JPH203-treatment group in each cell line, 10 ng/ml of recombinant TGF-β1 protein and the corresponding concentration of JPH203 (cat. no. TQ0081; TargetMol Chemicals Inc.) were added simultaneously to the cell culture media. For control cells, an equal volume of dimethyl sulfoxide (DMSO; cat. no. KGL2329-100; Jiangsu KeyGen Biotech Co., Ltd.) was added to the culture media, with the total concentration of DMSO not exceeding 1%.

HFL1 and MRC-5 were transfected with siRNAs using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, the final concentration of siRNAs is 30 nM. Cells were maintained at 37°C in a 5% CO₂ incubator. The transfection medium was removed after 8 h and replaced with fresh culture medium. For verification of siRNA knockdown efficiency, cells were harvested at 24 h post-transfection for RNA extraction and at 48 h post-transfection for protein extraction. For wound healing assays, cells were harvested at 48 h post-transfection for both RNA and protein analyses. For all other experiments, cells were harvested at 96 h post-transfection for RNA and protein extraction. The siRNA sequences used were synthesized by Seven Innovation (Beijing) Biotechnology Co., Ltd. The specific sequences of siRNAs were as follows: siRNA sequence targeting SLC7A5 (si-SLC7A5), forward GGGUGAUGUGUCCAAUCUA, reverse UAGAUUGGACACAUCACCC; and negative control siRNA (si-NC), forward UUCUCCGAACGUGUCACGU, reverse ACGUGACACGUUCGGAGAA.

The coding sequence for human SLC7A5 was cloned into the pCDNA3.4 vector to create a pCDNA3.4-SLC7A5 overexpression construct. The unmodified pCDNA3.4 plasmid was used throughout the present study as an empty vector control; both the blank vector and SLC7A5 overexpression plasmid were synthesized by Wuhan MiaoLing Biotech Science Co., Ltd. HFL1 cells were transiently transfected with the overexpression plasmid using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A total of 4 µg plasmid DNA and 10 µl Lipofectamine^® 2000 were diluted separately in Opti-MEM^® (cat. no. 31985062; Thermo Fisher Scientific, Inc.), gently mixed and incubated at room temperature for 5 min to form a complex. Then, this complex was added to the cells. Cells were maintained at 37°C in a 5% CO₂ incubator. The transfection medium was removed after 8 h and replaced with fresh culture medium. Total RNA and proteins were extracted from transfected HFL1 cells 48–72 h after transfection and subjected to RT-qPCR and western blotting assays respectively to verify the overexpression of the target protein. For cells in the JPH203 co-treatment group, cells were incubated with JPH203 (20 µM) 24 h after transfection.

A tightly packed monolayer of HFL1 cells was scratched in a straight line (when cells were completely attached), and siRNAs or JPH203 were added as appropriate. After the scratch, the cells were cultured in 1% serum (341506; Sigma-Aldrich; Merck KGaA) medium. Images were captured at the same position under an optical microscope (TH4-200; Olympus Corporation) after incubation at 37°C for 0, 24, and 48 h. The scratch area was calculated using the ImageJ 1.54d image analysis software (National Institutes of Health).

Cells were treated with the EdU-555 Cell Proliferation Assay Kit (Beyotime Biotechnology) according to the manufacturer's instructions. Cells were observed and images were subsequently captured under a fluorescence microscope (TH4-200; Olympus Corporation).

Cell viability in HFL1 cells was detected using a Cell Counting Kit [Seven Innovation (Beijing) Biotechnology Co., Ltd.] according to the manufacturer's instructions. A blank well containing culture medium and CCK-8 solution without cells was used for background correction. The optical density value of each well was measured using an enzyme detector (SpectraMax M5; Molecular Devices, LLC) at a wavelength of 450 nm. The following formula was used to calculate cell viability: Cell viability (%)=[A (with drug)-A (blank))/(A (no drug)-A (blank)] ×100 (A=optical density).

HFL1 cells were divided into three groups: DMSO, TGF-β1 + DMSO and TGF-β1 + 20 µM JPH203. After processing the cells, the same number of cells (1×10⁴) were collected from each treatment group. According to the manufacturer's instructions (cat. no. C1062; Beyotime Biotechnology), annexin V-fluorescein isothiocyanate and propidium iodide were used to stain the cells. The apoptotic rate of the cells was detected using a flow cytometer (BD FACSCelesta; BD Biosciences), and the results were analyzed using FlowJo_v10.8.1 software (BD Biosciences).

PMSF solution (cat. no. ST506; Beyotime Biotechnology) and phosphatase inhibitors (cat. no. HY-K0021; MedChemExpress) were added to RIPA lysis buffer (cat. no. P0013B; Beyotime Biotechnology) to prepare the cell lysis solution. A total of 1×106 cells were lysed in approximately 100 µl of cell lysis solution, and cells were harvested by gently scraping with a cell scraper. For mouse tissues, 20–30 mg of lung tissue was added to 250 µl lysis solution and subsequently ground on ice using an electric grinder. Cells or tissues were lysed on ice for 30 min and subsequently centrifuged at 14,000 × g for 10 min at 4°C. Protein concentration was measured using a BCA kit (cat. no. P0010; Beyotime Biotechnology). The standard curve formula for protein concentration measurements was as follows: y=0.397× + 0.191; R², 0.998. Proteins in each group were adjusted to reach the same volume and mass (15 µl; 20 µg) using the cell lysis solution and loading buffer (cat. no. LT101S; Shanghai Epizyme Biopharmaceutical Technology Co., Ltd.; Ipsen Pharma) and were subsequently heated at 100°C for 10 min.

PAGE gel (7.5%) was prepared according to the instructions of the PAGE Gel Rapid Preparation Kit (cat. no. PG111; Shanghai Epizyme Biopharmaceutical Technology Co., Ltd.; Ipsen Pharma). Denatured protein samples were added to the wells of the gel and pre-stained protein markers (cat. no. WJ103; Shanghai Epizyme Biopharmaceutical Technology Co., Ltd.; Ipsen Pharma) were added at both ends of the protein sample. Electrophoresis was performed using fresh electrophoretic solution (cat. no. P0561; Beyotime Biotechnology) at a constant voltage of 80 V until the samples entered the stacking gel (~30 min), and then at 120 V until the bromophenol blue dye front reached the bottom of the separating gel (approximately 1–2 h). Proteins were transferred onto PVDF membranes (cat. no. 03010040001; Roche Diagnostics (Shanghai) Co., Ltd.) at 300 mA for 90 min. The membranes were blocked with 5% non-fat milk [SW128; Seven Innovation (Beijing) Biotechnology Co., Ltd.] in TBS with 0.05% Tween [TBST; SW142; Seven Innovation (Beijing) Biotechnology Co., Ltd.] for 1 h at room temperature. The membrane was washed with TBST and then incubated with primary antibodies at 4°C overnight. The membranes were subsequently incubated with the appropriate secondary antibodies. For mouse primary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000; cat. no. RS0001; Immunoway) was used. For rabbit primary antibodies, HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. RS0002; Immunoway) was used. Incubation was performed at room temperature for 1 h with gentle shaking. After washing with TBST, the membranes were exposed to the MeilunBio^® fg super sensitive ECL luminescence reagent (cat. no. MA0186, MeilunBio) for protein band visualization. For target proteins with similar or identical molecular weights, the same membrane was used for sequential detection by stripping and reprobing. After imaging the first target protein, the membrane was incubated with a stripping buffer (cat. no. CW0056M; Jiangsu CoWin Biotech Co., Ltd.) at room temperature for 15 min to remove the primary and secondary antibodies, washed thoroughly with TBST, blocked with 5% non-fat milk in TBST for 1 h at room temperature, and then re-probed with the antibody against the second target protein. Band intensity was detected using the Tanon 5200 (Tanon Science and Technology Co., Ltd.) and the gray values of the bands were analyzed using the ImageJ 1.54d software (National Institutes of Health). Primary antibodies used in the present study included: GAPDH (1:5,000, cat. no. 10494-1-AP; Proteintech Group, Inc.), β-actin (1:5,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.), human SLC7A5 (1:1,000; cat. no. ab305251; Abcam), mouse SLC7A5 (1:1,000; cat. no. DF8065; Affinity Biosciences), fibronectin 1 (FN1; 1:2,000; cat. no. 66042-1-Ig; Proteintech Group, Inc.), collagen type 1 α1 (COL1A1; 1:1,000; cat. no. 14695-1-AP; Proteintech Group, Inc.), α-smooth muscle actin; (α-SMA; 1:1,000; cat. no. ab5694; Abcam), Bcl-2 (1:1,000; cat. no. F0125; Selleck Chemicals), Bax (1:1,000; cat. no. F0037; Selleck Chemicals), phosphorylated-(p-)PI3K (1:1,000; cat. no. AP0427; ABclonal Biotech Co., Ltd.), PI3K (1:1,000; cat. no. ab191606; Abcam), p-AKT (1:1,000; cat. no. 4060; CST Biological Reagents Co., Ltd.), AKT (1:1,000; cat. no. 4691; CST Biological Reagents Co., Ltd.), p-mTOR (1:1,000; cat. no. 5536; CST Biological Reagents Co., Ltd.) and mTOR (1:1,000; cat. no. 2983; CST Biological Reagents Co., Ltd.).

Total RNA was extracted from HFL1 cells, MRC-5 cells and mouse lung tissues using the total RNA extraction kit [Seven Innovation (Beijing) Biotechnology Co., Ltd.; cat. no. SM132]. cDNA was synthesized according to the instructions of the Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech Co., Ltd.; cat. no. AU341-02-V2). RT-qPCR was performed using the PerfectStart^® Green qPCR SuperMix (TransGen Biotech Co., Ltd.). The thermocycling conditions were as follows: initial denaturation at 94°C for 30 sec, followed by 40 cycles of denaturation at 94°C for 5 sec and annealing/extension at 60°C for 30 sec. The qPCR was performed on the SLAN-96S using the SLAN 8.2.2.7z software (Shanghai Hongshi Medical Technology Co., Ltd.). Gene expression was calculated using the following formulae: ΔCq=Cq(target gene)-Cq(reference gene); ΔΔCq=ΔCq(study sample)-ΔCq(control sample); total fold-change=2^−ΔΔCq (30). The sequences of the primers used are detailed in Table I.

HFL1 cells were subjected to immunofluorescence analysis in the following groups: si-NC group, si-NC + TGF-β1 group, si-SLC7A5 + TGF-β1 group, DMSO group, DMSO + TGF-β1 group, and DMSO + 20 µM group. The tissue was embedded in paraffin and the section thickness was 3 µm. Cells and tissue sections were fixed with 4% paraformaldehyde at room temperature for 15 min. After fixing the cells and tissue sections, 0.1% Triton permeabilization solution was added. After washing, the samples were blocked with 5% goat serum at room temperature for 1 h. The cells were then incubated with primary antibodies overnight at 4°C. The samples were incubated with CoraLite488-conjugated goat anti-rabbit IgG (H+L) (1:250, cat. no. SA00013-2; Proteintech Group, Inc.) and CoraLite594-conjugated goat anti-mouse IgG (H+L) (1:250, cat. no. SA00013-3; Proteintech Group, Inc.) for rabbit and mouse primary antibodies, respectively, at room temperature for 1 h in the dark. Nuclei were counterstained with DAPI at room temperature for 10 min. After counterstaining with DAPI, images of samples were captured under a fluorescence microscope (TH4-200; Olympus Corporation). The primary antibodies used in the present study included: COL1A1 (1:200; cat. no. 14695-1-AP; Proteintech Group, Inc.) for analysis of HFL1 cells, SLC7A5 (1:100; cat. no. DF8065; Affinity Biosciences) and α-SMA (1:100; cat. no. F2514; Selleck Chemicals) for analysis of mouse lung tissues.

The tissue was embedded in paraffin and sectioned at 3 µm. After tissues were dewaxed and hydrated, an endogenous peroxidase inhibitor (cat. no. PV6001; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was added and the sections were rinsed with PBS. Subsequently, sections were placed in immunohistochemical antigen retrieval buffer (EDTA method; cat. no. MVS0099; Fuzhou Maixin Biotech Co., Ltd.) and boiled for 10–20 min. After sections were cooled to room temperature, 5% BSA (Wuhan Servicebio Technology Co., Ltd.) was used for blocking for 30 min at room temperature. After blocking, sections were incubated with the primary SLC7A5 antibody (1:50; cat. no. DF8065; Affinity Biosciences) at 4°C overnight. The sections were then washed with PBS and incubated with the secondary antibody (cat. no. PV6001; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), which is conjugated to horseradish peroxidase (HRP) via a polymer backbone, for 30 min at room temperature. Color development was performed according to the instructions of the DAB color development kit (cat. no. DAB0031; Fuzhou Maixin Biotech Co., Ltd.) and sections were counterstained with hematoxylin for 30 sec at room temperature. After dehydration and clearing, the sections were sealed, before sections were observed and imaged under a light microscope (VS200; Olympus Corporation). Images were processed using the Olympus OlyVIA 4.1 software. The percentage of the positive area was calculated using ImageJ 1.54d software (National Institutes of Health).

The BCAA detection kit (Beyotime Biotechnology) was used to detect BCAA content within HFL1 cell samples and was used in accordance with the manufacturer's instructions. The optical density value of each well was measured using an enzyme detector (SpectraMax M5; Molecular Devices, LLC) at a wavelength of 450 nm. A standard curve was established based on the standard sample: y=0.0014× + 0.1765; R², 0.9989. Subsequently, the concentration of BCAA in each sample was calculated based on the standard curve.

The stained tissues included the lungs, heart, liver, spleen and kidneys. Fresh tissues were fixed in 4% paraformaldehyde at room temperature for 24 h. The tissue was embedded in paraffin and sectioned at 3 µm. Samples were stained using the improved H&E staining kit [Seven Innovation (Beijing) Biotechnology Co., Ltd.; cat. no. SI107] at room temperature for 5 min in hematoxylin and 2 min in eosin. Masson's trichrome staining was performed at room temperature according to the manufacturer's protocol (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. G1346), with the following key steps: Hematoxylin staining for 2 min, Ponceau-acid fuchsin staining for 5 min, phosphomolybdic acid treatment for 5 min and aniline blue staining for 5 min. Samples were observed under a light microscope and images were captured (VS200; Olympus Corporation). Images were processed using the Olympus OlyVIA 4.1 software.

All experiments were repeated at least three times. GraphPad Prism 10 software (Dotmatics) was used for the visualization and statistical analysis of the experimental results in the present study. For comparisons between two groups, unpaired t-tests were used for analysis. Pearson's correlation analysis was used to evaluate the linear correlation between two continuous variables. Two-way ANOVA was used to analyze the influence of two independent variables on the dependent variable, followed by Bonferroni's post-hoc test. One-way ANOVA was used to analyze the influence of one independent variable on the dependent variable, for which Bonferroni's post-hoc test was selected. Data are shown as mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

In order to explore the relationship between the expression level of SLC7A5 and IPF, differential gene analysis was conducted on the GSE49072, GSE40839, GSE135065 and GSE150910 datasets, which were retrieved from the GEO database and contained data regarding the clinical samples of patients with IPF. Differential genes identified in the four datasets were integrated and visualized in a Venn diagram (Fig. 1A). The results showed that three genes, SLC7A5, versican (VCAN) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), were significantly differentially expressed simultaneously in all four datasets, and the expression levels of these genes were increased in IPF. Notably, VCAN and MTHFD2 have been reported to be associated with pulmonary fibrosis in several studies (31–34). However, the relationship between the high expression of SLC7A5 and the occurrence and development of IPF remains to be fully elucidated. Therefore, a deeper exploration of the correlation between SLC7A5 and patients with IPF was conducted in the present study. Compared with the healthy control group, SLC7A5 was significantly upregulated in alveolar macrophages (Fig. 1B), lung fibroblasts (Fig. 1C and D) and lung tissues (Fig. 1E) in patients with IPF, suggesting that the upregulation of SLC7A5 expression may have been associated with IPF. To explore the correlation between the expression level of SLC7A5 and related proteins in pulmonary fibrosis, transcriptomic data from the GSE40839 dataset was analyzed. Pearson's correlation analysis showed that the expression level of SLC7A5 was significantly positively correlated with that of α-SMA, COL1A1, connective tissue growth factor (CTGF) and serpin E1 (SERPINE1) (Fig. 1F-I), further indicating the correlation between SLC7A5 expression and pulmonary fibrosis.

The results of pulmonary function tests in patients with IPF frequently show a series of characteristic changes, primarily including restrictive ventilation disorder, which manifests as a decline of indicators such as forced vital capacity (FVC) and total lung capacity, as well as reduced diffusion function, which manifests as a decline in diffusing capacity for carbon monoxide (DLCO) (35). Pearson's correlation analysis was conducted on the lung function test data of patients with IPF from two platforms (GPL6480 and GPL14550) in the GSE47460 dataset. The results of this analysis showed that in the GPL6480 platform, the expression level of SLC7A5 was negatively correlated with DLCO %pred (percent of predicted value) and FVC %pred in patients with IPF (Fig. 1J and K). In the GPL14550 platform, the expression level of SLC7A5 also exhibited a negative correlation with the DLCO %pred and FVC %pred values of patients with IPF (Fig. 1L and M). This indicated that the upregulation of SLC7A5 was correlated with the decline of pulmonary function in patients with IPF.

To explore whether the expression level of SLC7A5 affected the survival of patients with IPF, the present study conducted survival analyses using the GSE70866 and GSE27957 datasets. The results showed that in the GSE70866 dataset, high expression levels of SLC7A5 in the bronchoalveolar lavage fluid of patients with IPF was associated with a significantly higher mortality rate than patients with low SLC7A5 expression (Fig. 1N). In the GSE27957 dataset, the high expression of SLC7A5 in the peripheral blood mononuclear cells of patients with IPF was also associated with a significantly increased mortality rate compared with patients with low SLC7A5 expression (Fig. 1O). These results demonstrated that high SLC7A5 expression associated with a poor prognosis in patients with IPF. Based on the results of these bioinformatics analyses, it can be concluded that SLC7A5 was highly expressed in patients with IPF, and that the expression level of SLC7A5 was closely related to IPF development.

To further investigate the effect of SLC7A5 on IPF, a mouse model of pulmonary fibrosis was established via intratracheal BLM administration. Compared with the NS control group, the lung tissues of mice in the BLM group showed notable morphological changes, including thickening of the alveolar walls, destruction of the alveolar septa and increased collagen content (Fig. 2A and B). The results of western blotting showed that compared with NS group mice, the expression levels of the pulmonary fibrosis markers FN1, COL1A1 and α-SMA, as well as those of the SLC7A5 protein, were significantly upregulated in the lung tissues of the BLM group mice (Fig. 2C). RT-qPCR also achieved consistent results (Fig. 2D). The results of immunohistochemical staining showed that SLC7A5 staining, as shown by the brown staining of lung tissues in the figure, was darker and covered a significantly higher area proportion in BLM group mice compared with control mice (Fig. 2E). To investigate the localization of SLC7A5 in the lung tissues of mice, the present study performed immunofluorescence co-staining for the lung myofibroblast marker α-SMA together with SLC7A5. The results showed that in the BLM group, the expression levels of both α-SMA and SLC7A5 were markedly upregulated, and the SLC7A5 protein was predominantly aggregated in proximity to the α-SMA positive regions (Fig. S1). This indicated that SLC7A5 was predominantly localized on lung myofibroblasts. The aforementioned results indicated that the SLC7A5 was upregulated in the lung tissues of the BLM group mice, and this upregulation was demonstrated in vivo, supporting the differential expression of SLC7A5 in the pulmonary fibrosis model.

In order to verify the expression of SLC7A5 in the in vitro experiments using human lung fibrosis cell models, the TGF-β1 recombinant protein was used to induce HFL1 and MRC-5 cell lines to transform into myofibroblasts. Western blotting and RT-qPCR results showed that in HFL1 cells, after treatment with the TGF-β1 recombinant protein, the protein and mRNA expressions of FN1, COL1A1 and α-SMA were significantly upregulated compared with untreated cells, indicating that fibroblasts transformed into myofibroblasts. The expression of SLC7A5 was also significantly upregulated (Fig. 3A and B). The aforementioned results were also demonstrated in the MRC-5 cell line (Fig. 3C and D), indicating that SLC7A5 was highly expressed in the TGF-β1-induced lung fibrosis cell model.

To explore the potential mechanisms by which SLC7A5 played a role in pulmonary fibrosis, transcriptome sequencing was performed on the mRNA of TGF-β1-induced HFL1 cells. As shown in Fig. 3E and F, different expression patterns of differentially expressed genes were observed between the two treatment groups. Compared with the control group, a total of 2,459 genes were differentially expressed in the TGF-β1 group. Some fibrosis markers showed increased expression levels in the TGF-β1 group, such as SERPINE1, FN1, α-SMA, IL-11, collagen type IV alpha 1 (COL4A1), TGF-β1 and matrix metallopeptidase 1 (MMP1) (Fig. 3G). Furthermore, the expression of SLC7A5 in cells in the TGF-β1 group was significantly upregulated (Fig. 3H). Further exploration of the relevant signaling pathways involved in the TGF-β1-mediated induction of pulmonary fibroblasts involved GO functional enrichment analysis and KEGG pathway enrichment analysis. These analyses were conducted on the genes showing differential expression between the two treatment groups. Several IPF-related pathways were significantly enriched, including the Cell adhesion, Extracellular matrix, and TGF-β signaling pathway (Fig. 3I and J). GSEA comparing the gene expression profiles between the two groups revealed three mTOR-related signaling pathways that were enriched in different gene sets (HALLMARK, C2 and KEGG), and all of these pathways were significantly upregulated in the TGF-β1 group (Fig. 3K). Notably, SLC7A5, as one of the core genes contributing notably to the enrichment score, was enriched in all three identified mTOR signaling pathways. To verify the activation of mTOR signaling in TGF-β1-induced pulmonary fibroblasts, western blotting was used to detect p-mTOR and mTOR expression. The results showed that the p-mTOR/mTOR ratio was significantly increased in the TGF-β1 group compared with the control group, suggesting the activation of this pathway (Fig. 3L). These results indicated that SLC7A5 may have regulated the mTOR signaling pathway in order to affect the formation of pulmonary myofibroblasts and the occurrence and development of IPF.

To investigate whether SLC7A5 had any effect on the occurrence and development of IPF, SLC7A5 was knocked down using siRNA in HFL1 cells. Initially, the knockdown efficiency of si-SLC7A5 was measured via RT-qPCR and western blotting. The results showed that, compared with the si-NC group, the mRNA and protein expression levels of SLC7A5 in the si-SLC7A5 group were significantly decreased (Fig. 4A and B). To investigate whether silencing the expression of SLC7A5 would affect the cell function of pulmonary myofibroblasts, the present study conducted wound healing assays and EdU proliferation tests on transfected HFL1 cells. The results showed that, compared with the si-NC group, the cell migration and proliferation ability of si-NC-transfected cells were significantly increased after the addition of TGF-β1. Furthermore, compared with the si-NC + TGF-β1 group, the cell migration and proliferation ability of the si-SLC7A5 + TGF-β1 group were significantly decreased (Fig. 4C-E). Western blotting analysis revealed that, compared with the si-NC + TGF-β1 group, the protein expression of FN1, COL1A1 and α-SMA in the si-SLC7A5 + TGF-β1 group was significantly reduced (Fig. 4F and G). These results were also supported at the mRNA level (Fig. 4H). Immunofluorescence staining of HFL1 cells indicated that the expression of COL1A1 in the si-SLC7A5 + TGF-β1 group was decreased compared with that in the si-NC + TGF-β1 group (Fig. 4I). These results suggested that silencing SLC7A5 expression affected the cellular functions of pulmonary myofibroblasts.

In order to verify the reliability of siRNA-mediated knockdown and the universality of subsequent changes in pulmonary fibrosis markers in different human lung fibroblast cell lines, the siRNA sequences in the present study were also used to knock down SLC7A5 in MRC-5 cells. At both the mRNA and protein levels, significant knockdown of SLC7A5 expression was observed (Fig. S2A and B). Similarly, western blotting and RT-qPCR results showed that after knocking down SLC7A5, the expression levels of FN1, COL1A1 and α-SMA, which were also induced by TGF-β1 treatment in MRC-5 cells, were also significantly reduced (Fig. S2C and D). These results suggested that the upregulation of SLC7A5 may be the key factor causing the formation and development of pulmonary fibrosis.

JPH203 is a selective inhibitor of SLC7A5 that predominantly exerts effects in cancers, such as gastric, colon and bile duct cancer, by inhibiting the uptake of leucine (36). However, to the best of our knowledge, there is currently no literature reporting whether this inhibitor can be used to treat pulmonary fibrosis. Primarily, CCK-8 assays were performed on HFL1 cells treated with various concentrations of JPH203. The results of these assays indicated that, compared with the control group, JPH203 significantly inhibited the cell viability of pulmonary myofibroblasts and exerted both concentration-dependent and time-dependent effects (Fig. 5A). This inhibitory effect was partially reversed via overexpression of SLC7A5 at 24 and 48 h (Fig. S3A-D). Based on the results of the CCK-8 assay, the working concentrations of JPH203 for subsequent experiments were set at 20 and 30 µM, as these concentrations elicited moderate inhibition without excessive cytotoxicity, while higher concentrations (40 µM) led to a marked reduction that could confound the interpretation of pathway-specific effects. Subsequently, BCAA content, including leucine, isoleucine and valine levels, was detected in HFL1 cells following treatment with JPH203. As shown in Fig. 5B, after incubating HFL1 cells with JPH203 for 48 h, the content of BCAA in the TGF-β1 + 20 µM and TGF-β1 + 30 µM groups were significantly reduced compared with the TGF-β1 + DMSO group. These results indicated that in lung fibroblasts, JPH203 specifically exerted its pharmacological effects by inhibiting the translocation of BCAA from the extracellular space to the intracellular space by targeting SLC7A5.

Based on the aforementioned results, 20 µM was selected as the working concentration of JPH203 for subsequent experiments. This concentration was chosen because both 20 and 30 µM yielded similar levels of target inhibition in the preliminary assays, and 20 µM was sufficient to produce a significant effect while minimizing potential off-target cytotoxicity associated with higher doses. In HFL1 cells, wound healing and EdU staining assays were used to determine the effects of JPH203 on the migration and proliferation abilities of human lung myofibroblasts. The results demonstrated that, compared with the DMSO group, the migration and proliferation of cells in the TGF-β1 + DMSO group were significantly enhanced. Furthermore, JPH203 inhibited the enhancement of cell migration and proliferation caused by TGF-β1 treatment (Fig. 5C and D). A study has shown that TGF-β1, as a cytokine responsible for promoting fibrosis, can enhance the resistance of lung fibroblasts to apoptosis (37), thereby inhibiting the apoptosis of lung myofibroblasts in the present study. The results of western blotting (Fig. 5E) demonstrated that the expression of Bcl-2 in the TGF-β1 + DMSO group was significantly elevated compared with the DMSO group, whereas the expression of Bax significantly decreased. Furthermore, compared with the TGF-β1 + DMSO group, the expression of Bcl-2 in the TGF-β1 + 20 µM group significantly decreased, yet the expression of Bax significantly increased.

In addition, the apoptotic rate of HFL1 cells treated with JPH203 was detected using flow cytometry. Compared with the DMSO group, the number of apoptotic cells in the TGF-β1 + DMSO group significantly decreased and the proportions of early and late apoptosis were reduced. However, compared with the TGF-β1 + DMSO group, the apoptotic rate of cells in the TGF-β1 + 20 µM group significantly increased, reversing the observed resistance to apoptosis induced by TGF-β1 treatment (Fig. 5F); this indicated that JPH203 inhibited the resistance of lung myofibroblasts to cell apoptosis. In order to investigate whether JPH203 affected the expression of lung fibrosis markers, immunofluorescence staining was performed; the results of this staining showed that JPH203 co-treatment markedly inhibited the upregulation of COL1A1 caused by TGF-β1 treatment (Fig. 5G). Furthermore, western blotting was used to detect the protein expression of FN1, COL1A1 and α-SMA in HFL1 lung fibroblasts following JPH203 treatment. The results showed that the protein expression levels of FN1, COL1A1 and α-SMA in the TGF-β1 + 20 µM group were significantly decreased compared with the TGF-β1 + DMSO group (Fig. 5H). These results were also supported at the mRNA level (Fig. 5I). The aforementioned results indicated that the SLC7A5 inhibitor JPH203 inhibited the cellular functions of pulmonary myofibroblasts, and may therefore have affected the formation and development process of pulmonary fibrosis.

In order to explore the mechanism by which SLC7A5 mediated the occurrence of IPF, transcriptome sequencing analysis was conducted on the mRNA of HFL1 cells treated with si-SLC7A5 + TGF-β1, with the si-NC + TGF-β1 group representing the control group. The volcano plot (Fig. S4A) and heatmap (Fig. S4B) generated from these data showed that, compared with the control group, 1,527 genes were differentially expressed in the si-SLC7A5 + TGF-β1 group, indicating that there were different expression patterns of genes between the two treatment groups.

Subsequently, a GO functional enrichment analysis was performed on the differentially expressed genes identified between the two treatment groups (Fig. 6A). The results demonstrated that the pathways inhibited in the si-SLC7A5 + TGF-β1 group included three PI3K signaling pathways. The KEGG functional enrichment analysis also demonstrated the significant downregulation of the ‘PI3K-Akt signaling pathway’, which plays an important role in regulating the occurrence and development of IPF (Fig. 6B). The differentially expressed genes downregulated in the si-SLC7A5 + TGF-β1 group were then subjected to PPI analysis; it was found that these key nodes were also enriched in the ‘PI3K-Akt signaling pathway’ in the KEGG dataset (Fig. 6C). Finally, GSEA was performed between the gene expression profiles of the two groups. In the C2 gene set database, two PI3K/AKT-related signaling pathways were found to be enriched, and both pathways were significantly downregulated in the si-SLC7A5 + TGF-β1 group (Fig. 6D). By analyzing transcriptomic data from the GEO datasets GSE101286 and GSE135065, the PI3K/AKT-related pathway was shown to be significantly upregulated in patients with IPF (Fig. 6E). This suggested that the knockdown of SLC7A5 inhibited certain genes in the PI3K/AKT pathway to affect the activation of the PI3K/AKT pathway.

According to the aforementioned results, SLC7A5 may have participated in the formation of myofibroblasts by activating the mTOR signaling pathway; notably, mTOR is a downstream effector of the PI3K/AKT signaling pathway (38). Therefore, it can be inferred that inhibiting the function of the SLC7A5 gene may have affected the occurrence of pulmonary fibrosis by inhibiting the PI3K/AKT/mTOR signaling pathway. The results of western blotting showed that, compared with the si-NC group, the p-PI3K, p-AKT and p-mTOR expression levels in the si-NC + TGF-β1 group were significantly upregulated. Furthermore, compared with the si-NC + TGF-β1 group, the p-PI3K, p-AKT and p-mTOR expression levels in the si-SLC7A5 + TGF-β1 group were significantly downregulated (Fig. 7A). On the other hand, western blotting showed that, the p-PI3K, p-AKT and p-mTOR expression levels in the TGF-β1 + DMSO group were significantly upregulated compared with the DMSO group. Furthermore, by treating TGF-β1-induced HFL1 cells with the JPH203 inhibitor (20 µM), the p-PI3K, p-AKT and p-mTOR expression levels in the TGF-β1 + JPH203 group were downregulated compared with the TGF-β1 + DMSO group (Fig. 7B). This indicated that downregulating the expression of SLC7A5 by siRNA-mediated knockdown or inhibiting the function of SLC7A5 via JPH203 treatment suppressed the activation of the PI3K/AKT/mTOR signaling pathway, thereby inhibiting the formation of pulmonary myofibroblasts and possibly suppressing the occurrence and development of IPF.

In order to verify the therapeutic effect of JPH203 on pulmonary fibrosis in vivo and to determine whether SLC7A5 could represent a potential therapeutic target for IPF, mice with BLM-induced pulmonary fibrosis were treated with JPH203 by intraperitoneal injection for 13 consecutive days, as shown in Fig. 8A. Intraperitoneal injection was used as the administration method of JPH203 in vivo, consistent with previous studies [e.g., JPH203 was administered intraperitoneally in a thyroid carcinoma mouse model (39)]. The present study selected 12.5 and 25 mg/kg as the experimental doses for JPH203 treatment. These two doses have demonstrated notable antitumor effects in multiple studies (40,41) and were tolerated well by mice in the present study.

The lung tissue sections of experimental mice were stained with H&E and Masson's trichrome, as shown in Fig. 8B. Compared with the BLM group, the pathological changes in the lungs of the BLM + 12.5 mg/kg group were notably alleviated, and the degree of pulmonary fibrosis in the BLM + 25 mg/kg group was further improved. Based on the tissue-staining results, the BLM + 25 mg/kg group was selected as the treatment group for the subsequent experiments. The results of western blotting showed that, compared with the BLM group, the expression levels of FN1, COL1A1 and α-SMA in the lung tissue of mice in the BLM + 25 mg/kg group were significantly downregulated (Fig. 8C). RT-qPCR also achieved consistent results (Fig. S5). Furthermore, the present study used western blotting to verify that the PI3K/AKT/mTOR pathway was activated in the lung tissues of mice with BLM-induced pulmonary fibrosis, whereas the activity of this pathway was significantly inhibited in the BLM + 25 mg/kg group (Fig. 8D). In addition, H&E staining pathological analysis of heart, liver, spleen and kidney tissue sections from BLM-induced mice was also conducted. The results showed that, compared with the BLM group, the experimental groups (BLM + 12.5 mg/kg and BLM + 25 mg/kg groups) demonstrated more intact tissue structures in the heart, liver, spleen and kidneys, without obvious inflammatory infiltration, cell necrosis, fibrosis or other signs of pathological damage (Fig. S6). The aforementioned results indicated that JPH203 improved BLM-induced pulmonary fibrosis in mice by inhibiting the activation of the PI3K/AKT/mTOR pathway. Furthermore, at the therapeutic dose used in the present study, JPH203 demonstrated notable safety (Fig. S6), which provided important implications for the clinical application of JPH203.