The present study used 24 healthy male Sprague-Dawley rats (age, 6–8 weeks; body weight, 180–220 g at the start of the study), which were purchased from SPF Biotechnology Co., Ltd. [license no. SCXK (Beijing) 2019–0010]. The rats were housed under standard environmental conditions (temperature, 22±2°C; humidity, 50±10%; 12-h light/dark cycle) with free access to food and water, and were acclimated for 1 week before the experiment. The rats were randomly divided into four groups: Control (n=6; intraperitoneal injection of an equal volume of saline every other day; injection volume, 0.2 ml per rat), model (n=6; urethral injury followed by intraperitoneal injection of saline every other day; injection volume, 0.2 ml per rat), model + rapamycin [n=6; 2.0 mg/kg of rapamycin (cat. no. HY-10219; MedChemExpress) injected intraperitoneally every other day after injury] and model + TDN (n=6; 10 nmol/day TDN administered via tail vein injection daily after injury; injection volume, 0.2 ml per rat). TDN was freshly prepared in TM buffer (10 mM Tris-HCl, 5 mM MgCl₂; pH 8.0), and its successful assembly and purity were confirmed. Animal experiments were approved by the Experimental Animal Welfare and Ethics Committee of Guizhou Medical University (approval no. 2502311; Guiyang, China).

The four single-stranded DNAs used for TDN assembly were chemically synthesized by Sangon Biotech Co., Ltd. TDN was synthesized by self-assembly of four single-stranded DNAs at a final concentration of 30 µM in TM buffer (10 mM Tris-HCl, 5 mM MgCl₂; pH 8.0; cat. no. T10420; Shanghai Shangbao Biotechnology Co., Ltd.), followed by denaturation at 95°C for 10 min and rapid cooling to 4°C. The sequences of the four single-stranded DNAs were as follows: S1, 5′-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3′; S2, 5′-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3′; S3, 5′-ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3′; and S4, 5′-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG-3′. The successful formation of TDN was verified by 1% agarose gel electrophoresis (cat. no. A8201; Beijing Solarbio Science & Technology Co., Ltd.) using SerRed nucleic acid stain (cat. no. G3606; Wuhan Servicebio Technology Co., Ltd.), with staining performed at room temperature for 20 min, and by UV-visible spectrophotometry (NanoPhotometer^® N50; Implen GmbH). A distinct absorption peak was observed at ~260 nm, with a concentration of 184.35 ng/µl, confirming successful tetrahedral assembly and high purity (Fig. S1).

The urethral injury model was established based on previous studies with slight modifications (27,29–31). Rats were anesthetized with 1% pentobarbital sodium (40 mg/kg; intraperitoneal; cat. no. P3761; MilliporeSigma) and then fixed on a surgical platform (RWD Life Science Co., Ltd.). The surrounding hair of the penis was shaved using an electric clipper (Flyco), and the surgical field was disinfected with iodine tincture (Wuhan Servicebio Technology Co., Ltd.). A longitudinal incision was made on the ventral side of the penis to expose the urethra. Mechanical injury was induced using an 18-G needle (BD Biosciences), forming an injury of ~5 mm in length. After inducing injury, the urethral sponge and skin were sutured (Fig. S2) using 6–0 absorbable sutures (Ethicon, Inc.; Johnson & Johnson). The wound was disinfected daily with povidone-iodine solution (Wuhan Servicebio Technology Co., Ltd.), and body weight and wound healing were monitored.

At the end of the experiment, all rats were euthanized by intraperitoneal injection of sodium pentobarbital at a dose of 100 mg/kg (cat. no. P3761; MilliporeSigma). Death was confirmed by the absence of thoracoabdominal movements, a lack of corneal reflex upon gentle touch and no withdrawal response to a firm toe pinch after loss of consciousness.

After successful anesthesia with 1% pentobarbital sodium (40 mg/kg, intraperitoneal), as aforementioned, rats were fixed on a digital fluoroscopic table (DRX-Ascend; Carestream Health, Inc.). Experimental staff wore protective clothing (Wuhan Servicebio Technology Co., Ltd.) and inserted a 24-G intravenous catheter (BD Biosciences) into the anterior urethra via the urethral opening (32). Using real-time X-ray monitoring, one hand stabilized the urethra and catheter, while the other hand slowly injected the iodinated contrast agent (iohexol; 300 mg iodine/ml; GE Healthcare) into the bladder via the catheter. X-ray images were obtained to assess the narrowing of the urethral lumen.

The urethral tissue was fixed in 4% paraformaldehyde solution [cat. no. NH250218; Nuohai Life Science (Shanghai) Co., Ltd.] at room temperature for 24–48 h. Following fixation, the tissues were rinsed with PBS (cat. no. G1101; Wuhan Servicebio Technology Co., Ltd.), dehydrated through a graded ethanol series [70, 80, 95% and absolute ethanol (CAS no. 64-17-5; analytical grade;; Chengdu Kelong Chemical Co., Ltd.)], and cleared with xylene (cat. no. 33535; Xilong Scientific Co., Ltd.). Samples were subsequently embedded in paraffin (Shanghai Huayong Paraffin Co., Ltd.) and sectioned at a thickness of 5 µm using a rotary microtome [HistoCore MULTICUT; cat. no. 149MULTIGC1; Leica Biosystems Co., Ltd.].

For H&E staining, paraffin-embedded sections were deparaffinized with xylene and rehydrated through descending concentrations of ethanol, followed by staining with hematoxylin solution (cat. no. G1004-100ML; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 5 min, rinsed with running water, and counterstained with eosin staining solution (cat. no. G1108; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1–2 min. After dehydration and clearing with xylene, the sections were mounted using neutral resin mounting medium (cat. no. WG10004160; Wuhan Servicebio Technology Co., Ltd.) and histological images were captured using an inverted light microscope (TS2; Nikon Corporation).

Masson's trichrome staining was conducted to evaluate collagen deposition in the urethral tissue (33). The urethral tissue was fixed in 4% paraformaldehyde solution (cat. no. G1101; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 24 h, dehydrated and embedded in paraffin. Sections with a thickness of 4 µm were prepared using a rotary microtome. Masson's trichrome staining was conducted at room temperature using a commercial kit (cat. no. G1006; Wuhan Servicebio Technology Co., Ltd.) according to the manufacturer's protocol, with the staining duration following the standard instructions. Images were captured at a magnification of ×200 using a light microscope. Collagen fibers were stained blue, whereas muscle fibers, fibrin and red blood cells appeared red.

Immunohistochemical staining (34) was conducted to evaluate the expression of fibrosis markers, including α-smooth muscle actin (α-SMA; cat. no. AF1032; Affinity Biosciences), TGF-β1 (1:5,000; cat. no. 21898-1-AP; Proteintech Group, Inc.), collagen I (cat. no. AF7001; Affinity Biosciences), collagen III (1:1,000; cat. no. AF5457; Affinity Biosciences) and Smad3 (cat. no. 87035-1-RR; Proteintech Group, Inc.). Briefly, paraffin-embedded tissue sections prepared as aforementioned were sectioned at a thickness of 4 µm. Sections were then deparaffinized, rehydrated through a descending ethanol series and subjected to heat-induced antigen retrieval using citrate buffer (pH 6.0; cat. no. G1202; Wuhan Servicebio Technology Co., Ltd.). Subsequently, the sections were incubated with 3% hydrogen peroxide (Wuhan Servicebio Technology Co., Ltd.) to quench endogenous peroxidase activity and were blocked with 5% BSA (cat. no. CR2302110; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature; after which, the sections were incubated overnight at 4°C with the corresponding primary antibodies diluted in 2% BSA at a dilution of 1:100. This was followed by incubation with a HRP-conjugated secondary antibody (cat. no. G1215; Wuhan Servicebio Technology Co., Ltd.) for 50 min at 37°C. Color development was achieved using a DAB chromogenic kit (cat. no. G1211; Wuhan Servicebio Technology Co., Ltd.), and cell nuclei were counterstained with hematoxylin (cat. no. G1040-500ML; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 2 min. Images were captured at a magnification of ×200 using a light microscope (CX23; Olympus Corporation). Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.) was employed to quantify the percentage and intensity of positive staining to assess the degree of fibrosis.

To evaluate the protein expression levels of α-SMA, TGF-β1, collagen I, Smad3 and collagen III, total protein was extracted from rat urethral tissues using RIPA lysis buffer (cat. no. G2002; Wuhan Servicebio Technology Co., Ltd.) containing protease and phosphatase inhibitor cocktail (cat. no. G2007; Wuhan Servicebio Technology Co., Ltd.). The protein concentration was determined using a BCA assay kit (cat. no. G2026; Wuhan Servicebio Technology Co., Ltd.) (35). Proteins (30 µg per lane) were separated using SDS-PAGE (10% gel; cat. no. P0012A; Beyotime Biotechnology) and transferred onto PVDF membranes (cat. no. IPVH00010; MilliporeSigma) using the wet transfer method. The membranes were blocked with 5% BSA for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: α-SMA (1:1,000; cat. no. AF1032; Affinity Biosciences), TGF-β1 (1:5,000; cat. no. 21898-1-AP; Proteintech Group, Inc.), collagen I (1:1,000; cat. no. AF7001; Affinity Biosciences), collagen III (1:1,000; cat. no. AF5457; Affinity Biosciences) and Smad3 (1:10,000; cat. no. 66516-1-Ig; Proteintech Group, Inc.), and β-actin (1:25,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.), which was used as a loading control.

After washing three times with TBS containing 0.1% Tween-20 (cat. no. G0004-500M; Wuhan Servicebio Technology Co., Ltd.), the membranes were incubated for 1 h at 37°C with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:3,000; cat. no. GB23303; Wuhan Servicebio Technology Co., Ltd.) or HRP-conjugated goat anti-mouse IgG secondary antibody (1:5,000; cat. no. GB23301; Wuhan Servicebio Technology Co., Ltd.), as appropriate. Protein bands were visualized using an enhanced chemiluminescence kit (cat. no. G2014; Wuhan Servicebio Technology Co., Ltd.) and images were captured with a ChemiDoc™ MP Imaging System (cat. no. 12003154; Bio-Rad Laboratories, Inc.). Band intensities were semi-quantified using Image-Pro Plus software (version 6.0).

To explore the molecular mechanisms underlying urethral injury and repair, total RNA was extracted from rat urethral tissues using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

The RNA concentration and purity were assessed using a NanoDrop™ 2000 spectrophotometer (cat. no. ND-2000; Thermo Fisher Scientific, Inc.), and RNA integrity was verified by 1% agarose gel electrophoresis (cat. no. A8201; Beijing Solarbio Science & Technology Co., Ltd.). High-throughput sequencing libraries were prepared using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina (cat. no. E7530L; New England BioLabs, Inc.) and sequencing was performed on an Illumina NovaSeq 6000 platform using the NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles; cat. no. 20028312; Illumina, Inc.) (36). Paired-end RNA sequencing (2×150 bp) was performed in a forward-reverse orientation, with the final libraries loaded at a concentration of 300 pM. The quality of raw sequencing data was evaluated using FastQC (version 0.11.9; Babraham Bioinformatics), and adaptor trimming and filtering were conducted using Trimmomatic (version 0.39; http://www.usadellab.org/cms/?page=trimmomatic). Clean reads were aligned to the rat reference genome (Rnor_6.0) using HISAT2 (version 2.2.1; http://daehwankimlab.github.io/hisat2/). Principal component analysis (PCA) was performed to evaluate global transcriptional differences among samples and to assess the consistency of biological replicates. Sample-to-sample correlation analysis was performed using Pearson correlation coefficients based on normalized gene expression values to examine intra-group repeatability and inter-group variability. The results were visualized using PCA for dimensionality reduction and correlation heatmaps generated using the pheatmap package (version 1.0.12; http://cran.r-project.org/package=pheatmap) in R software (version 4.2.2; The R Foundation for Statistical Computing; http://www.r-project.org/). Differential gene expression analysis was performed using the DESeq2 package (version 1.38.0; http://bioconductor.org/packages/DESeq2/) in R software, with thresholds of |log2(fold change)|≥0.5 and P<0.05 to identify significantly differentially expressed genes (DEGs). Functional enrichment analyses were conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.kegg.jp) to identify pathways related to fibrosis, inflammation, immune regulation, metabolism, cell cycle regulation and tissue repair. Specifically, pathways were selected based on their direct relevance to immune regulation, inflammation, metabolism, cell cycle regulation and tissue repair, which are key processes associated with urethral injury and TDN-mediated therapeutic effects. Disease-related pathways with limited mechanistic relevance to urethral pathology were excluded from the final analysis. Statistical analyses and visualization were conducted in R software using the pheatmap package and ggplot2 package (version 3.4.0; http://cran.r-project.org/package=ggplot2) to display gene expression patterns and enrichment results.

To validate the transcriptome sequencing results, total RNA was extracted from rat urethral tissues using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScript™ RT reagent kit with gDNA eraser (cat. no. RR047A; Takara Bio, Inc.) to synthesize cDNA. Reverse transcription was performed at 42°C for 15 min, followed by enzyme inactivation at 85°C for 5 sec. qPCR was conducted using TB Green^® Premix Ex Taq™ II (cat. no. RR820A; Takara Bio, Inc.) on a QuantStudio™ 5 Real-Time PCR System (cat. no. A28574; Applied Biosystems; Thermo Fisher Scientific, Inc.). Each reaction (20 µl) contained 10 µl SYBR Green mix, 1 µl cDNA template, 0.4 µl of each primer (10 µM) and 8.2 µl nuclease-free water. The PCR amplification conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The expression levels of the cell cycle-related genes cyclin B1 (Ccnb1), ribonucleotide reductase regulatory subunit M2 (Rrm2), polo-like kinase 1 (Plk1) and cyclin-dependent kinase 1 (Cdk1), as well as the inflammatory cytokines IL-6, IL-1β and TNF-α, were analyzed, with GAPDH used as the internal control. The relative gene expression levels were calculated using the 2^−ΔΔCq method for quantification, as previously described by Livak and Schmittgen (37). Primer sequences are listed in Table SI.

After identifying DEGs from the transcriptome data, protein-protein interaction (PPI) network analysis was performed using the Search Tool for the Retrieval of Interacting Genes/Proteins database (version 12.0; http://string-db.org/) to evaluate gene-gene associations. A confidence score threshold of 0.7 (high confidence) was applied to filter significant interactions and the resulting interaction data were imported into Cytoscape software (version 3.10.0; Cytoscape Consortium) for visualization. The PPI network was optimized using the cytoHubba plug-in (version 0.1; http://apps.cytoscape.org/apps/cytohubba) to identify hub genes with the highest degree of connectivity.

These key genes were analyzed in the context of fibrosis, extracellular matrix remodeling and cell cycle regulation to explore their biological significance and potential roles in the pathogenesis of urethral injury.

The serum concentrations of IL-6, IL-1β and TNF-α were measured using ELISA kits: IL-6 (cat. no. SEA079Ra; CLOUD-CLONE CORP), IL-1β (cat. no. SEA563Ra; CLOUD-CLONE CORP) and TNF-α (cat. no. SEA133Ra; CLOUD-CLONE CORP). The assays were conducted according to the manufacturer's protocols. Absorbance was read at 450 nm using a SpectraMax^® iD3 microplate reader (iD3; Molecular Devices, LLC). Cytokine levels were quantified based on standard curves and expressed as pg/ml. These data were used to evaluate the systemic immune and inflammatory responses following urethral injury.

ssGSEA was performed to evaluate the immune microenvironment and quantify immune cell infiltration in rat urethral tissues.

The analysis was based on the normalized transcriptome expression matrix. The GSVA package (version 1.46.0; http://bioconductor.org/packages/GSVA/) in R software was used to calculate the enrichment scores of immune-related gene sets for each sample. Immune cell-related gene sets representing 24 immune cell types were derived directly from the immune cell signature gene sets defined in a previous study by Bindea et al (38) and were curated for application in ssGSEA. These gene sets included signatures for macrophages, T cells, natural killer cells, dendritic cells and neutrophils. The enrichment scores were visualized using the ggplot2 package and the pheatmap package n R., enabling quantitative assessment of immune cell infiltration and characterization of immune landscape alterations associated with urethral injury and TDN treatment.

Primary fibroblasts were isolated from rat urethral tissues under sterile conditions. Briefly, freshly harvested urethral tissues were rinsed 3–5 times with PBS supplemented with 5% penicillin-streptomycin solution (Dalian Meilun Biology Technology Co., Ltd.), with each wash lasting 5 min. The tissues were then minced into small fragments using sterile scissors and digested with 0.25% trypsin (Dalian Meilun Biology Technology Co., Ltd.) at 37°C in a humidified incubator with 5% CO₂ for 60 min.

Following enzymatic digestion, 6 ml complete Dulbecco's modified Eagle's medium (DMEM; Dalian Meilun Biology Technology Co., Ltd.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin was added to terminate digestion. The cell suspension was collected and centrifuged at 1,000 rpm for 5 min. The resulting cell pellet was resuspended in complete DMEM and transferred to culture flasks for incubation at 37°C in a humidified atmosphere containing 5% CO₂.

The culture medium was replaced every 2–3 days. When cells reached ~80% confluence, fibroblasts were passaged using 0.25% trypsin digestion. Cells were washed twice with PBS, digested until most cells became rounded and detached, and digestion was terminated with complete DMEM. After centrifugation at ~200 × g for 5 min at room temperature, the cells were resuspended in fresh complete medium and passaged at a ratio of 1:2. Primary rat urethral fibroblasts were cultured in complete DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator containing 5% CO₂. When cells reached 70–80% confluence, in vitro treatments were performed. Cells were divided into the following three groups: Control, TGF-β-treated and TGF-β + TDN-treated groups. For induction of vimentin expression, fibroblasts were treated with TGF-β (5 ng/ml; cat. no. 80116-R08H; Beijing Sino Technology Co., Ltd.) for 24 h. For the intervention group, TDN (1 µM) was added simultaneously with TGF-β, and the cells were co-treated for 24 h at 37°C. After treatment, the cells were collected for immunohistochemical analysis of vimentin expression, as described in the Materials and methods section.

To evaluate vimentin expression in fibroblasts, immunocytochemical staining was performed. After treatment, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature overnight. Following fixation, the cells were washed with PBS, subjected to antigen retrieval using pepsin solution at 37°C for 30 min, and endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 20 min at room temperature. The cells were then blocked with 5% bovine serum albumin (cat. no. CR2302110; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at 37°C and incubated overnight at 4°C with a primary antibody against vimentin (1:150; cat. no. AFRM0062; Hunan Aifang Biotechnology Co., Ltd.), diluted in 2% bovine serum albumin. After washing, the cells were incubated with a polymer-based secondary antibody using a universal two-step detection kit (cat. no. PV-9000; OriGene Technologies, Inc.) according to the manufacturer's instructions. Color development was achieved using a DAB chromogenic kit, and cell nuclei were counterstained with hematoxylin for 5 min at room temperature. Representative images were captured using a light microscope, and quantitative analysis was performed using Image-Pro Plus software, as aforementioned.

Primary rat urethral fibroblasts were isolated from urethral tissues and cultured under standard conditions as aforementioned. After expansion, fibroblasts were enzymatically dissociated and seeded onto glass coverslips for cell attachment. Following culture, fibroblast samples (n=6) were provided for histological evaluation, including H&E staining and immunocytochemical staining. H&E staining was performed as aforementioned to assess fibroblast morphology, and representative images were acquired using a light microscope.

To confirm the purity and identity of the fibroblasts isolated from rat urethral tissues, immunofluorescence staining for α-SMA and CD90 was performed. Cells were fixed with 4% paraformaldehyde (cat. no. G1101; Wuhan Servicebio Technology Co., Ltd.) for 15 min at room temperature, permeabilized with 0.3% Triton X-100 (cat. no. G1203; Wuhan Servicebio Technology Co., Ltd.) and blocked with 5% BSA for 30 min at room temperature. The cells were incubated with primary antibodies against α-SMA (1:100; cat. no. AF1032; Affinity Biosciences) and CD90 (1:100; cat. no. DF4804; Affinity Biosciences) overnight at 4°C. After washing with PBS, cells were incubated with goat anti-rabbit IgG H&L (Alexa Fluor™ 488-conjugated; 1:200; cat. no. ab150077; Abcam) secondary antibodies for 40 min at 37°C. Nuclei were counterstained with DAPI (cat. no. G1012; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 8 min in the dark. Fluorescence images were captured using a Nikon TS2-S-SM inverted fluorescence microscope (Nikon Corporation). The results showed negative α-SMA staining together with positive CD90 expression, indicating that the isolated cells were predominantly fibroblasts and excluding myofibroblast contamination.

All quantitative data are presented as the mean ± standard deviation from at least three independent experiments. Statistical analyses were performed using GraphPad Prism software (version 9.0; Dotmatics). This approach was used for multi-group quantitative analyses presented in the figures. Comparisons among three or more groups were performed using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Transcriptome data were analyzed using R software. The ggplot2 (version 3.4.0), pheatmap (version 1.0.12) and ClusterProfiler (version 4.6.0; http://bioconductor.org/packages/clusterProfiler/) packages were used for data visualization and functional enrichment analysis. Sample correlation analysis was performed using Pearson correlation analysis based on normalized gene expression levels, and the results are presented as a sample-to-sample correlation heatmap. All figures were generated using GraphPad Prism and R and assembled in Adobe Illustrator (version 2023; Adobe Systems, Inc.) for final presentation.

To ensure reliability, the urethral injury model was validated from multiple aspects. Fig. 1A shows the tissue collection images of rat urethras in different groups. Postoperative body weight recordings showed that the control group gradually gained weight, whereas the model group experienced marked weight loss. Notably, body weight in the rapamycin and TDN groups exhibited a slight, non-significant decrease and remained relatively stable overall (Fig. 1B). Urethrography showed that the urethras of rats were healthy in the control group, while urethral stenosis was successfully developed in the model group. Urethral stenosis improved after treatment with rapamycin and TDN (Fig. 1C). H&E staining showed that in the model group, the mucosal layer of the urethra was notably thickened and the lumen was narrower, with marked inflammatory cell infiltration compared with the control group. Consistent with the results of urethrography, the rapamycin and TDN groups showed notable improvements in urethral histological structure, with reduced luminal narrowing and improved tissue organization, suggesting an attenuation of urethral injury following rapamycin and TDN treatment (Fig. 1D).

These results suggested that the urethral injury model was successfully established, and different treatment groups exhibited notable differences in terms of urethral damage and fibrosis. Particularly, the results supported the therapeutic effects of Rapamycin and TDN, further supporting their efficacy in alleviating urethral injury.

Transcriptome sequencing revealed the relationship between different treatments and gene expression. PCA (Fig. 2A) showed that samples from the same group clustered closely together, whereas samples from different groups were clearly separated, indicating distinct transcriptomic profiles among the control model, and treatment groups. Consistently, sample correlation analysis (Fig. 2B) revealed high correlation coefficients among biological replicates within each group and lower correlations between different groups, demonstrating good intra-group reproducibility and clear inter-group differentiation. Differential expression analysis identified 3,008 DEGs, including 1,248 upregulated and 819 downregulated genes between the model and control groups, 194 upregulated and 45 downregulated genes between the Rapamycin and model groups, and 206 upregulated and 496 downregulated genes between the TDN and model groups (Fig. 2C and D). All three comparisons shared 41 genes (Fig. 2E). The slight discrepancy in gene numbers between the Venn diagram (Fig. 2E) and the DEG counts shown in Fig. 2C and D is due to differences in gene inclusion criteria, as one gene located at the significance threshold was included in the Venn analysis but excluded from the final DEG count. These genes, including genes related to metabolism, keratin, acute-phase response, inflammation and immune function, exhibited differential expression patterns after different treatments (Fig. 2F).

KEGG pathway enrichment analysis of all differential genes revealed that differential genes between the model and control groups were predominantly enriched in: i) Immune-related and inflammation-related pathways, including ‘Cytokine-cytokine receptor interaction’, ‘Chemokine signaling pathway’, ‘NOD-like receptor signaling pathway’ and ‘TNF signaling pathway’; ii) survival, proliferation and metabolism-related pathways, such as ‘PI3K-Akt signaling pathway’, ‘MAPK signaling pathway’, ‘cAMP signaling pathway’ and ‘FoxO signaling pathway’; and iii) cardiovascular and metabolic pathways, including the ‘Lipid and atherosclerosis’ and ‘Oxidative phosphorylation’ pathways. Differential genes between the Rapamycin and model groups were mainly enriched in: i) Immune-related and inflammation-related pathways, such as ‘Cytokine-cytokine receptor interaction’, ‘Chemokine signaling pathway’, ‘Toll-like receptor signaling pathway’, ‘NF-κB signaling pathway’, ‘TNF signaling pathway’ and ‘IL-17 signaling pathway’; and ii) antigen presentation and immune recognition pathways, including the ‘Antigen processing and presentation’ and ‘Phagosome’ pathways. Differential genes between the TDN and model groups were predominantly enriched in: i) Immune-related and inflammation-related pathways, such as ‘Cytokine-cytokine receptor interaction’, ‘NF-κB signaling pathway’ and ‘NOD-like receptor signaling pathway’; ii) metabolism-related and cell function-related pathways, such as the ‘Nucleotide metabolism’, ‘Glutathione metabolism’ and ‘Glycine, serine and threonine metabolism’ pathways; and iii) cell cycle-related pathways such as the ‘p53 signaling pathway’ (Fig. 2G).

Masson's trichrome staining showed a notable increase in collagen fibers in the urethral tissue of the model group compared with the control group. By contrast, the content of collagen fibers decreased after treatment with rapamycin and TDN. Immunohistochemical staining also demonstrated upregulated expression of fibrosis markers, such as α-SMA, TGF-β1, collagen I, collagen III and Smad3, in the model group compared with the control group, but their expression was subsequently decreased in the rapamycin and TDN groups compared with that in the model group (Fig. 3A).

Quantitative analysis of Masson's trichrome-stained sections using Image-Pro Plus software revealed a significant increase in collagen fiber content in the model group compared with that in the control group (P<0.001), along with a significant increase in the expression of fibrosis markers based on immunohistochemical analyses (P<0.01 or P<0.001). By contrast, the rapamycin and TDN groups exhibited a significant decrease in collagen content and reduced expression of fibrosis markers compared with the model group (P<0.05, P<0.01 or P<0.001), suggesting that rapamycin and TDN treatment exerted inhibitory effects on fibrosis (Fig. 3B).

Western blot analysis supported these findings, with significant increases observed in the protein expression levels of α-SMA, TGF-β1, collagen I, Smad3 and collagen III in the model group compared with the control group (P<0.01 or P<0.001). Compared with the model group, significant decreases in the expression of these markers were observed in the Rapamycin and TDN groups (P<0.05, P<0.01 or P<0.001) (Fig. 3C and D).

Overall, TDN effectively mitigated the progression of urethral fibrosis in the rat model by downregulating the expression levels of the fibrosis markers α-SMA, TGF-β1, collagen I, collagen III and Smad3, as well as by inhibiting collagen deposition as evidenced by Masson's trichrome staining Masson's trichrome staining and immunohistochemical staining indicated that TDN significantly suppressed the accumulation of collagen fibers and downregulated fibrosis markers in the model group, suggesting that TDN displayed protective effects against fibrosis. These results provide a theoretical basis for exploring the potential application of TDN.

Immune infiltration scores in rats were analyzed by ssGSEA to unravel the relationship between urethral injury and the immune system. The model group showed marked immune cell infiltration of innate and adaptive immune cells, particularly macrophages, monocytes, and activated CD4⁺ and CD8⁺ T cells, whereas neutrophil infiltration was reduced in the model group compared with in the control group, indicating that urethral injury induced a shift from acute neutrophil-dominated inflammation toward a macrophage- and lymphocyte-driven immune response, suggesting the development of a sustained inflammatory and immune remodeling process (Fig. 4A). Furthermore, the immune infiltration score heatmap showed marked differences in immune cell infiltration across the groups, with TDN treatment markedly reducing immune cell infiltration compared with that in the model group (Fig. 4B).

RT-qPCR showed that the mRNA expression levels of the inflammatory cytokines IL-6, IL-1β and TNF-α were significantly increased in the model group compared with the control group (P<0.001). Compared with those in the model group, rapamycin and TDN significantly downregulated the levels of these inflammatory cytokines (P<0.01 or P<0.001), indicating that after urethral injury, both rapamycin and TDN can modulate the immune response at the transcriptional level (Fig. 4C). An ELISA also supported these findings. The serum levels of inflammatory cytokines were significantly higher in the model group compared with the control group (P<0.001). Compared with those in the model group, the serum levels of inflammatory cytokines were significantly decreased in the treatment groups (P<0.01 or P<0.001) (Fig. 4D).

These results suggested that urethral injury significantly enhanced the immune response, while TDN effectively inhibited immune cell infiltration and lowered the expression levels of inflammatory cytokines, reducing the immune response triggered by urethral injury. TDN modulated the immune response at the transcriptional level and prevented the release of inflammatory cytokines, indicating its potential anti-inflammatory and immunoregulatory properties after urethral injury.

Transcriptomic analysis revealed that the expression levels of cell cycle-related genes, such as Ccnb1, Rrm2, Plk1 and Cdk1, were notably upregulated in the model group compared with the control group, suggesting that urethral injury may have accelerated cell proliferation by promoting cell cycle progression Compared with those in the model group, the expression levels of most of these genes were decreased in the rapamycin and TDN groups, whereas Rrm2 showed no marked reduction in the rapamycin group, indicating that rapamycin and TDN may have regulated cell proliferation by inhibiting the expression of cell cycle-related genes. Heatmap and boxplot analyses (Fig. 5A and B) visualized between-group differences in the expression of these genes, showing that after urethral injury, the expression of cell cycle-related genes was effectively controlled in the intervention groups.

RT-qPCR further supported the results of the transcriptomic analysis. The analysis (Fig. 5C) showed that, compared with those in the control group, the mRNA expression levels of Ccnb1, Rrm2, Plk1 and Cdk1 were significantly enhanced in the model group (P<0.05 or P<0.001). Furthermore, the expression levels of cell cycle genes were significantly lower in the rapamycin and TDN groups compared with the model group (P<0.05 or P<0.001), providing evidence that rapamycin and TDN regulated the expression of genes related to the cell cycle to inhibit cell proliferation.

Differential gene network analysis revealed the relationships between genes in the rat model of urethral injury. The network illustrated genes with significant interactions after urethral injury, with gene nodes connected by edges. The density of the network reflected the strength of gene interactions. By analyzing the interaction networks of these genes, the present study divided them into several functional modules, each representing a specific biological characteristic or function (Fig. 6).

This module contained genes related to cell energy metabolism, such as phosphoglycerate kinase 1 (Pgk1) and lactate dehydrogenase A (Ldha), indicating that urethral injury reprogrammed cell metabolism to support the repair and recovery of damaged cells. The changes in genes in this module suggested that cell energy metabolism may have served a key role after injury, providing sufficient energy for the repair process.

Changes in the expression of genes related to tissue repair and fibrosis, such as biglycan (Bgn) and matrix metallopeptidase 3 (Mmp3), revealed that urethral injury may have triggered a fibrotic response, changing the tissue structure. The initiation of fibrosis may have affected normal urethral function, with gene regulatory changes indicating that the repair process after injury may have been accompanied by unwanted fibrotic reactions.

This module involved immune-related genes, such as IL-1β complement C1q A chain (C1qa) and complement C1q B chain (C1qb), whose expression was significantly upregulated after urethral injury. Activation of these immune genes supported the important role of the immune system in the repair process after injury.

Genes related to cell proliferation and migration, such as Cd68 and actin γ1 (Actg1), may have promoted cell proliferation and migration at the injury site, thereby facilitating tissue repair after urethral injury. Regulation of cell proliferation and migration is a critical step in the tissue repair process, as it directly influences cellular turnover and structural remodeling during wound healing (28), and changes in gene expression in this module may reflect the activation of repair-associated cellular programs. Cellular stress response. Genes involved in cellular stress responses, such as peroxiredoxin 1, and heat shock protein family A member 5, may have helped cells to cope with injury and maintain normal function by regulating stress responses. The regulation of stress responses is important for cell survival and functional recovery after injury (27).

After urethral injury, activation of the immune response was supported by the differential expression of genes, such as IL-1β, C1qa and C1qb, suggesting that the immune response may have served an important role in the repair process. The immune system may have regulated inflammation and served a key role in fibrosis and tissue repair. Differential expression of Cd68 and Actg1 suggested that cell proliferation and migration served key roles in the repair process after injury. Changes in the expression of genes involved in cell energy metabolism, such as Pgk1 and Ldha, suggested that cell metabolism was important for repair after injury by providing sufficient energy to support cellular activities involved in tissue repair. The differential expression of genes associated with fibrosis, such as Bgn and Mmp3, suggested that fibrosis may have occurred after urethral injury and affected tissue repair.

To explore the effect of TDN on vimentin expression, fibroblasts were successfully isolated from rat urethral tissue and cultured using an enzymatic digestion method. The cells exhibited typical adherent growth characteristics (Fig. 7A). H&E staining revealed that these fibroblasts displayed typical morphological features, including deeply stained nuclei, lightly stained cytoplasm and clearly defined cell boundaries (Fig. 7B). To ensure the purity of fibroblasts used for vimentin regulation assays, immunofluorescence staining of α-SMA and CD90 was performed. Cells exhibited negative α-SMA staining together with positive CD90 expression, confirming fibroblast identity and excluding myofibroblast contamination (Fig. 7C). Immunohistochemical analysis showed high expression of vimentin in all three repeats (Fig. 7D and E).

Subsequently, three experimental conditions were established: Control, TGF-β-induced vimentin expression and TGF-β-induced vimentin expression + TDN treatment. Cells showed satisfactory growth in all groups (Fig. 7F). Immunohistochemical analysis was conducted to detect vimentin expression after different treatments. Cell images showed that the nuclei were stained blue, with vimentin stained brownish-yellow (Fig. 7H). Quantitative analysis indicated that, compared with the control group, treatment with TGF-β significantly increased the expression of vimentin (P<0.01). Treatment with TDN significantly reduced the expression of vimentin compared with that in the TGF-β group (P<0.05) (Fig. 7G). These results suggested that TDN may have regulated urethral fibrosis by inhibiting TGF-β-induced expression of vimentin.