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Article Open Access

Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats

  • Authors:
    • Caifen Guo
    • Jian Li
  • View Affiliations / Copyright

    Affiliations: Department of Urology, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China, Department of Sports Medicine, The Beijing Jishuitan Hospital Guizhou Hospital, Guiyang, Guizhou 550014, P.R. China
    Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 105
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    Published online on: February 2, 2026
       https://doi.org/10.3892/mmr.2026.13815
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Abstract

Urethral injury is a common type of traumatic damage to the urinary system, often leading to urethral stricture, fibrosis and dysfunction, which significantly impair physiological function and quality of life. The present study aimed to investigate the therapeutic efficacy of the novel immune‑regulatory molecule tetrahedral DNA nanostructure (TDN) in a rat model of urethral injury and explore the underlying mechanisms of action. A rat model of urethral injury was established through mechanical injury. Animals were divided into four groups: Control, model, model + rapamycin and model + TDN. Therapeutic effects and associated mechanisms were assessed via retrograde urethrography, Masson's trichrome staining, immunohistochemistry, western blotting, reverse transcription‑quantitative PCR (RT‑qPCR) and transcriptomic analysis. The results revealed that TDN markedly alleviated the immune response after urethral injury, reduced immune cell infiltration, downregulated the expression of inflammatory cytokines, including IL‑6, IL‑1β and TNF‑α, and effectively inhibited the progression of fibrosis. Masson's trichrome staining and western blotting provided evidence of reduced collagen deposition and decreased expression of fibrosis markers, including α‑smooth muscle actin, TGF‑β1, collagen I, collagen III and Smad3, after treatment with TDN. Transcriptomic analysis revealed that TDN modulated multiple immune‑related pathways, including the NF‑κB signaling pathway, NOD‑like receptor signaling pathway and cytokine‑cytokine receptor interaction, accompanied by a decrease in immune‑inflammatory responses, such as reduced inflammatory cytokine production and immune cell infiltration. Additionally, the results suggested that TDN may improve cellular metabolism and inhibit cell proliferation by downregulating the expression of cell cycle‑associated genes, as demonstrated by transcriptomic analysis and RT‑qPCR validation of cyclin B1, ribonucleotide reductase regulatory subunit M2, polo‑like kinase 1 and cyclin‑dependent kinase 1. In conclusion, TDN notably promoted tissue repair after urethral injury in rats by regulating the immune response, inhibiting fibrosis and enhancing cellular metabolism. These findings highlight TDN as a promising therapeutic candidate for urethral injury and offer novel insights into immune-regulatory strategies for the treatment of other fibrotic diseases.

View Figures

Figure 1

(A) Urethral tissue collection images
from the control, model, model + rapamycin, and model + TDN group.
(B) Body weight monitoring curves for rats in all groups over time.
(C) Urethrographic imaging results for each group, with red arrows
indicating the location of urethral injury model establishment. (D)
Hematoxylin and eosin staining results for urethral tissues from
each group. Red arrows indicate urethral lumen narrowing and
associated fibrotic changes in the injured urethra. Scale bar, 500
µm (left) and 50 µm (right). TDN, tetrahedral DNA
nanostructure.

Figure 2

(A) PCA results. Different colors
represent different treatment groups. (B) Sample correlation
heatmap. The color intensity corresponds to correlation values. (C)
Combined volcano plot showing the distribution of FCs in
differentially expressed genes in the three group comparisons
(model vs. control; model + rapamycin vs. model; model + TDN vs.
model), with yellow dots representing upregulated genes and green
dots representing downregulated genes. (D) Bar chart of
differential gene counts showing the number of differential genes
in the three group comparisons. (E) Venn diagram of differential
genes displaying the distribution of differential genes in the
three group comparisons. The numbers in different areas represent
specific intersections or unique differential genes. (F) Heatmap
showing the expression patterns of 25 common differentially
expressed genes identified from three pairwise comparisons,
displayed across four experimental groups (Control, Model, Model +
rapamycin, and Model + TDN). Each row represents one gene and each
column represents an individual sample. Color gradients indicate
normalized gene expression levels. (G) KEGG pathway enrichment
analysis of differentially expressed genes from the three pairwise
comparisons (model vs. control; model + rapamycin vs. model; model
+ TDN vs. model). Enrichment results are presented as dot plots.
The x-axis represents the GeneRatio, and the size of each dot
reflects the proportion of genes enriched in the corresponding
pathway. Dot color indicates the statistical significance expressed
as -log10(P-value). KEGG pathways are displayed consistently across
the three comparisons to facilitate direct visual comparison. KEGG,
Kyoto Encyclopedia of Genes and Genomes; TDN, tetrahedral DNA
nanostructure; FC, fold change; PCA, principal component
analysis.

Figure 3

(A) Masson's trichrome staining and
immunohistochemistry results. Masson's trichrome staining detected
changes in collagen fibers in urethral tissues, with collagen
fibers appearing blue and muscle fibers, fibrin and red blood cells
appearing red. Immunohistochemistry staining showed the cell nuclei
in blue and positive protein staining for the fibrosis markers
α-SMA, TGF-β1, collagen I, collagen III and Smad3 in brown. Images
are presented at a magnification of ×200. (B) Statistical analysis
of Masson's trichrome staining and immunohistochemistry results.
Staining results were analyzed with Image-Pro Plus software(version
6.0; Media Cybernetics, Inc.), followed by bar chart construction
using GraphPad Prism. *P<0.05, **P<0.01 and ***P<0.001.
(C) Western blot analysis results. Western blot analysis was used
to detect the protein expression of fibrosis markers in urethral
tissue. (D) Statistical analysis of western blotting results. Band
densities were analyzed using Image-Pro Plus software(version 6.0;
Media Cybernetics, Inc.), and bar charts were drawn with GraphPad
Prism. *P<0.05, **P<0.01 and ***P<0.001. α-SMA, α-smooth
muscle actin; TDN, tetrahedral DNA nanostructure.

Figure 4

(A) Immune infiltration score
boxplot. Single-sample gene set enrichment analysis was used to
calculate the immune cell infiltration scores in each group and
results were displayed as a boxplot, with the box representing the
interquartile range, the median indicated by a horizontal line and
the whiskers showing the distribution range of the data. (B) Immune
infiltration score heatmap. After standardization, the heatmap
displays the infiltration scores of different immune cells across
groups. Color changes represent high to low immune infiltration
scores, with red indicating high scores and blue indicating low
scores, rows represent different immune cell types, and columns
represent experimental groups. (C) RT-qPCR detection of
inflammatory cytokines. RT-qPCR analysis was used to measure the
mRNA expression levels of IL-6, IL-1β and TNF-α. **P<0.01 and
***P<0.001. (D) ELISA detection of inflammatory cytokines in
serum. An ELISA was used to measure the levels of IL-6, IL-1β and
TNF-α in serum. **P<0.01 and ***P<0.001. RT-qPCR, reverse
transcription-quantitative PCR; TDN, tetrahedral DNA nanostructure;
NK, natural killer; NKT, natural killer T cell; DC, dendritic cell;
pDC, plasmacytoid dendritic cell; MDSC, myeloid-derived suppressor
cell; Act., activated; Imm., immature; CM, central memory; EM,
effector memory; Treg, regulatory T cell; Tfh, T follicular helper
cell; Th1, type 1 T helper cell; Th2, type 2 T helper cell; Th17,
type 17 T helper cell; Mono, monocyte; Macro, macrophage; Neut,
neutrophil; Eos, eosinophil; Mast, mast cell; Plasma, plasma
cell.

Figure 5

(A) Gene expression heatmap and (B)
boxplots for Ccnb1, Rrm2, Plk1 and Cdk1. The heatmap
shows the expression level changes of Ccnb1, Rrm2, Plk1 and
Cdk1 across groups, with color changes reflecting the
difference in gene expression, where orange indicates high
expression and green indicates low expression. The boxplot
illustrates the distribution of gene expression levels among
groups. (C) RT-qPCR detection of gene expression. RT-qPCR was used
to measure the mRNA expression levels of Ccnb1, Rrm2, Plk1
and Cdk1 in urethral tissue, with results displayed in a bar
chart. *P<0.05 and ***P<0.001. RT-qPCR, reverse
transcription-quantitative PCR; TDN, tetrahedral DNA nanostructure;
Ccnb1, cyclin B1; Rrm2, ribonucleotide reductase
regulatory subunit M2; Plk1, polo-like kinase 1;
Cdk1, cyclin-dependent kinase 1; FPKM, fragments per
kilobase million.

Figure 6

Differential gene network analysis.
The nodes represent differentially expressed genes across the four
groups, and the edges represent the interactions between these
genes. The network analysis involved multiple biological processes,
including tissue repair and fibrosis, cell proliferation and
migration, energy metabolism and cell function, immune response and
cellular stress response. Each node represents an individual
differentially expressed gene in the network, and the depth of the
color of the node reflects the frequency of gene interaction in the
network. The darker the color, the more frequently the gene
participates in interactions.

Figure 7

(A) Fibroblast isolation and
cultivation. Representative phase-contrast images of primary rat
urethral fibroblasts following enzymatic digestion are shown. The
two images represent different microscopic fields from the same
primary culture, illustrating progressive cell adhesion and the
typical spindle-shaped, adherent morphology with elongated cell
bodies and prominent cytoplasmic extensions. (B) H&E staining.
Cell nuclei were stained darkly, with the cytoplasm stained lightly
and clear cell boundaries visible, with cells displaying the normal
morphological characteristics of fibroblasts after H&E
staining. (C) Immunofluorescence staining identification of
fibroblasts. Immunofluorescence staining for α-SMA (green) and CD90
(red) was performed to verify fibroblast purity. Nuclei were
counterstained with DAPI (blue) and imaged at a magnification of
×200 using a Nikon TS2-S-SM fluorescence microscope. Cells
exhibited strong CD90 expression, α-SMA staining was negative,
whereas CD90 staining was clearly positive, confirming fibroblast
identity and excluding myofibroblast contamination. (D)
Immunohistochemical detection of vimentin. Vimentin expression in
fibroblasts was detected by immunohistochemistry. The nuclei were
stained blue, and vimentin-positive cytoplasm appeared
brownish-yellow. Images were obtained at a magnification of ×200
using a digital slide scanner. (E) Immunohistochemical detection of
vimentin. Quantitative analysis among three replicates was
performed using Image-Pro Plus, and statistical evaluation with
GraphPad Prism 9.0 showed no significant differences among groups
(P>0.05), confirming reproducibility. (F) Cell culture under
different treatments. Representative phase-contrast images
(magnification, ×100) showed fibroblasts in the control, TGF-β and
TGF-β + TDN groups, all displaying sufficient adherence and
morphology. (G) Quantitative analysis of vimentin expression under
different treatments. Vimentin expression levels in fibroblasts
from the control, TGF-β and TGF-β + TDN groups were quantified
based on immunohistochemical staining using Image-Pro Plus.
Statistical analysis was performed using GraphPad Prism 9.0
(*P<0.05, **P<0.01). (H) Immunohistochemical detection of
vimentin under different treatments. Representative
immunohistochemical images showed vimentin expression in
fibroblasts from the Control, TGF-β and TGF-β + TDN groups. α-SMA,
α-smooth muscle actin; TDN, tetrahedral DNA nanostructure.
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Copy and paste a formatted citation
Spandidos Publications style
Guo C and Li J: <p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>. Mol Med Rep 33: 105, 2026.
APA
Guo, C., & Li, J. (2026). <p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>. Molecular Medicine Reports, 33, 105. https://doi.org/10.3892/mmr.2026.13815
MLA
Guo, C., Li, J."<p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>". Molecular Medicine Reports 33.4 (2026): 105.
Chicago
Guo, C., Li, J."<p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>". Molecular Medicine Reports 33, no. 4 (2026): 105. https://doi.org/10.3892/mmr.2026.13815
Copy and paste a formatted citation
x
Spandidos Publications style
Guo C and Li J: <p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>. Mol Med Rep 33: 105, 2026.
APA
Guo, C., & Li, J. (2026). <p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>. Molecular Medicine Reports, 33, 105. https://doi.org/10.3892/mmr.2026.13815
MLA
Guo, C., Li, J."<p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>". Molecular Medicine Reports 33.4 (2026): 105.
Chicago
Guo, C., Li, J."<p>Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats</p>". Molecular Medicine Reports 33, no. 4 (2026): 105. https://doi.org/10.3892/mmr.2026.13815
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