Urine-Derived Stem Cells Attenuate Post-Injury Urethral Fibrosis in Association With Reduced Smad3/JunD Signaling Activation, Inflammation, and Extracellular Matrix Remodeling

Background: Urethral stricture is characterized by persistent inflammation and excessive extracellular matrix deposition, which contribute to progressive urethral fibrosis and narrowing. Urine-derived stem cells (USCs) have shown anti-inflammatory and anti-fibrotic potential, but their role in post-injury urethral fibrosis remains unclear. This study aimed to investigate whether early administration of USCs attenuates post-injury urethral fibrosis and subsequent stricture formation, and whether these effects are associated with changes in Smad3/JunD signaling activation, inflammation, and extracellular matrix remodeling.Methods: The adipogenic and osteogenic differentiation potential of USCs was evaluated by lineage-specific staining, including Oil Red O staining for lipid droplet formation and Alizarin Red staining for mineralized matrix deposition. A rat model of urethral stricture was established, and the animals were divided into five groups: Sham, US, USCs, US + USCs, and US + USCs + TGF-β1. Changes in tissue structure and collagen accumulation were determined using hematoxylin and eosin (H&E) staining and Masson’s trichrome staining, and urethral diameter was measured. Immunohistochemistry was performed to detect alpha-smooth muscle actin (α-SMA) expression. Urethral scar fibroblasts were isolated and indirectly co-cultured with USCs. Cell proliferation and fibrosis-related protein expression were assessed using the5-ethynyl-2′-deoxyuridine (EdU) assay and immunofluorescence staining, respectively. Expression levels of key mediators associated with Smad signaling and fibrotic remodeling, including phosphorylated Smad3 (p-Smad3), Smad family member 3 (Smad3), JunD proto-oncogene (JunD), collagen type I (COL-1), collagen type III (COL-3), fibronectin (FN), thrombospondin-1 (TSP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were determined using Western blot analysis. The levels of inflammatory cytokines, namely tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10), were assessed by enzyme-linked immunosorbent assay (ELISA).Results: USCs exhibited multilineage differentiation potential. At day 14 after urethral injury, rats showed a significant reduction in urethral diameter accompanied by epithelial hyperplasia, inflammatory cell infiltration, and increased collagen deposition. Early administration of USCs significantly increased urethral diameter, reduced collagen accumulation, and decreased α-SMA expression (p < 0.001). In vitro experiments demonstrated that USCs inhibited the proliferation of urethral scar fibroblasts and reduced the expression of α-SMA, COL-1, and TSP-1 (p < 0.001). USC treatment was accompanied by reduced p-Smad3/Smad3 and JunD expression in urethral tissues and fibroblasts. TGF-β1 treatment partly attenuated the anti-inflammatory and anti-fibrotic effects after USC treatment (p < 0.001).Conclusion: Early administration of USCs attenuates post-injury urethral tissue damage and fibrosis, and these effects may be partly associated with reduced Smad3/JunD signaling activation, decreased inflammation, and lower extracellular matrix deposition. These findings provide preclinical evidence for the early intervention of post-injury urethral fibrosis.