Human umbilical cord mesenchymal stem cell-derived exosomes attenuate fibrotic stricture remodeling through the let-7i -5p/TGFBR1 axis

Introduction Urethral stricture is a common and debilitating urological condition in which fibrosis narrows the urethral lumen. Recurrence after surgical repair is frequent, and no antifibrotic pharmacotherapy has been established. Extracellular vesicles from human umbilical cord mesenchymal stem cells (hUC-MSC-EVs) show antifibrotic activity in several organ systems, but whether they act in urethral fibrosis, and by what mechanism, had not been examined. Methods We isolated and characterized hUC-MSC-EVs and tested them in primary human urethral fibroblasts (UFBs) stimulated with TGF-β1 and in a rat model of trauma-induced urethral stricture. Small RNA sequencing was used to profile the EV miRNA cargo, and the role of let-7i-5p was examined by gain- and loss-of-function experiments, dual-luciferase reporter assays, and let-7i-5p-depleted EVs (KD EVs), both in vitro and in vitro . Results hUC-MSC-EVs dose-dependently suppressed TGF-β1-induced UFB proliferation, migration and myofibroblast differentiation and lowered α-SMA and Collagen I expression, with reduced Smad2/3 phosphorylation. Periurethral injection in rats significantly reduced urethral wall hyperechogenicity, luminal narrowing, submucosal collagen deposition and profibrotic protein expression at the 4-week postoperative endpoint. Small RNA sequencing placed let-7i-5p among the most abundant miRNAs packaged in hUC-MSC-EVs, and this miRNA was efficiently transferred to recipient UFBs. let-7i-5p bound the 3′-UTR of TGFBR1 and suppressed TGFBR1 expression at both mRNA and protein levels. Depleting let-7i-5p from EVs with an inhibitor largely abolished their antifibrotic activity in vitro and in vitro , linking EV-delivered let-7i-5p to suppression of the TGFBR1/Smad2/3 signaling axis. Conclusion These results identify hUC-MSC-EVs as an injectable, cell-free antifibrotic candidate for urethral stricture and let-7i-5p as a cargo on which their activity depends.