Exosomal MiR?146a Contributes To The Enhanced Therapeutic Efficacy Of TNF-?-Primed Mesenchymal Stem Cells Against Urethral Stricture In Rats

[Objective] Currently,the treatment of urethral stricture is still a problem that bothered urologists,because the intraluminal treatment and drugs are ineffective.Mesenchymal stem cells(MSC)have a certain effect in the treatment of urethral stricture,but the mechanism remains unknown.Studies have shown that the proinflammatory cytokines can enhance the effect of MSC on urethral stricture.In this study,we illustrate the effect of human umbilical cord-derived MSC exosomes pretreated with tumor necrosis factor alpha(TNF-?)on urethral stricture in rats and its mechanism.[Methods] MSCs were pretreated with TNF-?.Both TNF-?-induced MSC exosomes(?MSC-exo)and untreated MSC exosomes(MSC-exo)were collected to detect the expression of mi R-146 a.Male Sprague-Dawley rats were induced by TGF-?1 and cut into the urethra to produce a model of urethral stricture.MSC-exo(30?g)and ?MSC-exo(30?g)were injected in the penis urethra respectively.There were four groups as follow,control group,TGF-?1 group(TGF-?1 injection),T+MSC-exo group(TGF-?1 and MSC-exo injection)and T+?MSC-exo group(TGF-?1 and TNF-? induced MSC-exo injection).Four weeks later,the urethral stricture were evaluated by ultrasound,and histological analysis of the urethra tissue was conducted after euthanasia.In vitro,MSC-exo and ?MSC-exo were applied to fibroblasts respectively,and the migration of fibroblasts were observed by cell wound scratch assay.q RT-PCR was used to detect the expression of mi R-146 a in fibroblasts.The effects of TNF-?-treated MSC-derived exosomes on fibroblast activation and collagen secretion were analyzed by immunofluorescence,quantitative RT-PCR and Western blotting.The expression of IRAK1/TRAF6/NF-?B-related pathway proteins were assessed by Western blot and q RT-PCR.Finally,mi R-146 a in ?MSC-exo was inhibited to observe whether it affects fibroblast activation and collagen secretion.[Results] Rat urethral ultrasound results showed that MSC exosomes pretreated with TNF-? inhibited urethral fibrosis and stenosis more effectively than the virgin MSC exosomes.Immunohistochemistry showed that the expression of ?SMA in T+?MSC-exo group was significantly lower than that in TGF-?1 group and T+MSC-exo group.Masson trichrome(MT)and H&E staining also showed that there was only mild submucosal urethra fibrosis in ?MSC-exo group.In vitro experiments showed that ?MSC-exo enhanced fibroblast migration ability(P < 0.05).Immunofluorescence,Western blot and q RT-PCR indicated that,in T+?MSC-exo group,the m RNAs and protein expression of ?SMA,Collagen I and Collagen III was significantly reduced(P < 0.05).We found that mi R-146 a is strongly upregulated in TNF-? stimulated MSC and selectively packaged into exosomes.In addition,Western blot and q RT-PCR showed that the expression of IRAK1,TRAF6 and NF-?B in T+?MSC-exo group were lower than those in T+MSC-exo group(P<0.05).As the expression of mi R-146 a was decreased in TNF-?-treated MSC exosomes,the protein and m RNA expression of ?SMA,Collagen I and Collagen III were significantly increased(P<0.05),which partially reduced TNF-? induced anti-fibrotic effects of MSC exosomes.[Conclusion] TNF-?-treated MSC can increase the expression of mi R-146 a in its exosomes,which can inhibit the IRAK1/TRAF6/NF-?B signaling pathway and improve the effectiveness for urethral stricture.