| Background: Post-traumatic urethral stricture is one of the common causes of urethral stricture in clinical practice,and it is also one of the intractable problems perplexing urologists.Glutaminase(GLS)is a key enzyme in regulating the metabolism of glutamine,and the process of urethral scar formation,which is crucial in fibroblast activation.Mesenchymal stem cells(MSCs)can be used to treat urethral stricture,but the mechanism of their regulation of fibroblast metabolism has not been clarified.Therefore,the study of the regulation of MSCs exosomes on the metabolism of glutamine in fibroblasts after urethral injury may bring new ideas for the diagnosis and treatment of urethral stricture.Objective: To investigate the effect of miR-23 a in human umbilical cord blood derived MSCS-Exo on glutamine metabolism of urethral fibroblast in New Zealand rabbits and its therapeutic effect on urethral stricture.Methods: The supernatant of MSCs culture was collected,exosomes were extracted and identified by hypervelocity centrifugation.TGF-β1 was used to construct a New Zealand rabbit urethral stricture model and exosome intervention was given,and the experiment was divided into three groups:Blank control group,TGF-β1 group(only TGF-β1 was injected into urethral injury site)and TGF-β1+MSCS-Exo group(TGF-β1+ MSCS-Exo was injected into urethral injury site).Two months later,urethral stricture was assessed by urethrography.Immunohistochemical and Masson staining were performed on urethral scar tissue.Deterine the degree of tissue fibrosis.In vitro experiments,LC-MS was used to clarify the differences in glutamine metabolism before and after activation of urethral fibroblasts,and the experiments were divided into four groups:Control group,TGF-β1 group,TGF-β1+BPTES group and TGF-β1+BPTES+α-KG group,Western blot analysis,The expression of GLS and collagen in fibroblasts in each group and their effects on the migration and proliferation of urethral fibroblasts were confirmed by q RT-PCR,scratch test and Transwell co-culture.The binding of miR-23 a to GLS was verified by dual-luciferase reporter gene detection and other techniques.The expression of GLS and collagen in each group was detected by cell transfection,metabolite detection,Western blot and q RT-PCR.To explore the regulation and mechanism of human umbilical cord blood-derived MSCS-Ex O on glutamine metabolism in urethral fibroblasts.Results: Nanosight detection,TEM morphological observation and WB marker protein detection indicated that the microvesicles obtained after superionization were consistent with the characteristics of exosomes.Urethrogram images of New Zealand rabbits showed that TGF-β1+ MSCS-Exo group was more effective in inhibiting urethral fibrosis and stricture than TGF-β1 group.Immunohistochemistry,Masson(Trichrome MT)staining and H&E(Hematoxylin-Eosin)staining also showed that TGF-β1+ MSCS-Exo group alleviated the degree of tissue urethral fibrosis more obviously.In vitro experiments,LC-MS detection was used to verify the increase of glutamine metabolites in urethral fibroblasts after TGF-β1stimulation.Western blot,q RT-PCR scratching test and Transwell co-culture results indicated that TGF-β1 stimulation promoted the expression of GLS.At the same time,the proliferation and migration ability of fibroblasts were increased.GLS inhibitor BPTES was used to reduce GLS level and collagen synthesis,and the proliferation and migration ability of fibroblasts were also decreased.Supplementation of α-KG could reverse the inhibitory effect of BPTES.Dual luciferase reporter gene assay confirmed that the exosome miR-23 a inhibited the translation of GLS m RNA 3’UTR by targeting GLS,thereby down-regulating the level of gluten metabolism,and inhibiting the expression of collagen,proliferation and migration of urethral fibroblasts.Conclusion: Umbilical cord blood-derived mesenchymal stem cells exosome miR-23 a can inhibit the proliferation and activation of urethral fibroblasts by regulating the expression of GLS in urethral fibroblasts,and at the same time reduce the expression of collagen in fibroblasts and reduce urethral scar formation. |
