Process Of Re-Epithelialization And Functional Mechanisms Of PDE5 Inhibitor In The Wound Healing After Transurethral Resection Of The Prostate

Objective:To investigate wound healing process after transurethral endoscopic resection of the prostate.To clarify the source of the regenerated urothelial cells in the wound and the importance of the prostate epithelial stem/progenitor cells in the process of re-epithelialization.To establish an in vitro induction model for trans-differentiation from the prostate epithelial stem/progenitor cells into urothelial-like cells.To study the dynamic changes of type 5 phosphodiesterase(PDE5)expression in wound tissue.To determine the effect and related mechanisms of PDE5 inhibitor(PDE5i)during wound healing.Methods:1.Adeno-associated virus(AAV)was used to label beagle canine prostate cells with EGFP.After that,an endoscopic prostat resection model was establish and the wound tissues were collected.HE staining,immunofluorescence detection,flow cytometry analysis,and scanning electron microscopy were carried out to study the process of postoperative wound healing and clearigy the source of the re-epithelial cells.Transcriptome change of the prostate epithelial stem/progenitor cells after urea treatment was investigated using gene chip and bioinformatics analysis.2.CD49f highly expressioned prostate stem/progenitor cells were sorted by magnetic strategy,and then the cells were induced to trans-differentiate into urothelial cells in vitro.Immunofluorescence(IF)and Western blotting(WB)were carried out to detect epithelial cell markers p63,CK5,CK8/18,prostate epithelial marker AR,PSA,Nkx3.1,urothelial marker Uroplakins(UPs).ELISA,transepithelial electrical resistance,proliferation and apoptotic assays were carried out to determine cell physiological function changes after the trans-differentiation.Cellular transcriptomics changes were analyzed using RNA-seq and bioinformatics.3.IF,WB,q PCR and other techniques were used to detect the expression of PDE5 protein in the wound tissue.Moreover,to determine effects of PDE5 inhibitor(PDE5i)on trans-differentiation of the prostate epithelial stem/progenitor cells,and fibrosis,autophagy and apoptosis of the prostate stromal fibroblasts respectively,the cells were treated with PDE5i and analyzed using i IF and WB.Additionally,canine model was established to study clinical effects of PDE5i,tadalafil,on the wound healing.Results:1.The canine prostate cells were successfully labeled with EGFP.The canine prostate resection model was further established.The EGFP positive regenerated urothelial cells gradually lined the wound surface in 1-4 weeks after the surgery,and formed integrated tight junction between the cells.The prostate epithelial cells were able to migrate toward the wound surface.Urothelial metaplasia and urothelial hallmarks could be observed among these prostate epithelial cells.Urea could penetrate into the wound tissue at the earlier phase of the wound healing because of the urine-tissue barrier destruction.Additionally,we revealed that gene expression profile of the prostate epithelial stem/progenitor cell changed obviously after urea stimulation.2.CD49f highly expressed prostate epithelial stem/progenitor cells were successfully sorted.flow cytometry identified that these cells expressed high level of epithelial stem cell marker p63 and CD49f but not the luminal cell marker CD26.The prostate epithelial stem/progenitor cells could be trans-differentiated into urothelial-like cells after in vitro culture with prostate-urothelium trans-differentiation medium.The urothelial-like cells expressioned high level of UPs,owned transmembrane resistance>3000?·cm~2,and tolerance to high concentration of urea.5949 differential expressed genes were determined using RNA-seq between prostate epithelial stem/progenitor cells and induced urothelial-like cells.3.IF staining showed that PDE5protein was highly expressed in the regenerated urothelial cells at 1-3 weeks after the surgery,and gradually decreased to normal level at week 4.WB and q PCR demonstrated that M1 macrophage could promote PDE5 expression in the prostate epithelial stem/progenitor cells.PDE5i was able to promote trans-differentiation of the prostate epithelial stem/progenitor cells into the urothelial-like cells as well as inhibit fibrosis and autophagy of the prostate stromal fibroblasts.Animal experiments demonstrated that PDE5i tadalafil promotes post-operative wound healing.Conclusion:1.The regenerated urothelial cells during prostatic urethral wound healing originate from epithelial stem/progenitor cells in the residual prostate tissue after BPH surgery.Urea in urine interferes differentiation of prostate epithelial stem/progenitor cells into normal prostate luminal cells.2.The prostate-urothelia trans-differentiation medium can induce CD49f highly expressed prostate epithelial stem/progenitor cells trans-differentiate into urothelial-like cells.3.PDE5i tadalafil can promote wound healing after prostate resection by accelerating re-epithelialization and inhibiting excessive fibrosis.