Determination The Role And Exploration The Mechanism Of Reactive Oxygen Species(ROS) In The Wound Inflammation Microenvironment On Wound Healing Of Benign Prostatic Hyperplasia After Prostatectomy

Objective:Thulium laser resection of the prostate（Tm LRP）,a major treatment for benign prostatic hyperplasia（BPH）,has several postoperative complications that affect the patients’quality of life.The aim of this study was to investigate the effect of the M1 macrophage-secreted reactive oxygen species（ROS）on prostatic wound healing,and the role of MAPK signaling in this process.Methods:Part I:An co-culture model in vitro was established between macrophages with prostate epithelial or stromal cells to simulate the microenvironment of the initial inflammatory phase of wound.The effect of defferent types of macrophages on prostate epithelial or stromal cells were analyzed by CCK-8,flow cytometry,wound healing assay and cell migration assay.The ROS probe identify the intracellular ROS levels in prostate epithelial or stromal cells after co-cultured with M1/M2macrophages,and Western blot measure the expression of EMT and MAPK-related proteins in prostate epithelial or stromal cells after co-cultured with M1/M2 macrophages.Part II:To simulate the effects of M1 macrophage-secreted ROS on prostate epithelial and stromal cells,we cultured the cells with H₂O₂and the M1 macrophages co-culture model with ROS blockers.We used some experiments as above to determine the impact of different ROS level to prostatic cells.The expression of MAPKs signaling pathway related proteins was detected by Western blot.PartⅢ:An in vivo model of prostatectomy was established in beagles by subjecting them to Tm LRP,and were either treated with N-acetyl-L-cysteine（NAC）and or placebo.The histological analysis,immunohistochemical and immunofluorescence were then carried out to study the wound healing and re-epithelialization of the prostatic wound,the proliferation and differentiation of basal cells and the polarization of M1 macrophages in wound tissues in each group.Results:Part I:Co-culture between M1 macrophages with prostate epithelial and stromal cells increased the levels of ROS and apoptosis in prostate epithelial and stromal cells and impeded the proliferation and migration of prostatic epithelial and stromal cells.M2 macrophage group is reversed,accelerating the proliferation and migration and promoting EMT.MAPKs signaling pathway-related proteins were up-regulated in M1macrophages co-culture group.Part II:We found that a dose and time-dependent decrease in the viability of the prostate cells which were exposure to H₂O₂.The opposite was apoptotic level.We also analyzed the impact of H2O2 on MAPK signaling and found a dose-dependent increase in the levels of MAPK pathway mediators.M1 macrophage-mediated prostatic epithelial and stromal cell damage was abolished by ROS inhibition.The experimental result showed that the proliferation and migration of prostate cells was increased in ROS blockers group.Adding ROS scavengers in M1 macrophage co-culture group could down-regulate MAPKs-related proteins expression.PartⅢ:In the NAC group,the healing of the prostatic urethra after a Tm LRP was superior to the control group.The number of basal cells in NAC group was significantly higher than that in the control group,NAC reduce the duration the infiltration of M1 macrophages in the wound healing process.Conclusion:Intracellular ROS levels were significantly increased in the non-ROS-producing cells（prostate epithelial and stromal cells）following co-culture with M1-like macrophages via MAPK activation,which affected their proliferation,migration,apoptosis and EMT levels,and delayed the wound healing process.Usage of ROS inhibitors could partially improve the affects of ROS.The beagle models have shown that the appropriate using of ROS inhibitors could accelerate the process of wound healing and re-epithelialization,promote the proliferation of basal cells and inhibit the continuous infiltration of M1 macrophages.