An Exploratory Study Of Platelet-rich Plasma In Improving Bladder Dysfunction Caused By Partial Bladder Outlet Obstruction In Rats

Objective:Bladder outlet obstruction is an increase in urinary resistance caused by various types of bladder neck and/or urinary tract lesions,manifested as urinary disorders.Currently,it is believed that BOO is an orderly process from inflammation to fibrosis,and there is no effective way to delay bladder dysfunction caused by obstruction.Platelet rich plasma is a platelet concentrate obtained after blood centrifugation.It contains a variety of growth factors and cytokines and is widely used in the field of regenerative medicine.Previous studies have shown that PRP can improve tissue inflammation by promoting M2 type transformation of macrophages,and its specific mechanism is still unclear.This study aims to investigate the effects of platelet rich plasma activated by different activators and stored for different times on the phenotype of human monocyte derived macrophages,in order to explore the optimal conditions for inducing M2 polarization of macrophages in platelet rich plasma treatment;Preliminary exploration of whether the optimal treatment conditions of PRP can improve bladder tissue inflammation and bladder function in rats with partial bladder outlet obstruction,and its mechanism.Methods:This study includes two parts:cytological study and animal study.In the first part:（1）PRP was collected by automatic blood component separator,and activated by thrombin,ADP or Ca Cl₂.（2）Monocyte were separated from the buffy coat by Ficoll density gradient centrifugation,then induced by 50ng/m L M-CSF to obtain monocyte derived macrophages.（3）Under different conditions,PRP was co cultured with macrophages for 24 hours.（4）Flow cytometry was used to detect the expression of macrophages CD86,CD163,and CD206.（5）The concentration of growth factor in supernatant of PRP with different storage time activated by Ca Cl₂were detected by ELISA.Part II:Establish a rat model of partial bladder outlet obstruction using partial bladder neck ligation method;After 2 weeks,urodynamic testing was used to evaluate bladder function,and observing the morphological characteristics of the bladder specimen with the naked eye;HE staining was used to observe the inflammatory infiltration status of the submucosa of the bladder,Masson staining was used to observe the fibrosis area of the submucosa of the bladder,immunofluorescence was used to detect the number of macrophage subtypes in the submucosa of the bladder,and ELISA was used to detect the level of inflammatory related factors in the bladder tissue.Results:（1）After PRP treatment of macrophages under different treatment condition,the expression level of CD86 in macrophages stimulated by PRP supernatant（thrombin and Ca Cl₂activated）was higher than that by PRP group,and the expression of CD163 in macrophages increased by Ca Cl₂activated PRP supernatant.（2）The concentrations of FGF and EGF in the groups with storage time more than 10 months were significantly higher than those in the groups with storage time less than 10 months,and the expressions of CD86,CD163 and CD206 of cocultured macrophages in both groups were increased.（3）The results of urodynamic tests in rats showed significant abnormalities in bladder function after 2 weeks of partial obstruction of the bladder outlet.After PRP injection treatment,the bladder function of rats improved.（4）The results of HE staining and Masson staining showed that the bladder tissue structure of p BOO rats was severely abnormal after 2 weeks of obstruction,with a large number of infectious inflammatory cells infiltrating the tissue.The bladder tissue structure and inflammatory cell infiltration in rats treated with PRP were significantly improved.（5）The result of ELISA detection of cytokines in rat bladder showed that the expression of IL-1β,TNF-αand IL-6 in the bladder of p BOO rats after 14 days of obstruction increased and decreased after PRP treatment,but only the expression of IL-6was statistically different.（6）The results of immunofluorescence detection of macrophage subpopulation in rat bladder showed that the number of M1macrophages in the bladder of p BOO rats increased significantly,while after PRP treatment,M1 macrophages decreased and M2 macrophages increased significantly.Conclusion:（1）Activation of PRP by CaCl₂may better promote M2polarization in macrophages.（2）After long-term storage at-80℃（>10months）,the anti-inflammatory effect of PRP increases,but the concentration of pro-inflammatory cytokines also increases accordingly.（3）This study is the first to use intravesical injection of PRP to treat bladder dysfunction secondary to p BOO in rats.The results showed that activation of PRP supernatant by Ca Cl2can improve the urination efficiency of p BOO rats,and the infiltration of inflammatory factors in bladder tissue is significantly reduced.（4）The beneficial role of PRP in tissue repair may not only be attributed to the large number of growth factors contained within it,but also depends on its regulation of macrophage polarization,thereby re improving inflammatory processes.