IL-1β-induced Mesenchymal Stem Cell Exosomes Regulate Macrophage Polarization In The Treatment Of New Zealand Rabbit Urethral Stricture

Objective:in recent years,one of the most difficult problems for urological surgeons is the treatment of urethral stricture.At present,neither endovascular resection or drug therapy is effective.Currently,there is a literature that has been used in the treatment of urethral stricture with mesenchymal stem cells（MSc）.However,the molecular mechanism of its effect has not been elucidated.This study is to explore the effect of human umbilical cord blood derived mesenchymal stem cells exocrine on the New Zealand rabbit urethral stricture model and its molecular mechanism.Methods:At the first,we extracted the exosin of MSC with ultra high speed centrifugation,then verified the exosomatic phenotype protein,particle size and electron microscopic observation of exosomatic morphology.Then we explored the effect of exosin on urethral stricture in New Zealand rabbits.We induced and cut the penile urethra model through the use of transforming factorβ1（TGF-β1）in male New Zealand rabbits.Injecting different reagents or drugs into the penile urethra of New Zealand rabbits,the experiment was divided into four groups,namely,blank control group,TGF-β1 group（only TGF-β1）,MSC-EXO+TGF-βgroup（injected TGF-β1 and untreated MSC-EXO）and MSC^IL-1β-exo group（TGF-β1 injection TGF-β1 and IL-1βinduced MSC-EXO）after a month of.2,urinary tract stenosis was evaluated by urography.Histological and cytological analysis was performed on the urethra tissue of New Zealand rabbits.Subsequently,in vitro experiments,the changes in the phenotypic markers of macrophages in different treatment groups and the changes of inflammatory related cytokines secretion in the microenvironment and the expression of fibrotic related proteins after fibroblasts were detected by Western blot,q RT-PCR and flow cytometry.Then,we identified the mechanism of macrophage polarization through Transwell co culture and cell scratch tests.Finally,we detected the expression of mi R-92a in the macrophages treated with MSC^IL-1β-exo by q RT-PCR,and Western blot and q RT-PCR were detected with or without mi R-92a inhibitors.The expression of PTEN,p-Akt and p-P65 pathway proteins and m RNA expression were determined,and the specific mechanism of macrophage polarization was defined.Results:first,we detected the marker protein of exocrine body by Western blot,detected the size of exocrine body by particle size and observed the morphology of exocrine under electron microscope.The results confirmed that we extracted the exosomatic body.Then,the urography showed that the effect of exocrine on the urethral stricture of New Zealand rabbits was compared with that of untreated MSC.The MSC derived exoses with IL-1βpretreatment were more effective in urethral fibrosis and reducing the degree of urethral stricture.In Masson trichrome staining and H&E staining,the degree of tissue fibrosis in theβMSC-exo group was significantly reduced.In immunohistochemical results,the expression ofαSMA in the MSC-exo group was significantly lower than that in the control group.QRT-PCR results showed that:compared with the control group,Arg-1expression increased significantly in the exocrine group and increased with the increase of exocrine concentration,while INOS and IL-6 decreased significantly.In flow cytometry,compared with control group,the proportion of CD206 expressing protein in exsome stimulation group and MSCs culture group was significantly lower than that in control group.Moreover,the cells expressing CD206 marker protein in exosome（500ug/ml）group were slightly higher than those in exosome（200ug/ml）group and MSCs co culture group.In Transwell co culture technique,we found that compared with control group,the number of cells migrating to the lower chamber in antagormi R-92a group and MSCIL-1β-exo culture group increased significantly,but in antagormi R-92a group.The migration of cells in group NC was similar to that in group control.Compared with mi R-92a analogues,the levels ofα-SMA,Collagen I and Collagen III in mi R-92a analogues negative control group were significantly lower than those in group A.Through the analysis of the results of Western blot and q RT-PCR,it was found that the expression of mi R-92a in the macrophages stimulated by IL-1βstimulated macrophages decreased significantly.The expression of p-Ikkα/β,mi R-92a,p-AKT,p-p65（PTEN downstream protein）was downregulated（P<0.05）,and the result was reversed in mi R-92a negative control group（PTEN）.Conclusion:IL-1βpretreatment of MSC exudates inhibits the PI3K/Akt/NF-k B signaling pathway by inhibiting the expression of endogenous miR-92a in macrophages and promoting the expression of PTEN.It promotes the polarization of macrophages to M2,weakens the aggregation of macrophages and fibroblasts,thus inhibits the activation of fibroblasts and reduces the production of collagen,and finally achieves the effect of inhibiting urethral stricture.