To Assess The Mechanism Of Microtrauma Of Penile Tunica Albuginea In Peyronie's Disease And The Effect And Mechanism Of Prevention And Treatment Of ADSCs To This Disease

Background and objectives:Peyronie's disease(PD)is a common disease which disrupting the physical and mental health of males.however,its etiology is still unclear and the effect of current treatment is not satisfied.As we all known,restoration and fibrosis are two opposite endings after tissue injury.If fibroblasts are persistent activated and escape from apoptosis,the tissue after injury will develop to irreversible fibrosis.Adipose tissue-derived stem cell(ADSCs)is a kind of easy acquisition and high purity stem cells.Current progression revealed that ADSCs could enhance the matrix metalloproteinases(MMPs),degrade the collagen and increase apoptosis of active fibroblasts(myofibroblasts,MFs).Therefore,we suppose that ADSCs could be a great effective treatment for PD.The project aims of this study:To establish the animal model of PD by repeated microtruama of penile tunica albuginea and to explore the pathophysiology mechanism of PD;to analysis the activation and regulation process of key cells(MFs)of fibrosis;to reverse the fibrosis micro-environment used ADSCs by increase MMPs and multiple differentiation;to observe the impact of myofibroblasts and ECM after co-cultured with ADSCs;to assess the treatment and mechanism of ADSCs for the PD animal model.Methods:1.To compare repaired rat penile tunica albuginea(PTA)after single local injections of chlorhexidine ethanol(ChE)into the PTA and after repeated local injections of ChE into the PTA,Forty-two rats were included and were divided into seven groups.Rats either served as the normal control group(1-5 injections of saline)or they received a single injection,two injections,three injections,four injections,or five injections of ChE(0.1%chlorhexidine gluconate(CG)plus 15%ethanol dissolved in saline);rats in the positive control group were injected with TGF-?1.At 60 days after the last injection,the intracavernous pressure,degree of penile curvature and histology were evaluated for comparison among the groups.2.Tunica albuginea-derived fibroblasts(TAFs)obtained from male Sprague-Dawley rats were stimulated with either transforming growth factor-?1(TGF-?1)or E2.Western blot analysis and immunofluorescence staining were used to assess changes in the expression levels of ?-smooth muscle actin(?SMA).The expression levels of additional proteins(GAPDH,p-Smad2,Smad2,Smad4,RhoA,Racl,ROCK1 and ROCK2)were also measured by western blot analysis.We used collagen gel assays to assess cell contractility.Additionally,the concentration of hydroxyproline in the TAF cell culture medium was detected using commercially available kits.3.Primary ADSCs extracted from SD rat mixed culture with MFs(from TAFs activated by TGF-?1)in different concentrations.Western blot was used to access the expression of aSMA and commercially available kits was used to detect the concentration of hydroxyproline of the cell culture medium.After transwell co-culture of MFs and ADSCs,Western blot?real time PCR and commercially hydroxyproline kits were used to detect the synthesis and secretion ability of collagen in MFs and the expression of related proteins in Smad pathway.Collagen gel contraction assay and western blot were used to assess the self contractile function and the expression of related proteins in Rho/ROCK pathway.Western blot,real time PCR and ELISA were used to determine the changes in the synthesis and secretion of matrix metalloproteinases(MMPs,MMP9,MMP13,MMP2,MMP3).MTT analysis and western blot were used to compare the proliferation and apoptosis of MFs and Caspase-3 and caspase-9 protein before or after co-cultured with ADSCs.4.The SD rats were divided into 5 groups,PD rats:five times injection of chlorhexidine ethanol(ChE);Prevention group 1:injection of ADSCs suspension at the same time with ChE;Prevention group 2:injection of conditioned medium of ADSCs(CM-ADSCs)at the same time with ChE;Treatment group 1:injection of ADSCs suspension in the penile of PD rats;Treatment group 2:injection of CM-ADSCs in the penile of PD rats.Experiments using the artificial erection,Masson stain,intracavernous pressure,Weigert's stain,Western blot,immunohistochemistry,and immunofluorescence were used to assess the preventive and treatment effect of ADSCs and CM-ADSCs for PD rats and the expression of proteins in related signaling pathways.Results:1.Compared with the single injection group,we found the following in the repeat damage(multiple injections)group:an increase in the degree of penile curvature,fibrous plaques in the local tunica albuginea and/or corpus cavernosum,broken elastic fibers,slightly decreased erectile function,and increased expression of TGF-?1 and aSMA(P<0.05).The PTA and corpus cavernosum,when repaired after a single injury,resemble normal tissue,whereas repeated injuries may lead to fibrosis(P<0.05).2.We found that E2 reduced aSMA expression which was induced by TGF-?1.E2 also suppressed the TGF-?1-induced increase in the concentration of hydroxyproline(a marker of collagen)in addition to suppressing the contraction of TAFs.The key processes affected by TGF-?1 treatment included the phosphorylation of Smad2,ras homolog gene family,member A(RhoA)and Rho-associated,coiled-coil containing protein kinase 2(ROCK2);this increase in phosphorylation was inhibited by treatment with E2.3.After co-culture with ADSCs and MFs,the expression of aSMA in MFs was decreased,the concentration of hydroxyproline in cell supernatant was decreased,the ability of collagen synthesis was decline and the expression of RhoA and Rock2 were decresed resulted in the cell contraction weakened.ADSCs could secret large amounts of cytokines and MMPs to degrade extracellular matrix(ECM)?inhibit proliferation and promote apoptosis of MFs(P<0.05).When MFs mixed culture with ADSCs and the number of ADSCs was lager than MFs,the expression of aSMA of cells and the concentration of hydroxyproline in cell supernatant were decreased(P<0.05).While the number of MFs was lager than ADSCs,the expression of ?SMA of cells and the concentration of hydroxyproline in cell supernatant were increased(P<0.05).4.ADSCs and CM-ADSCs are play the effective roles of treatment and prevention in PD rats.ADSCs and CM-ADSCs could reduce the penis bending angle,inhibit the local collagen fibers deposition,repair the damaged elastic fibers,enhance the erectile function by increasing the expression of inducible nitric oxide synthetase(iNOS)and von Willebrand factor(VWF),reduce the expression of aSMA in penile tunica albuginea and inhibit the activation and collage secretion of MFs(P<0.05).It will be noteworthy that the effect of ADSCs is better than CM-ADSCs,and the effect of prevention is better than treatment(P<0.05).Conclusions:The penile TA and corpus cavernosum will be fully restored after a single injury,while repeated injuries may lead to fibrosis.This represents an excellent model of PD that involves repeated injections of ChE into the local penile TA as well as reveals the pathophysiologic mechanism of PD.In vitro,TGF-?1 could induced conversion of primary TAFs into myofibroblasts(MFs),promote synthesis and secretion of collagen through Smad dependent pathway and achieve the ability of contraction by the Rho/ROCK signaling pathways.ADSCs could attenuate the activity of MFs,inhibit synthesis and secretion of collagen,degradate collagen which has been formed already,inhibit MFs contraction and promote MFs apoptosis.ADSCs and CM-ADSCs have better prevention and treatment effect for PD rats.Howerver,the effect of ADSCs is better than CM-ADSCs,and the effect of prevention is better than the treatment.