Mechanism Of Schwann Cell-derived Exosomes In Process Of Electrical Stimulation Treating Female Rats With Stress Urinary Incontinence

Objective:1.To explore the effect of pudendal nerve electrical stimulation(PNES)on stress urinary incontinence(SUI)in vivo/at the histological level by etablishing a SUI rat model using pudendal nerve crush(PNC).2.To investigate the effects of electrical stimulation on rat dorsal root ganglion(DRG)cells and the possible role and related mechanisms of rat sciatic nerve Schwann cells(RSC96)-derived exosomes in this process by constructing a direct electrical stimulation DRG cell model,a cyclic mechanical stretching injured DRG cell model and a co-culture model of DRG cell and RSC96 cell.Materials and Methods:Part I: Female non-pregnant SD rats were randomly divided into four groups: CON group(untreated group),Sham group(only separated the pudendal nerve by surgery and no other treatment was performed),PNC group(bilateral pudendal nerves were surgically isolated,then were crushed three times of 30 seconds each side with micro-clamps),PNC+ES group(one hour after PNC modeling,a self-made electrode needle was used to give bilateral pudendal nerves electrical stimulation for one hour).There were 15 rats in each group.After 7 days of modeling,sneeze test and urodynamic examination(including maximum bladder(MBC)volume and leak point pressure(LPP))were performed to evaluate the effect of PNES on SUI.Then the rats were sacrificed.The pudendal nerve tissue and the tissue around the bladder neck of the urethra were taken.The changes of muscles and collagens in the peri-urinary tissue were observed by Masson staining.The pudendal nerve injury and repairment were observed by HE staining.Part II:(1)DRG cells were stimulated by direct electrical stimulation device.After different electrical stimulation(ES)parameters(size: 100 m V/mm,200 m V/mm,time: 0.5 h,1 h,2 h)were intervened to DRG cells,the cell activity(CCK-8 method),the incidence of cell senescence(?-galactosidase staining)and the cell apoptosis(Hoechst staining)were detected to evaluate the most suitable ES parameters for DRG cells.(2)According to previous studies,fixed cyclic mechanical loading(CMS)parameters(5333 ?strain,8 h,1 Hz)were selected to construct a DRG cells injury model.Combined with DRG cells ES model,the changes of DRG cell viability(CCK-8 method,Ed U staining method),cell cycle(PI staining,flow cytometry)and cell apoptosis(Annexin V/PI double labeling method)were detected,to study the effects of appropriate parameters ES on injured DRG cells.(3)The co-culture model of DRG cells and RSC96 cells was constructed by transwell 0.4 ?m pore membrane six-well cell culture plate.Combined with CMS-induced DRG cell injury model and cell ES model,the changes of DRG cell activity(CCK-8 method)and cell apoptosis(Annexin V/PI double labeling method)in each group were detected,to investigate the indirect effect of RSC96 cells in the process of ES influences DRG cells.Part III:(1)The effects of ES on glutamate secretion in DRG cells were detected by glutamate detection kit,and the effects of glutamate on exosome secretion and intracellular calcium concentration in RSC96 cells were detected by transmission electron microscopy and fluo-4 AM probe.Meanwhile,the inhibition of glutamate by NMDA ionic glutamate receptor inhibitor MK801 was used to study the relationship between glutamate,intracellular calcium concentration and exosome secretion of RSC96 cells.(2)In co-culture system,exosome secretion inhibitor GW4869 was used to detect the changes of DRG cell activity and apoptosis in each group,to explore the role of RSC96 cell-derived exosomes in the process of RSC96 cells indirectly influence the DRG cells with ES.(3)RSC96 cell-derived exosomes were extracted and directly added into DRG cells to detect the changes of DRG cell cycle(PI staining flow cytometry)and Wnt/?-catenin pathway related proteins(Western blot,RT-PCR).(4)DRG cells were treated with Wnt/?-catenin pathway inhibitor XAV939 firstly,and then treated with RSC96 cell-derived exosomes.The activity,cell cycle,apoptosis and downstream protein changes of DRG cells were detected to investigate the role of Wnt/?-catenin pathway in the process of RSC96-derived exosome affecting DRG cells.Results:Part I:(1)Compared with the rats in the CON group,there was no significant difference in the positive rate of sneeze test,MBC value and LPP value in the Sham group(P>0.05).Compared with the CON group,the MBC value and LPP value of the PNC group were significantly lower(P<0.001),and the positive rate of sneeze test was significantly higher(P<0.001).Compared with PNC group,the MBC and LPP values increased significantly(P<0.001)and the positive rate of sneeze test decreased(P<0.05)after PNES.(2)HE staining of pudendal nerves showed that the nerves in CON and Sham groups were arranged neatly,without twists and pull-off.In PNC group,the pudendal nerves were arranged sparsely and the nerves were in disorder,and obvious nerve breakage and defect were observed.Compared with PNC group,the pudendal nerve of PNC+ES group showed obvious nerve repair marks,most of the nerve tissue healed,and a few nerves did not heal completely.(3)Masson staining of tissue in bladder neck-urethral junction showed that the tissue around the urethra of rats was intact,surrounded by complete and thick muscle tissue,and the interstitial space of muscle tissue was filled with dense and long collagen fibers in CON group and Sham group.In the PNC group,the smooth muscle around the urethra was sparse,the content of collagen fibers was slightly reduced,and the appearance of denervation occurred.After PNES,the smooth muscle of the peri-urethral tissue recovered well,and collagen fibers also increased to some extent.Part II:(1)Compared with the non-ES group,the activity of DRG cells increased first and then decreased with the prolongation of time(0 h,0.5 h,1 h,2 h),while the senescence rate and apoptotic cell number of DRG cells decreased first and then increased.Under the electrical stimulation parameters of 100 m V/mm for 1 h,DRG cells had the highest activity,the lowest rate of cell senescence and the lowest number of apoptotic cells.(2)Compared with the control group(normal DRG cells),the cell viability in the ES group(100 m V/mm for 1 h)increased significantly(P<0.001),the G2 phase cells increased significantly(P<0.001),and the total apoptotic rate decreased significantly(P<0.001).Compared with the control group(normal DRG cells),the cell viability in the injury group(CMS-induced DRG cells)decreased significantly(P<0.001)and the total apoptotic rate increased significantly(P<0.001),in addition,the S phase cells increased(P<0.001)and the G2 phase cells decreased(P<0.05)significantly.At the same time,compared with CMS group,the cell viability of DRG cells in CMS + ES group increased significantly(P<0.001),cells in S phase decreased(P<0.001),cells in G2 phase increased(P<0.01),and the total apoptotic rate decreased significantly(P<0.001).(3)Compared with the ES group(the normal/injured DRG cells were treated with ES only),the cell activity of co-culture group(the normal/injured DRG cells were stimulated with ES and then cocultured with RSC96 cells)increased significantly(P<0.001,P<0.001),and the total apoptotic rate of DRG cells decreased significantly(P<0.001,P<0.01).Part III:(1)The concentration of glutamate at each time point(4 h,6 h,8 h,10 h,12 h)in the ES group were higher than that in the non-ES group(P<0.001).The glutamate secretion of DRG cells reached its peak at 6-8 hours after ES.When treated with exogenous glutamate(50?mol/L?100?mol/L?200?mol/L),the results showed that exosome secretion increased(P<0.001,P<0.001,P<0.001)and intracellular calcium concentration of RSC96 increased(P<0.05,P<0.001,P<0.01)compared with the control group.Besides,when the concentration of glutamate was 100?mol/L,the exosome secretion and the intracellular calcium concentration was the highest.MK801 decreased intracellular calcium concentration by inhibiting NMDA ionotypic glutamate receptor.In the presence of MK801,glutamate-induced exosome secretion of RSC96 cells decreased significantly(P<0.001).(2)When exosome secretion inhibitor GW4869 was added to the co-culture system,the activity of the normal/injured DRG cells was lower than that of the groups without GW4869(P<0.001,P<0.001),and the total apoptotic rate was increased(P<0.001,P<0.001).(3)Compared with the non-exosome groups(the Con group and the CMS group),the Sphase cells of normal/injured DRG cells(Exo group and CMS+Exo group)after exosome treatment were significantly reduced(P<0.001),and the G2-phase cells were significantly increased(P<0.001).The results of wnt/?-catenin pathway detection showed that,in the Exo group,the expression of ?-catenin and p-GSK3? increased,the expression of GSK3? decreased,the expression of C-myc and cyclin D1 increased compared with the Con group(all P value <0.001).Meanwhile,compared with the Con group,the expression of ?-catenin and p-GSK3? decreased,the expression of GSK3? increased,and the expression of C-myc and cyclin D1 increased in CMS group(all P value <0.001).Compared with CMS group,the expression of ?-catenin and p-GSK3? in DRG cells increased,the expression of GSK3? decreased,and the expression of C-myc and cyclin D1 increased when CMS group was treated with exosomes(all P value <0.001).(4)There were no significant changes in cell activity,cell cycle and apoptosis after treating the normal/injured DRG cells with the Wnt/?-catenin pathway inhibitor XAV939(P>0.05).Compared with the groups treated with exosome alone,the activity of the normal/injured DRG cells decreased significantly(P<0.001,P<0.05),the S-phase cells increased(P<0.01,P<0.01),the G2-phase cells decreased(P<0.001,P<0.01),and the total apoptotic rate increased(P<0.001,P<0.001)when exosomes and XAV939 were given at the same time.Detection of downstream proteins in Wnt/?-catenin pathway showed that treating with XAV939 alone had no effect on the expression of C-myc,cyclin D1,Bcl-2 and Bax in normal DRG cells(P>0.05),but decreased the expression of C-myc,cyclin D1 and Bcl-2(P<0.001,P<0.001,P<0.001),and increased the expression of Bax(P<0.001)in injured DRG cells.When treating the normal/injured DRG cells with exosomes and XAV939 at the same time,the expression of C-myc,cyclin D1 and Bcl-2 decreased(P<0.001,P<0.001,P<0.001),and the expression of Bax increased(P<0.001)compared with groups treated with exosomes only.Conclusion:1.Pudendal nerve injury is an important factor in the occurrence of stress urinary incontinence.Pudendal nerve electrical stimulation can significantly improve the urodynamic function of rats with stress urinary incontinence induced by pudendal nerve crush,and repair the injured pudendal nerve,periurinary muscles and collagen tissue.2.Cell direct electrical stimulation model can effectively establish DRG cell electrical stimulation model,and electrical stimulation parameters of 100 m V/mm 1 h can be used as the most appropriate parameters for electrical stimulation of DRG cells in vitro.At the same time,electrical stimulation can increase the activity of DRG cells and reduce apoptosis in normal or injured DRG cells.The indirect effect of RSC96 cells on DRG cells was confirmed by co-culture of DRG cells and RSC96 cells.3.Electrical stimulation promotes the secretion of glutamate by DRG cells.Glutamate promotes the secretion of exosomes by increasing the concentration of calcium ions in RSC96 cells.The exosome secreted by RSC96 cells can feedback to DRG cells,activate the Wnt/?-catenin signaling pathway in DRG cells,improve cell viability,reduce apoptosis and promote cell cycle process,thus achieving the repairment of injured DRG cells.