The Protective Role Of Schwann Cells In Bladder Smooth Muscle Cell Fibrosis

BackgroundNeurogenic bladder(NB)is a common disease in children.Currently,various treatment methods can only partially improve the clinical symptoms of NB patients and cannot fundamentally restore the normal function of NB bladder.One important reason is that the pathological changes of bladder fibrosis may occur in most children with the progress of NB,leading to a significant reduction of the bladder capacity and compliance,but the increase of bladder trabeculization,thus the normal bladder wall tissue is replaced by the abnormal proliferative connective tissue infiltration,and the bladder eventually changes to a pouch with a small capacity and high pressure,which cannot achieve its normal urine storage and urination function.Therefore,it is of great significance to explore the main factors that cause bladder fibrosis and to intervene effectively to prevent the progression of the NB disease.Previous studies have confirmed that the deposition of collagen and other extracellular matrix components in different organs,such as heart,liver and lung,will lead to organ fibrosis under different pathological conditions.Similar to these organs,the characteristic of bladder fibrosis is the deposition of collagen in the bladder wall.Connective tissue growth factor(CTGF)may play an important role in mediating the occurrence and development of tissue and organ fibrosis.After denervation,fibrosis will take place in the bladder,which may result from the failure of functional nerve cells to provide necessary nutrients for bladder smooth muscle cells,leading to the dysfunction of bladder,and eventually lead to bladder fibrosis.Previous studies found that the dysfunction of bladder detrusor muscle was correlated with the function of innervating detrusor nerve.We hypothesized that innervation and neurotrophins play significant roles in the process of bladder fibrosis.Schwann cell is a kind of glial cell with important functions in the peripheral nervous system.It supports neurons and promotes the regeneration of nerve tissue by secreting a variety of neurotrophins including brain-derived neurotrophic factor(BDNF).At present,no relevant studies have revealed the role of Schwann cells in the bladder fibrosis of NB.This study is composed of two parts.The first part is the establishment of rat denervated bladder fibrosis model and the role of CTGF in bladder fibrosis.The second part is the role of Schwann cells and BDNF in bladder smooth muscle cell fibrosis and the possible mechanism.Part ?Establishment of rat model of denervated bladder fibrosis and the effect of CTGF in bladder fibrosisObjectiveTo establish a NB bladder fibrosis model by denervation in rats.The histological changes of bladder were observed at different time points.Observe the change trend of CTGF with the development of bladder fibrosis.Bladder smooth muscle cells(BSMCs)were isolated and cultured.The above cultured cells were treated with CTGF to induce the occurrence of bladder fibrosis.Observe the differences of ?-smooth muscle actin(?-SMA),type ? and type ? collagen(Collagen ?,Col ?;Collagen ?,Col ?)and other indexes to determine whether CTGF can promote bladder fibrosis.MethodsThe NB fibrosis model was established by denervating female SD rats.Thirty-two adult female SD rats weighing 160-200g were randomly divided into four groups(n=8/group),and the NB denervation model was established by destroying bilateral pelvic ganglia with electric cauterization.One group received sham surgery and was sacrificed 10 days later.The other three groups were sacrificed at 10th day,20th day and 30th day after denervation.The bladder tissues of rats were extracted and stained with H&E to observe the changes of bladder wall tissue structure,and then stained with Masson method to further evaluate the degree of fibrosis in bladder tissues.The expression of CTGF in bladder tissue was detected by immunohistochemistry.The bladder tissue of SD rats was excised,and BSMCs were isolated and cultured.After treated with CTGF,the expression of ?-smooth muscle actin(?-SMA)in the BSMCs was observed by immunofluorescence staining.Western blot was used to detect the expression of type ? and ? collagen.Results1.H&E staining characteristics of bladder tissue.After denervation in the rat NB model,HE staining showed that the BSMCs were disordered and inflammatory cells were infiltrated in submucosa and muscular layer.In addition,the smooth muscle tissue of bladder significantly decreased,while the fibrous connective tissue of bladder wall pronouncedly proliferated over time after modeling.2.Masson staining results:consistent with H&E staining,the red stained smooth muscle cells significantly decreased in a time-dependent manner,while the blue stained collagen fiber remarkably increased in the rat NB model(compared with the control group,NB 10th day,P<0.05;NB 20th day,P<0.01;NB 30th day,P<0.01).These results suggested that tissue fibrosis was gradually aggravating,and the rat NB model was successfully established.3.CTGF immunohistochemical staining results:after denervation in the rat NB model,the smooth muscle and the transitional epithelium of the mucosa showed positive yellow stained of CTGF,and its thick brown particle deposition was observed in the BSMCs.Furthermore,the staining particles of CTGF were more obvious as the longer the time after NB modeling(compared with the control group,NB 10th day,P>0.05;NB 20th day,P<0.0001;NB 30th day,P<0.001),but there was no obvious brown particle deposition in the bladder submucosa.These results indicated a significant increase of CTGF expression in the rat NB model group,suggesting that it may play a crucial role in the development of NB bladder fibrosis.4.Effect of CTGF on cultured BSMCs.To confirm the effect of CTGF on the occurrence of bladder fibrosis,primary BSMCs were isolated and cultured with CTGF.Immunofluorescence staining showed a significant increase in a-SMA after treatment with CTGF(P<0.0001).Western blot analysis revealed a significant increase in Col ?and Col ?(P<0.01).This result suggested that CTGF may contribute the fibrosis of BSMCs.ConclusionThe rat NB bladder fibrosis was successfully constructed using denervation,and CTGF may play a crucial role in promoting the development of NB bladder fibrosis.Part ?The protective effect of Schwann cells on bladder smooth muscle cell fibrosis and its possible mechanismObjectiveThe expression level of BDNF in bladder tissue of rats with bladder fibrosis was detected at different time points,and the change trend of BDNF content in denervated rat bladder tissue was observed.BSMCs and Schwann cells were isolated.Bladder smooth muscle cells were cultured separately or co-cultured with Schwann cells.CTGF was used to induce fibrosis in the cell culture system.The expression levels of ?-SMA,type ? and type ? collagen were detected to observe whether Schwann cells had inhibitory effect on bladder smooth muscle cell fibrosis.Supplementary experiments were performed to further demonstrate the protective effect of Schwann cells on bladder smooth muscle cell fibrosis and to verify that this effect is mediated by BDNF.The expression of matrix metalloproteinase(MMPs)mRNA of different experimental groups including MMP-1,MMP-2 and tissue inhibitor of matrix metalloproteinases 1(TIMP-1)were measured to reveal the possible mechanism of the inhibiting effect of Schwann cells and BDNF on the fibrosis of bladder smooth muscle cells.MethodsEnzyme linked immunosorbent(ELISA)was used to detect BDNF in bladder tissues on day 10,20 and 30 after denervating,and the change trend of BDNF content in bladder tissues of rats with denervating was observed.Under aseptic conditions,Schwann cells were isolated from sciatic nerve of newborn SD rats and co-cultured with bladder smooth muscle cells.The co-cultured cell system was exposed to CTGF and the expression of a-SMA was observed by immunofluorescence staining,and Col? and Col ? were detected using western blot.To evaluate the effect of Schwann cells on fibrosis,the above results of the co-culture system were compared with BSMCs under the intervention of CTGF.Furthermore,the expression level of BDNF was also compared between BSMCs and the co-culture system using an ELISA method,so as to explore the possible mechanism of the protective effect of Schwann cells on bladder fibrosis.The senescence of Schwann cells was detected by SA-glycosidase staining to exclude the disturbance of their survival on the co-culture system.Different experimental groups were set up according to the differences of factors such as whether to co-culture with Schwann cells and the application of BDNF.Group A:bladder smooth muscle cells control group;Group B:bladder smooth muscle cells+CTGF;Group C:bladder smooth muscle cells+CTGF;Group D:bladder smooth muscle cells+BDNF+CTGF.Immunofluorescence staining was used to observe the changes of-SMA,Col ? and Col ? expression,further verifying the protective effect of Schwann cells on bladder smooth muscle cell fibrosis through BDNF.Quantitative real-time PCR(qPCR)was used to detect the mRNA expressions of MMP-1,MMP-2 and TIMP-1 in each group,revealing the possible mechanism of BDNF's inhibitory effect on vesical smooth muscle cell fibrosisResults1.The content of BDNF in the fibrotic bladder tissues was significantly reduced in the rat NB model.ELISA results indicated that the BDNF in the bladder tissues of NB denervated rats was significantly decreased in a time-dependent manner(compared with the control group,NB 10th day,P<0.01;NB 20th day,P<0.001;NB 30th day,P<0.0001),suggesting that BDNF may play a role in protecting bladder tissues from fibrosis in the NB model.2.Schwann cells can significantly reduce the fibrosis of BSMCs.When compared with CTGF-treated BSMCs,BSMCs showed reduced expressions of ?-SMA,Col ? and Col ? after CTGF intervention in the co-culture system with Schwann cells(a-SMA,P<0.0001;Col ?,P<0.001;Col ?,P<0.01).These results suggested that Schwann cells significantly reduced CTGF-induced BSMCs' fibrosis and may play a protective role in the NB bladder fibrosis.3.ELISA results showed that BDNF was significantly increased in co-culture system when compared with the BSMC group after CTGF exposure,which indicated that the protective effects of Schwann cells on bladder fibrosis may be mediated by releasing BDNF4.SA-?-gal staining showed that the positive density of Schwann cells was similar in each group,indicating that Schwann cells did not undergo senescence in the entire experiment.5.Immunofluorescence staining showed increased expression of-SMA,Col ? and Col ? in group B compared with group A(P<0.0001),which further verified that CTGF could promote the occurrence of bladder smooth muscle cell fibrosis.Compared with group B,the expression of-SMA was decreased in group C(P<0.0001),Col ?was decreased(P<0.05)and Col ? was decreased(P<0.01).Compared with group B,the expression of-SMA,Col ? and Col ? in group D was decreased(P<0.0001).It suggests that both Schwann cells and BDNF can inhibit CTGF induced fibrosis of bladder smooth muscle cells,and the protective effect of Schwann cells on fibrosis may be generated through BDNF6.Compared with group A,mRNA expression of MMP-1 and MMP-2 was decreased and that of TIMP-1 was increased in group B(P<0.0001).Compared with group B,mRNA expression of MMP-1 was increased(P<0.01),MMP-2 was increased(P<0.05),and TIMP-1 mRNA was decreased(P<0.0001)in group C.Compared with group B,mRNA expression of MMP-1 increased(P<0.0001),MMP-2 increased(P<0.01)and TIMP-1 decreased(P<0.0001)in group D.It can be inferred that CTGF could promote the occurrence of fibrosis by down-regulating the expression of MMP-1 mRNA and MMP-2 mRNA in bladder smooth muscle cells and upregulating the expression of TIMP-1 M RNA.This effect can be inhibited by Schwann cells or BDNF Schwann cells may regulate the expression levels of MMP-1,MMP-2 and TIMP-1 in bladder smooth muscle cells by secreting BDNF,thereby inhibiting the occurrence of fibrosisConclusionSchwann cells significantly inhibit vesical smooth muscle cell fibrosis,which is related to the function of BDNF production.Schwann cells may regulate the expression levels of MMP-1,MMP-2 and TIMP-1 in bladder smooth muscle cells by secreting BDNF,thereby inhibiting the occurrence of fibrosis.