SB431542 Blocks TGF-?1/Smad Signaling Pathway And Delays Neurogenic Bladder Fibrosis In Rats

Background and ObjectiveNeurogenic bladder(NB)is caused by central or peripheral nervous system diseases that dominate bladder and urethra,its etiology and clinical symptoms are complex,and it is a worldwide medical problem.With the prolongation of the disease,most NB patients will have varying degrees of bladder fibrosis.It is reported that bladder fibrosis is an important factor affecting the treatment efficacy of NB.Therefore,finding the mechanism of bladder fibrosis caused by NB and ways to prevent or delay the occurrence and development of fibrosis,has attracted more and more attention from clinicians and researchers.The typical pathophysiological changes of NB fibrosis is the deposition of extracellular matrix(ECM).Therefore,exploring how to inhibit the deposition of ECM may help prevent the occurrence of NB fibrosis and delay its progress.Transforming growth factor ?1(TGF-?1)and TGF-?1 / Smad signaling pathway have been proved to be the key factors for various tissue and organ fibrosis.The role of TGF-?1 / Smad signaling pathway in bladder fibrosis caused by partial bladder outlet obstruction model in rats has been reported,but the change of this signaling pathway has not been reported in bladder fibrosis caused by NB.SB431542 is a small molecule inhibitor,it can inhibit the phosphorylation of Smad2 and Smad3 by inhibiting activin receptor-like kinase(ALK)4,5 And 7.Animal and cell experiments have found that SB431542 has the role of preventing and delaying fibrosis in organs such as lungs,kidneys,and eyes.However,whether it has an effect on bladder fibrosis caused by NB has not been reported in the literature.ObjectivesIn this study,we established the NB model by transecting the lumbosacral spinal nerves and intervened them with SB431542 to explore the relationship between TGF-?1 / Smad signaling pathway and NB fibrosis,and the effect of SB431542 on NB fibrosis.Materials and methods1.Sixty healthy adult female Sprague Dawley(SD)rats were randomly divided into Sham group(SG,n=20),Spinal nerve disconnection group(SNDG,n=20)and Spinal nerve disconnection + SB431542 intervention group(SSIG,n=20).After the rats in SNDG and SSIG groups were anesthetized,the spinal canal was opened to expose L5-L6,the cauda equina spinal nerve was transverse in this place.In SG group,the spinal canal was opened,but the cauda equina spinal nerve was not transverse as control.SSIG rats were intraperitoneally injected with SB431542(10mg / kg)every 3 days,and SNDG and SG rats were intraperitoneally injected with SB431542 co-solvent every 3 days.2.At 4 weeks and 8 weeks after NB model surgery,10 rats were randomly selected and the bladder pressure volume of each group of rats was measured using rat urodynamic instruments.After the cystometry,rats' bladder tissues was retained,we took photos of rats' bladder to record the general morphology of bladder specimens.Then microbalances were used to determine the bladder weight of each rats.1/2 of the bladder tissue was used to Hematoxylin and eosin,Masson and immunohistochemical staining.The other half of the bladder tissue was frozen for Western blot experiments on related proteins.3.Statistical analyses were performed using SPSS 20.0 statistical software.The measurement data was expressed as mean ± standard deviation.T test was used for comparison between the two groups,and ANOVA was used for comparison between the three groups and more than three groups.P < 0.05 were considered statistically significant.Results1.Urodynamic parameters: At 4 and 8 weeks,the maximum cystometric capacity of SNDG and SSIG rats was significantly higher than that of SG(P < 0.001).The bladder leak point pressure of rats in SG,SNDG and SSIG had no significant difference at 4 weeks;however the bladder leak point pressure of rats in SNDG was higher than that of SG and SSIG(P < 0.001;P < 0.01).The bladder compliance of rats in SNDG and SSIG was higher than that of SG at 4 and 8 weeks(P < 0.001);The bladder compliance of rats in SNDG was lower than that in SSIG at 8weeks(P < 0.01).2.Bladder weight and morphology: The bladder wet weight of SNDG rats was higher than SG and SSIG at 4 and 8 weeks(P < 0.001).The abnormality of bladder tissue of SNDG rats was higher than that of SG and SSIG rats at 4 and 8 weeks.The bladder wall thickness of SNDG rats was higher than those of SG and SSIG rats at 4 and 8 weeks(P < 0.001).3.Bladder tissue fibrosis indexes: The bladder fibrosis degree and area of SNDG rats were higher than SG and SSIG rats at 4 and 8 weeks(P < 0.01).The immunohistochemical staining intensity,average IOD value and relative expression level of Collagen I and III in SNDG rats were higher than SG and SSIG rats at 4 and 8 weeks(P < 0.001).4.TGF-?1 / Smad signaling pathway-related protein expression: The immunohistochemical staining intensity,average IOD value and relative expression level of TGF-?1 in SNDG and SSIG rats were higher than those in SG rats at 4 and 8 weeks(P < 0.001).At 4 and 8 weeks,the immunohistochemical staining intensity,average IOD value and relative expression level of P-Smad2 in SNDG rats were higher than those in SG and SSIG rats(P < 0.001).Conclusion1.The NB model can be successfully established by transsecting rat L6 segment spinal nerve,and the bladder gradually fibrosis with the extension of modeling time.2.Bladder fibrosis in NB rats is closely related to the activation of TGF-?1 / Smad signaling pathway3.SB431542 can delay bladder fibrosis in NB rats by blocking TGF-?1 / Smad signaling pathway.