The Mechanism Of MiR-133 Mediated TGF Beta 1/CTGF Signaling Pathway In Neurogenic Bladder Caused By Multiple Sclerosis

Background and PurposeMultiple sclerosis(MS)is a common white matter demyelinating disease of the central nervous system,and it is the second leading cause of neurogenic bladder.Bladder dysfunction in MS patients with urinary frequency,urgency of urine and retention of urine is one of the most common clinical diseases in the clinical work of urinary system.About 75% of MS patients may have lower urinary tract symptoms at all stages of the disease,which seriously affect the life quality of patients and bring heavy psychological and economic burden to patients.The pathophysiological mechanism associated with lower urinary tract symptoms such as urinary frequency and urinary retention in MS patients is not clearly elucidated.The structure and function of bladder smooth muscle will changed with impaired of related nerve cells that govern micturition,resulting in some microscopic changes,such as the impaired detrusor contractility(IDC).For now,the treatment of neurogenic bladder-related symptoms caused by MS is still very intractable.Previous treatment methods such as some drugs that inhibiting bladder overactivity have some effect in some patients,but still far from the expected effects.The fibrosis and remodeling process of bladder local tissue plays an important role in the occurrence and development of lower urinary tract symptoms caused by neurogenic bladder.However the specific molecular mechanism of bladder tissue remodeling and fibrosis is still not clear.TGF?1 and connective tissue growth factor(CTGF)may be closely related to this process.In recent years,many studies have gradually realized that miRNAs are closely related to multiple fibrotic diseases.miR-133 includes two members,miR-133 a and miR-133 b.Some scholars have found that miR-133 a is closely related to liver fibrosis and myocardial fibrosis.However,little is known about its regulatory and regulatory mechanisms in the process of neurogenic bladder remodeling caused by MS.Therefore,our study intends to explore the molecular mechanism of miR-133-mediated TGF?1/CTGF signaling pathway in neurogenic bladder caused by MS,find a new target of molecular action against bladder fibrosis,inhibit or slow the process of bladder remodeling,improve bladder compliance in patients with MS and provides new ideas for the further treatment of neurogenic bladder caused by MS.Method1.Thirty,female,7 ~ 9 weeks old(18 ~ 22g),C57BL/6J black mice were purchased from Beijing Weitong Lihua Animal Company,and construction of neurogenic bladder animal model caused by MS,that is,EAE mouse model. The black mice were divided into two groups,the Blank group(10)and the model group(20).After successful establishment of EAE mouse model, clinical scores(CS)was performed according to the severity of the symptoms in mice.2.Regularly weigh the mouse and record it.The time of onset of mice in the model group was approximately concentrated at the 13 th to 19 th days after the establishment of the model.At this time,the onset of the mice was the acute stage of the disease.On the 49 th day after modeling,the mice in model group with a CS score of 3 and all mice in blank group were anesthetized with 10% chloral hydrate.And the bladder tissues were removed under aseptic conditions and rinsed in a 1×PBS buffer,weigh empty bladder wet weight and record,and then half of the bladder tissue immediately placed in a liquid nitrogen tank,the other half placed in a prepared specimen fixative,all specimens marked,retained for subsequent use.3.Paraffin sections were made from two groups of fixed mouse bladder tissues and stained with HE and Mason.The number and morphology of smooth muscle in the bladder wall of mice were observed.4.The total RNA of bladder tissue was extracted from two groups of black mice respectively.The expression of mi R-133 in bladder tissue was detected by QRT-PCR.The difference between the two groups was observed.5.The expression of TGF?1 and CTGF in the bladder tissues of two groups was qualitatively and quantitatively detected by immunofluorescence and Western blot.The differences between the two groups were observed.6.Primary cultured bladder smooth muscle cells of C57BL6/J mice.After identification,smooth muscle cells were transfected with mi R-133 analogues respectively,and their transfection efficiency was measured,then the miR-133 overexpression group was set up.The blank group was also set up.7.Total RNA and protein of the Blank group and the miR-133 overexpression group were extracted,and the expression changes of TGF?1 and CTGF were further observed by QRT-PCR and Western blot to analyze the regulatory relationship between miR-133 and CTGF genes.Results1.EAE model mice were established successfully,the incidence rate was 87.9%, the peak incidence period was 13~19 days after the establishment of the model,and the CS score was 3 points for a total of 13 mice.2.There was no significant difference in the total weight of the mice between the Blank group and the model group,but the bladder weight of the model group increased significantly(P<0.001)and the ratio of the bladder body weight increased significantly(P<0.001).3.The results of HE and Masson staining showed that the smooth muscle cells in the bladder wall of EAE mice were hypertrophiy and hyperplasia.Collagen fibers in lamina propria and myofilament were significantly higher in model group than that in the Blank group.And model mice developed bladder fibrosis.4.Animal-level studies showed that miR-133a/mi R-133 b expression in the bladder of the model group was significantly down-regulated(P<0.05) compared with the Blank group,and TGF?1 and CTGF were up-regulated at the mRNA and protein levels(P<0.05).5.The results of cell-level studies showed that mi R-133 was transfected into smooth muscle cells,and total RNA was extracted after 48 hours.The expression of miR-133 was significantly higher than that of the Blank group (P<0.05).Further examination of TGF?1 and CTGF molecules showed that compared with the Blank group,there was no significant difference in mRNA and protein levels of TGF?1 in the miR-133 overexpression group(P>0.05), but the expression of CTGF was significantly downregulated(P<0.05).Conclusion1.There is a bladder remodeling phenomenon in the bladder tissue of MS induced neurogenic bladder mice.The TGF beta 1 and CTGF molecules may play an important role in bladder remodeling.2.MiR-133 may targets the regulation of CTGF gene and prevent further development of bladder remodeling.It can provide a new idea and target for the treatment of neurogenic bladder in MS patients.