The Effect And Mechanism Of TGF-β1 Regulating The Contraction And Proliferation Of Bladder Smooth Muscle Through LIMK

【Background】Lower urinary tract symptoms（LUTS）^[1]are the most common urinary system symptoms,whose incidence rate and severity increase significantly with age.With the intensification of aging,the number of patients with LUTS will increase,and the economic and social burden will also increase^[2].LUTS includes symptoms during urination and symptoms during storage.Currently,it is believed that benign prostatic hyperplasia（BPH）is the most common cause of LUTS in middle-aged and elderly men,with BPH-related LUTS（hereinafter referred to as BPH/LUTS）occurring in up to 80%of men over the age of 70^[3].More than 60%of patients with BPH have secondary bladder involvement and symptoms of overactive bladder（OAB）^[4].Although symptoms such as difficulty urinating and dripping water caused by bladder outlet obstruction（BPH）caused by BPH are most common in LUTS,OAB symptoms such as frequent urination,urgency urination,and increased nocturnal urination caused by involuntary contractions of the detrusor muscle have the most serious impact on the quality of life of patients.Frequent urination to and from the toilet during the night can significantly increase the risk of falls and fractures in patients^[11].It is also the most important reason for patients to seek medical treatment and the most urgent demand to be addressed^[12].Currently,M receptor antagonists^[14][15]orβ3 Receptor agonists^[16][17],but due to their low efficacy and significant side effects,approximately 40%of patients fail to adhere to medication^[18][19].After combination withα1 receptor blockers^[20],patients’tolerance and compliance decrease due to the superimposed side effects^[21][22].Therefore,searching for new signaling pathways in the pathogenesis of OAB caused by BPH（BPH/OAB）,as well as common therapeutic targets for BPH and secondary OAB,is of great significance for improving the therapeutic efficiency of LUTS and reducing social and personal burdens.Transforming growth factors-β（TGF-β）play an important role in regulating cell growth and differentiation.In a study using botulinum neurotoxin type A to treat spinal cord dysplasia-associated OAB,TGF-β1 may enhance the contractile function of bladder smooth muscle in OAB^[30].As an inducer of cell contraction^[31],TGF-β1can promote the contraction of airway smooth muscle^[32].Studies have found that TGF-β1 can regulate fibrosis^[33]and cytoskeletal remodeling^[34][35]through the LIMK/cofilin signaling pathway,but the contraction of bladder smooth muscle by TGF-β1 and its downstream LIMK/cofilin signaling pathway,as well as its role and mechanism in the pathogenesis of BPH/OAB,have not been clarified.Our team previously confirmed that LIMK,as a key enzyme for phosphorylating cofilin,plays an important role in regulating the contraction of prostate smooth muscle and is a potential therapeutic target for BPH/LUTS^[36].LIMK is also involved in regulating the contraction and proliferation of bladder detrusor muscle,and its role is achieved by regulating cofilin phosphorylation^[37].But the expression level of LIMK is influenced by upstream TGF-β1 impact,further research and validation are needed.【Objective】1.Explore the role of TGF-β1 in the occurrence and development of BPH/OAB patients in clinical practice.2.Explore the role and mechanism of TGF-β1,a key component of the TGF-β1/LIMK signal axis,in the contraction and proliferation of bladder smooth muscle.【Methods】1.Expression level of TGF-β1 in urine of BPH/OAB patientsThe morning urine of patients with BPH or BPH/OAB was collected,and the concentration of TGF-β1 in the urine of each group of patients was measured using an ELISA kit.The ratio of TGF-β1 to urinary creatinine was calculated.2.Effects of TGF-β1 and LIMK on the contraction of bladder smooth muscle tissue in ratsThe organ bath experiment was used to verify the contraction strength of bladder smooth muscle.Grouped into control group and TGF-β1 Group,LIMK inhibitor group（SR7826 or LIMKi3）,and TGF-β1+LIMK inhibitor groups.The muscle contraction tension in each group of experiments is expressed as the percentage（%）of contraction induced by a high molar concentration of KCl.Inducers including carbachol,acetylcholine chloride,and acetylcholine-β-Methylcholine chloride induced contraction were compared quantitatively with KCl.3.Effects of TGF-β1 and LIMK on myofilament of human bladder smooth muscle cells（HBSMCs）HBSMCs were cultured and incubated with TGF-β1 or LIMK inhibitors（SR7826or LIMKi3）for 24 hours.The control group was treated with solvent BSA or solvent BSA+DMSO.Then,HBSMCs were stained with fluorescein isothiocyanate（FITC）labeled phalloidin,and the labeled cells were photographed using a laser scanning microscope.4.Effects of TGF-β1 and LIMK on the proliferation of HBSMCsHBSMCs were incubated in a 6-well plate at a density of 50000/well for 24 hours.In a fetal bovine serum-free culture dish,add TGF-β1 or LIMK inhibitors（SR7826 or LIMKi3）to a 6-well plate.After 24 hours of incubation,the culture medium was replaced with EdU solution with a concentration of 10 m M;After another 24 hours,the cells were fixed,stained,and analyzed using a fluorescence microscope.【Results】1.The expression level of TGF-β1 in the urine of BPH/OAB patients induced by BPH was significantly higher than that of BPH patients.After measuring the concentration of TGF-β1 in the morning urine of clinical patients using an ELISA kit,the ratio of TGF-β1 to urinary creatinine showed that compared with the BOO group,the expression level of TGF-β1 in the urine of patients in the BPH/OAB group increased,and the difference was statistically significant.2.TGF-β1 promotes contraction of bladder smooth muscle in rats,and its effect can be inhibited by LIMK inhibitors.The results of the organ bath experiment showed that the contractile tension of bladder smooth muscle in the TGF-β1 group was significantly higher than that in the BSA group;While the LIMK inhibitor group inhibited the contraction of bladder smooth muscle,the TGF-β1+LIMK inhibitor groups exhibited both promoting and inhibiting effects on bladder contractile tension,which were related to the concentration of LIMK inhibitors.3.TGF-β1 promotes skeletal remodeling in HBSMCs,and its effect can be inhibited by LIMK inhibitors.Phalloidin staining results showed that compared with the BSA group,the myofilament protein skeleton of HBSMCs in the TGF-β1 group increased significantly,while the myofilament protein skeleton of HBSMCs in the TGF-β1+LIMK inhibitor group showed both promoting and inhibiting effects,which were related to the concentration of LIMK inhibitor.As the concentration increased,the inhibitory effect significantly increased.4.TGF-β1 promotes the proliferation of HBSMCs,and its effect can be inhibited by LIMK inhibitors.EdU staining results showed that compared with the control group,the proliferation of HBSMCs in the TGF-β1 group was significantly increased,while in the TGF-β1+LIMK inhibitor group when the concentration of LIMK inhibitor was low,both groups showed a role in promoting the proliferation of HBSMCs;At higher concentrations of LIMK inhibitors,the SR7826 group still exhibited a proliferative effect,while the LIMKi3 group exhibited an inhibitory effect.【Conclusion】TGF-β1 may be involved in the occurrence and development of BPH/OAB patients,and its effect may be achieved through LIMK.The elevated expression level of TGF-β1 in urine may serve as an auxiliary diagnostic indicator for OAB.LIMK inhibitors（SR7826 and LIMKi3）can block the contraction and proliferation of human bladder smooth muscle promoted by TGF-β1,making them potential therapeutic targets for OAB.