| Background and purpose:Diabetic cystopathy(DCP)mainly was manifested as decompensated bladder excitability and detrusor underactivity(DUA),resulting in dysuria,increased residual urine,chronic urinary retention and urinary incontinence and so on.The existing theory suggests that the mechanism may include: the nerve innervated bladder damage,bladder detrusor dysfunction,changes in urothelial function,the proportion of collagen fibers within the bladder and urethral coordination imbalance.And now more and more studies have confirmed that detrusor cell dysfunction and number reduction are the key reason of underactive bladder leading into a series of corresponding clinical symptoms.The purpose of this study is to study and elucidate the pathology and molecular mechanism of the detrusor underactivity of DCP,and to observe the therapeutic effect of urine-derived stem cells.Methods:Part I: Dysfunction of ICC cells in detrusor underactivity of diabetic cystopathyWe established rat diabetic model by intraperitoneal injection of streptozotocin plus high fat and high sugar diet.The rats were divided into two groups,normal rats for NC,DCP rats for DCP.The rat diabetic model was verified by insulin resistance test,cystometry and bladder fibrosis analysis.The nucleic acid and protein expression of HCN channel were analyzed by q RT-PCR,western blot and immunofluorescence staining.After isolating ICC cells successfully,the effect of HCN-mediated Ih current and channel activity were measured by patch clamping technique.The bladder detrusor contractility and intracellular calcium activity were tested by the method of isolated detrusor muscle strips tension recording and intracellular calcium concentration measurement.The number of ICC cells,expression of caveolin-3 and HCN channel proteins were detected by transmission electron microscope and western blot.The interaction between Caveolin and HCN was confirmed by using immunofluorescence staining and immunoprecipitation.siRNA-Caveolin-3 were transfected into the primary isolated ICC cells to down-regulate the expression of Caveolin-3,then the effects of down-regulating Caveolin-3 on HCN channel expression and channel functional activity were observed.Part II: To investigate the effect of AGEs on detrusor cell apoptosis of DCPThe expression of AGEs and RAGE was detected by collecting bladder samples from patients with DCP.Rats were divided into four groups,normal rats for NC,DCP rats for DCP,DCP rats were injected intraperitoneally with ALT-711 for DCP + ALT-711,DCP rats were treated with AG orally for DCP + AG.The therapeutic effects of ALT-711 and AG were evaluated.The contents of serum,pancreatic insulin and AGEs in bladder were measured by immunohistochemistry and Elisa test.The Body weight growth curve and fasting plasma glucose curve were drew.The bladder micturition function and detrusor contractility were tested by the method of cystometry and in vitro isolated detrusor muscle strips tension recording.The expression of SOD-2,Caspase-3 and RAGE,oxidative stress and apoptosis regulating protein,p38-MAPK,JNK and ERK1/2 were detected by western blot.The numbers of apoptotic cells and fibrosis degree were detected by TUNEL staining and Masson staining.The bladder detrusor cells were isolated and cultured in vitro.After treated with AGEs-BSA,the concentration and time dependence of AGEs were detected.The expression of RAGE and SOD-2 were detected by western blot.The si RNA-RAGE and siRNA-p38-MAPK were used to down-regulate the expression level of target genes,and the percentage of intracellular ROS concentration and the percentage of apoptotic cells were measured.It was confirmed that the role of this signal pathway in AGEs induced oxidative stress and apoptosis.Part III: Therapeutic effect of human urine-derived stem cells on DCP detrusor underactivity of ratsThe human urine-derived stem cells(hUSCs)were isolated and cultured,and their characteristics of proliferation and differentiation were observed.The isolated h USCs were transfected with eukaryotic lentiviruses carrying e GFP,and the cells injected into DCP rats by tail vein injection to observe the homing and therapeutic effect of hUSCs in vital organs.Rats were divided into three groups,normal rats for NC,DCP rats for DCP,DCP rats were injected with human urinary stem cells for DCP + USCs.The changes of blood glucose,body weight,bladder wet weight,blood biochemical indexes and glucose metabolism in rats were observed.The bladder voiding function and detrusor contractility were tested by cystometry and in vitro isolated detrusor muscle strips tension recording.HE staining,Masson staining and TUNEL staining were carry out to evaluate the alteration of percentage of apoptotic cells and fibrosis degree.Western blot was used to quantify the expression of α-SMA and Caspase-3.Results:1.Low-dose streptozotocin intraperitoneal injection combined with high-fat and high-sugar diet can induce a good model of type 2 diabetes mellitus.12 weeks after injection,a typical manifestations of underative detrusor was observed in the STZ injected rats.The nucleic acid and protein expression of HCN channel,HCN-mediated Ih current density were decreased.The HCN channel protein was located in the caveolae domain.The expression of caveolae and its structural protein caveolin were decreased.Caveolin-3 was the main function subtype,which can interact with the four subtypes of HCN channel and positively regulate the HCN channel-mediated Ih current.2.The levels of AGEs and RAGE in bladder tissue of DCP patients were significantly increased.After treatment with DCP rats by oral AG and intraperitoneal injection with ALT-711,both of them could block the production of AGEs in vivo and inhibit the oxidative stress and apoptosis to improve bladder voiding function and detrusor muscle contractility in DCP rats.AGEs can induce ROS production and apoptosis in detrusor cells in a time-dependent and concentration-dependent manner.Transfecting with si RNA to interfering the expression of RAGE and p38-MAPK can decrease the expression of Caspase-3 and SOD-2,and significantly inhibit the AGEs-induced intracellular ROS production and the ratio of cell apoptosis.3.The hUSCs were isolated and extracted from human urine,and can effectively differentiate into epithelial cells and smooth muscle cells by adding PDGF 5ng/ml plus TGFβ 2.5ng/ml and VEGF 30ng/ml respectively.The h USCs carrying the eGFP gene can homing into the pancreas but not bladder after tail vein injection every week.However,after injection of stem cells,DCP bladder fibrosis degree and ratio of detrusor cell apoptosis were effectively decreased,and bladder voiding function and detrusor contractility were also improved to varying degrees.Conclusions:1.The decrease in the number of ICC cells and HCN channel protein expression and functional activity may be an important molecular basis for DCP detrusor underactivity.Caveolin-3 in the cave domain can interact with and regulate the four subtypes of HCN expression and channel activity.2.Endogenous AGEs could accumulate in the bladder and activate RAGE/p38-MAPK signaling pathway to induce detrusor cell oxidative stress and apoptosis.3.The primary isolated hUSCs can inhibit the bladder fibrosis and detrusor cell apoptosis and improve the detrusor contractility and bladder voiding function of in the later phrase of DCP rats.In brief,the mechanism of DCP was complicated and difficult to clarify.It may be involved in the changes of ion channles,nerve receptor and several cell pathological processes.Homologous urine-derived stem cell may be a kind of new therapeutic resource. |
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