ABSTRACT
Objective:To investigate the effects of 7-difluoromethy-5,4:dimethoxygenistein (DFMG) on stress urinary incontinence (SUI) model in Sprague Dawley (SD) rats and its possible mechanisms.Methods:SD rat model of SUI was established through simulating pregnancy,birth trauma and ovarian castration.The rats were divided into a normal control group,a SUI group,and a DFMG group at 10 or 20 mg/kg.They were treated with 10 mg/kg normal saline (NS),10 mg/kg NS,10 mg/kg DFMG and 20 mg/kg DFMG,respectively,via gastric gavage every other day.Maximal bladder capacity (MBC),leak point pressure (LPP),abdominal leak point pressure (ALPP),hematoxylin-eosin (HE) staining,and Masson staining were performed to detect the index for the model.MiR-26b and its down-stream gene phosphatase and tensin homolog deleted on chromosome 10 (PENT) mRNA in urethral sphincter muscles cells (USMCs) were analyzed by RT-PCR.The protein levels of PENT,phosphatidylinositol 3-kinase (PI3K),protein kinaseB (AKT),B-cell lymphoma 2 (Bcl-2),Bcl-2 associated X protein (Bax),cytochrome C(Cyt-c) and caspase-3 were examined by Western blot.The apoptotic rate of USMCs was determined by flow cytometry (FCM),and the proliferative rate of USMCs was examined by MTT assay.Results:The SD rat model of SUI was successfully established.HE staining and Masson staining showed that the pathological features of urethral sphincter were improved in the DFMG-treated groups compared with the SUI group.The urine dynamics indexes of model rats,such as MBC,LPP and ALPP,were improved (all P＜0.05).The results of RT-PCR showed that the miR-26b mRNA was up-regulated (P＜0.05) and PENT mRNA was down-regulated (P＜0.05) in the DFMG-treated groups compared with the SUI group.Simultaneously,compared with the SUI group,the protein levels of PENT,Bax,Cyt-c and caspase-3 were down-regulated (all P＜0.05) and the protein levels of PI3K,AKT and Bcl-2 protein were up-regulated (all P＜0.05),accompanied by the decreased apoptotic rate of USMCs (P＜0.05) and the increased proliferative rate of USMCs (P＜0.05) in the DFMG-treated groups.Conclusion:The DFMG can significantly improve the symptoms of urinary dynamics,which might be related to the up-regulation of miR-26b expression and the regulation of PI3/AKT-Bcl-2/ Bax signaling pathways.
ABSTRACT
Objective: To investigate the effects of 5-hydroxy-4'-nitro-7-propionyloxy-isoflavone (HNPI), a new derivative of genistein, on proliferation and apoptosis of human cervical cancer HeLa cells in vitro , and to explore the possible molecular mechanism. Methods: HeLa cells were treated with different concentrations of HNPI (5, 10, 20, 40, 60 and 80 μmol/L) for 48 h, while the cells were treated with 0.9% sodium chloride solution as the control group. The proliferation inhibitory rate of HeLa cells was detected by MTT assay. The alterations of HeLa cells on surface morphology, height, width, root-mean-squared roughness (Rq) and average roughness (Ra) were measured by atomic force microscopy. The changes of nuclear morphology of HeLa cells stained with DAPI were detected by laser scanning confocal microscopy. The apoptosis rate, cell cycle distribution, the level of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in HeLa cells were detected by FCM. Results: The proliferation of HeLa cells was significantly inhibited by HNPI in a dosedependent manner, and there was statistical significance as compared with the control group when the concentration of HNPI was greater than 10 μmol/L (all P < 0.05). The value of half maximal inhibitory concentration (IC50) of HNPI on HeLa cells was 46.83 μmol/L. After treatment with 20 and 60 μmol/L HNPI for 48 h, HeLa cells became round and shrinkage, the cytoplasm condensed, the surface roughness decreased, the filopodia structure was shrunk and even completely destroyed; the surface ultrastructure of HeLa cells became smooth and homogeneous, and merged into large protuberance or bulk; the height of single cell improved, but the width, Rq and Ra of cells down-regulated in a dose-dependent manner as compared with the control group (all P < 0.05); the nucleus indented, the chromosomes gathered, and the apoptosis bodies formed. Furthermore, the apoptosis of Hela cells was induced and the cell cycle was arrested at G0/G1 phase by HNPI in a dose-dependent manner, and there were statistical differences as compared with the control group when the concentration of HNPI was greater than 10 μmol/L (all P < 0.05). Meanwhile, the level of intracellular ROS in HeLa cells was increased, and MMP was decreased in a dose-dependent manner as compared with the control group (both P < 0.05). Conclusion: HNPI can significantly inhibit the proliferation and induce the apoptosis of HeLa cells in vitro , which may be related to the cell damage and ultrastructure change caused by HNPI, resulting in accumulation of intracellular ROS, reduction of MMP, and the blockage of cell cycle at G0/G1 phase.
ABSTRACT
Objective To investigate the effects of human umbilical mesenchymal stem cells (hUCMSCs) on Wistar rat models of stress urinary incontinence and its possible mechanism.Methods hUCMSCs cells were separated and extracted from human umbilical cord of cesarean delivery of full-term pregnancy women.The models of stress urinary incontinence (SUI) of Wistar rats were established by imitating delivery damage and ovary removing surgery,and then randomly divided into three groups:model group,transforming growth factor-β1 (TGF-β31) group,hUCMSCs group,and normal rats as normal group;and every group included ten rats.Maximal bladder capacity (MBC),leak point pressure (LPP),abdominal leak point pressure (ALPP),and sneezing experiment of rats including normal rats,model rats and model rats accepted TGF-β1 or hUCMSCs treatment were examined.When the treatment accomplished,the rats were killed and urethral sphincter were separated and examined by hematoxylin eosin (HE) staining and the proteins of troponin Ⅰ (TnI),troponin C (TnC),troponin T (TnT),tropomyosin (Tm),actin (AT),myosin heavy chain (MHC) and myosin light chain (MLC) of urethral sphincter was detected with Western blot method.Results The cells of hUCMSCs were extracted and authenticated,SUI models of Wistar rats were successfully established and authenticated.Compared to model group and TGF-β1 group,the MBC,LPP and ALPP of hUCMSCs group dramatically improved (P ＜ 0.05),while the positive rate of sneezing experiment significantly dropped (P ＜ 0.05).HE staining showed urethral sphincter became attenuation,fracture,sparse and disorder,simultaneously the proteins of TnI,TnC,TnT,Tm,MHC and MLC of urethral sphincter reduced (P ＜ 0.05).While the damaged urethral sphincter basically restored the normal structure and the proteins of TnI,TnC,TnT,Tm,MHC and MLC of urethral sphincter up-regulated through hUCMSCs treatment (P ＜ 0.05).Conclusions The treatment of hUCMSCs translation could observably improve the clinic symptoms of SUI Wistar rat models,and its mechanism may be related to enhancement of the protein expression of TnI,TnC,TnT,Tm,MHC and MLC of urethral sphincter,and were involved in the rehabilitation and reconstruction of urethral sphincter.