7-二氟亚甲基-5,4'-二甲氧基金雀异黄素对大鼠压力性尿失禁模型的疗效及其机制

摘要

目的:探讨7-二氟亚甲基-5,4'-二甲氧基金雀异黄素(7-difluoromethy-5,4'-dimethoxygenistein,DFMG)对SD大鼠压力性尿失禁(stress urinary incontinence,SUI)模型的疗效及其机制.方法:采用模拟妊娠、难产产伤及卵巢去势建立SD大鼠SUI模型,分为正常对照组、SUI组、DFMG(10,20mg/kg)组,模型鼠行DFMG隔日灌胃治疗.采用膀胱最大容积(maximal bladder capacity,MBC)、漏尿点压力(leak point pressure,LPP)、腹部漏尿点压力(abdominal leak pointpressure,ALPP)以及HE染色和Masson染色检测建模效果;采用RT-PCR检测尿道括约肌细胞(urethral sphincter muscles cells,USMCs)miR-26b及其下游靶基因磷酸酶和肌腱同源染色体(phosphatase and tensin homolog deleted on chromosome 10,PENT)mRNA表达;用Western印迹检测USMCs细胞PENT,磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K),蛋白激酶B(protein kinaseB,AKT),B细胞淋巴瘤/白血病-2(B-cell lymphoma 2,Bcl-2),Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax),细胞色素C(cytochrome C,Cyt-C)和含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase-9)的蛋白表达;采用流式细胞仪(flow cytometry,FCM)检测USMCs细胞凋亡率,采用噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]检测尿USMCs细胞增殖率.结果:成功建立SD大鼠SUI模型.HE染色和Masson染色显示:与SUI组比较,DFMG组尿道括约肌病理症状显著改善,MBC,LPP和ALPP均有显著提高(均P＜0.05).RT-PCR结果显示:与SUI组比较,DFMG组USMCs细胞miR-26b mRNA表达升高(P＜0.05),PENT mRNA表达下调(P＜0.05).Western印迹显示:与SUI组比较,DFMG组PENT,Bax,Cyt-C和caspase-3蛋白均下调(均P＜0.05);而PI3K,AKT和Bcl-2蛋白表达均上调(均P＜0.05);并伴随USMCs细胞凋亡率降低(P＜0.05),增殖率增高(P＜0.05).结论:DFMG可以显著改善SUI模型鼠尿动力学症状,其机制可能与上调miR-26b表达、调控PI3/AKT-Bcl-2/Bax信号通路而抑制细胞凋亡有关.%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.