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

Effects of 7-difluoromethy-5,4'-dimethoxygenistein on stress urinary incontinence model in rats and its mechanisms

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(10,20 mg/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 point pressure,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)mi R-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 chromosome10,PENT)m RNA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 kinase B,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mi R-26b m RNA表达升高(P﹤0.05),PENT m RNA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mi R-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） mKNA 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 MqT 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 ofUSMCs （P〈0.05） and the increased proliferative rate ofUSMCs （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 ofmiR-26b expression and the regulation of PI3/AKT-Bcl-2/ Bax signaling pathways.