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

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)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 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。

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

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.