富马酸二甲酯对压力性尿失禁疾病模型中细胞外基质重构的影响及其机制

Dysfunction of periurtheral connective tissue is one of the core etiology for stress urinary incontinence (SUI), and extracellular matrix (ECM) remodeling induced by mechanical injury has been identified as an important molecular pathology. Previous studies have revealed that mechanical strain can induce ECM remodeling, and oxidative stress is considered a potential regulatory mechanism. Recent studies have shown that dimethyl fumarate (DMF) can act as an effective antioxidant and against oxidative damage by activating Nrf2, however, role of DMF in repairing post pelvic floor injury has not been reported. The preliminary study of this project has confirmed that DMF has a protective effect in ECM remodeling induced by cyclic mechanical stretch. This research project consists of two parts: the first part is animal experiment, Nrf2 knockout mice and TGFβ1 knockout mice are used to establish SUI disease mouse model through the method of vaginal balloon dilatation (VD), to study the effects of DMF and its related mechanism in the repairing process secondary to pelvic floor damage caused by vaginal dilation in SUI mice. In the second part, L929 cells are transfected with lentivirus carrying Nrf2 overexpression plasmid or (and) TGFβ1 shRNA plasmid to screen cell lines with Nrf2 overexpression, TGFβ1 silencing, Nrf2 overexpression + TGFβ1 silencing. Construct disease cell model through the method of cyclic mechanical stretch (CMS) using abovementioned cell lines with designated phenotype, to investigate the exact role of DMF on CMS-induced ECM remodeling and its molecular mechanism. This research project aims to demonstrate the pathogenesis and to explore a new therapeutic target for SUI.

尿道周围组织支持功能减退是压力性尿失禁（SUI）的核心病因学说之一，机械性损伤诱导细胞外基质(ECM)重构是尿道周围结缔组织支持功能减退的分子基础，我们预测氧化应激是潜在的调节机制。最新研究显示富马酸二甲酯（DMF）可高效激活Nrf2，对抗氧化损伤，其在盆底损伤修复过程中的作用尚无报道。本项目组前期研究初步证实DMF在机械损伤诱导ECM重构过程中具有保护作用。本研究分为两个部分：第一部分，运用Nrf2基因敲除小鼠、TGFβ1基因敲除小鼠，采用阴道球囊扩张法（VD）建立SUI疾病小鼠模型,研究DMF在SUI小鼠盆底组织损伤修复过程中的作用及相关机制；第二部分，通过携带特定基因载体的慢病毒转染并筛选具有Nrf2过表达、TGFβ1沉默、Nrf2过表达+TGFβ1沉默特性的L929细胞株，建立机械损伤细胞模型，探讨DMF对机械性损伤诱导ECM重构的影响及其分子机制。旨在为SUI的防治提供理论参考。

本课题涉及在体动物水平和离体细胞水平两个层面的研究内容。动物实验部分，应用野生型、Nrf2基因敲除型小鼠，采用阴道球囊扩张法模拟盆底机械损伤建立SUI疾病小鼠模型，研究DMF在SUI小鼠盆底组织损伤修复过程中的作用及相关机制。细胞实验部分，应用小鼠结缔组织成纤维细胞株，通过携带Nrf2过表达质粒或（和）TGFβ1 shRNA质粒的慢病毒体系转染，筛选出Nrf2过表达、TGFβ1沉默、Nrf2过表达+TGFβ1沉默三种稳定转染细胞株，采用周期性机械牵张作用诱导建立机械损伤细胞模型，探讨DMF对机械性损伤诱导ECM重构的影响及其分子机制。研究结果表明，盆底组织机械性损伤（阴道扩张）可以诱导小鼠发生SUI，其致病机制与尿道括约肌功能减退及盆底结缔组织ECM代谢异常有关；Nrf2激活剂DMF可活化TGF-β1/Smad3信号通路，上调PAI-1、α-SMA、TIMP2等纤维代谢调节因子的表达，促进盆底组织ECM沉积、减少降解，降低SUI发生率，提示Nrf2参与了SUI发生机制。机械性损伤可使L929细胞ECM合成减少、降解增加，上调Nrf2基因可缓解机械损伤诱导的ECM代谢异常，其作用机制与TGF-β1/Smad3信号通路及 PAI-1等纤维代谢调节因子的激活相关；Nrf2基因可通过缓解线粒体等细胞器损伤、减少细胞凋亡，进而在机械损伤过程中发挥保护作用。本课题揭示了机械性损伤（阴道扩张）对小鼠控尿功能的影响，证实DMF对SUI小鼠的治疗作用，阐明Nrf2在成纤维细胞损伤修复过程中的保护作用及相关机制，为探寻SUI防治靶点提供线索。其次，本研究成功将阴道扩张法建立的压力性尿失禁（SUI）小鼠模型和机械损伤细胞模型引入PFD相关的基础研究，为后续研究提供了值得借鉴的思路。