Nrf2通过抑制TGFβ1、NF-κB信号通路对糖尿病肾病具有保护作用的实验研究

The renal tissue injury induced by oxidative stress is one of the important mechanisms in the development of diabetic nephropathy. The abnormal production of reactive oxygen species (ROS) is a key reason accounting for the oxidative stress. Therefore, how to scavenge the excessive ROS is one of the crucial strategies for diabetic nephropathy therapy, as severe renal injury is induced by ROS. NFE2L2(Nrf2) has emerged as one of the most important cellular defense mechanisms to cope with oxidative stress. It regulates intracellular antioxidants, phase II detoxifying enzymes, and many other proteins that detoxify xenobiotics and neutralize ROS to promote cell survival and maintain cellular redox homeostasis. Based on our pilot study, we set up a hypothesis that Nrf2 also has a protective role in diabetic nephropathy, but it need to be confirmed and the further molecular mechanism need to be explored. Accordingly, we try to observe the roles of Nrf2 pathway inducer in diabetic nephropathy model, including type I and II diabetes. Meanwhile, considering the important functions of NF-κB and TGFβ1 signal pathways in diabetic nephropathy, we try to analyze the cross-talks between them and Nrf2 signal pathway in cultured renal mesangial cells. We expect to confirm the protective role of Nrf2 signal pathway in diabetic nephropathy by performing the well-designed experiments, moreover, to uncover the molecular mechanism accounting for the protective roles of Nrf2. It is worthful for clinical therapy on diabetic nephropathy since this study will widen our knowledge of Nrf2 and provide new ideas to therapeutic actions.

氧化应激造成的组织损伤是糖尿病肾病(DN)发生发展重要的分子机制之一，因此通过有效抑制氧化应激，减轻肾组织损伤是治疗DN的重要策略之一。申请者前期工作己证实，Nrf2信号通路是体内(包括DN病人)重要的防御信号通路，其激活后可带来多种下游基因的表达，从而有效清除体内的活性氧自由基(ROS)，拮抗氧化应激。由此，本课题拟通过观察Nrf2激活剂(Sulforaphane)在I、II型糖尿病模型中的作用，并对体外培养的系膜细胞(MsC)的Nrf2信号通路与NF-κB信号通路、TGFβ1信号通路之间的串话(cross-talk)的研究，探索Nrf2-ARE对DN动物模型的可能保护作用，并探讨其可能的作用机制。这将有助于进一步明确Nrf2信号通路在DN发病过程中的作用及其分子机制，从而为将来更有效的治疗DN提供新的思路以及实验依据。

随着经济社会的发展和生活水平的提高，糖尿病的发病率也呈现上升趋势。糖尿病是一组以血糖升高为主要特征的代谢性综合征，累及至肾脏的病变-糖尿病肾病的后果最为严重。其中氧化应激水平的异常升高是糖尿病肾病发生发展的基本原因，最终诱导致纤维因子TGFβ1的分泌以及细胞外基质的合成和沉积，从而导致肾小球硬化和肾功能的丧失。转录因子NF-E2相关因子2（Nrf2）能够结合到下游抗氧化应激的酶类启动子上，促进抗氧化应激蛋白和II相解毒酶的表达，进而增强细胞的抗氧化能力。然而Nrf2在糖尿病肾病中的具体作用还不是很明确。本课题将通过对II型糖尿病小鼠模型的体内研究，用高糖刺激肾系膜细胞模拟糖尿病的病理变化的体外研究，应用慢病毒构建、RT-PCR、Western Blot、结合位点定点突变实验、siRNA干扰和质粒转染过表达实验、荧光素酶报告基因实验、染色质免疫共沉淀等实验方法，从小鼠模型和培养的系膜细胞两个角度分别观察Nrf2在糖尿病肾病中的保护性作用。研究发现经腹腔注射Nrf2特异性激活剂SFN的小鼠呈现出明显上升的Nrf2水平，同时小鼠的糖尿病肾病的进展也得到缓解。在糖尿病肾病的小鼠模型以及体外培养的系膜细胞经高糖刺激后Nrf2的表达都是显著上调的，从而抑制了致纤维因子TGFβ1及其下游基因的表达。Nrf2通过转录因子c-Jun和SP1结合到TGFβ1的启动子上调控其转录，进而抑制下游细胞外基质成分的沉积。C-Jun和SP1两个蛋白之间存在自我激活和相互调控的作用，并且受其磷酸化水平的调控。在TGFβ1启动子上将c-Jun和SP1的结合位点定点突变后，Nrf2对TGFβ1的抑制作用消失。本研究具体阐述了抗氧化应激蛋白Nrf2在糖尿病肾病中的保护性作用，并且深入探讨了具体的作用机制，将会为临床上糖尿病肾病的治疗提供新的理论依据。