丁苯酞钾盐经由Nrf2信号通路调控阿尔茨海默病氧化应激损伤的机制研究

Accumulating evidence has shown that oxidative stress-induced damage may play an important role in the initiation and progression of Alzheimer’s disease (AD) pathogenesis. Combating oxidative stress is dependent on the cell’s ability to maintain cellular redox homeostasis. A critical pathway in this regard is the Nrf2 (nuclear factor erythroid 2-related factor 2)-antioxidant response element (ARE) pathway. .Potassium 2-(1-hydroxypentyl)-benzoate (PHPB), is designed as a pre-drug of NBP. NBP is widely used in clinic for treatment of ischemic stroke in China. PHPB is hydrosoluble and can be quickly converted into NBP. Recent studies showed that PHPB improved the learning and memory deficits, and reduced oxidative stress in the chronic cerebral hypoperfused rats, senescence-accelerated mouse prone 8 (SAMP8) mice and APP/PS1AD transgenic mice. PHPB might protect neurons against H2O2-induced apoptosis. These results suggested that PHPB might be a potential drug candidate for treatment of AD by inhibiting oxidative stress. Our late data showed that PHPB up-regulated Nrf2, HO-1 and NQO-1 expressions in the cortex and hippocampus of LPS i.p. injected mice. Furthermore, PHPB reversed Nrf2 decrease in APP/PS1 AD transgenic mice. NBP treatment significantly reduced cataract scores and the levels of DNP, 4-HNE and MDA, and enhanced the expressions of Nrf2, TRX and catalase in the lens of diabetic rats in the lens. It indicated that NBP might delay the onset and progression of diabetic cataract by inhibiting the oxidative stresses. In our preliminary study, we found that Nrf2 level was increased in neuroblastoma SK-N-SH transfected with APP. Based on these findings, we hypothesized that targeting Nrf2 this pathway might involve in the effect of PHPB on protecting neurons from degeneration in AD treatment. .In the present study, we will investigate whether PHPB can up-regulate the expression and function of Nrf2 (nuclear translocation) and its downstream antioxidant genes to prevent oxidative stress in AD mouse model (APP/PS1 and APP/PS1-Nrf2 KO transgenic mice) and in cultured neuronal cells. Moreover, the upstream pathways which control Nrf2 activation will be detected. The project will propose three specific aims: 1) to define the pivotal role of Nrf2 in the oxidative stress during AD progression; 2) to define whether PHPB protects neurons against oxidative stress-induced injury via modifying Nrf2 in AD; 3) to define the mechanism of PHPB-induced Nrf2 up-regulation. The study will provide strong scientific evidences for targeting Nrf2 to prevent AD. The result will contribute to further clarify the mechanism of PHPB in the prevention and/or treatment of AD.

氧化应激在阿尔茨海默病（AD）的发病机制中扮演着至关重要的角色，影响着AD的发生和发展。核因子E2相关性因子2 (Nrf2）是细胞内源性抵抗氧化应激及细胞毒防御机制的关键转录因子，调控200多个基因转录表达，在神经保护方面有重要意义。研究发现丁苯酞钾盐（PHPB）能改善多种AD模型的学习记忆障碍，减少氧化应激损伤，并上调Nrf2的表达。由此提出科学假设：Nrf2是治疗AD的有效靶点，PHPB通过调控Nrf2信号通路，激活其下游的多种抗氧化酶，抑制氧化应激损伤进而发挥AD治疗作用。本课题的主要目标是以Nrf2为靶点，利用多种动物和细胞模型（基因过表达或基因敲除），系统考察PHPB是否通过激活Nrf2信号通路对AD起到治疗作用，并解析PHPB调控Nrf2激活的分子机制。为PHPB开发成为新一代抗AD药物，早日进入临床应用奠定理论基础。并为将来临床以Nrf2为靶点，开发AD治疗药物提供科学依据。

氧化应激在阿尔茨海默病（AD）的发病机制中扮演着至关重要的角色，影响着AD的发生和发展。核转录E2相关因子（Nrf2）是细胞内源性抵抗氧化应激及细胞毒防御机制的关键转录因子，调控200多个基因转录表达，在神经保护方面有重要意义。我们的前期研究发现丁苯酞钾盐（PHPB）能改善多种AD模型的学习记忆障碍，减少氧化应激损伤，并上调Nrf2的表达。由此提出科学假设：Nrf2是治疗AD的有效靶点，PHPB通过调控Nrf2信号通路，激活其下游的多种抗氧化酶，抑制氧化应激损伤进而发挥AD治疗作用。为验证这一假说，我们采用多种转基因小鼠和细胞模型，从整体和离体水平分别考察PHPB改善学习记忆障碍，抑制氧化应激损伤，缓解AD特征性病理改变，促进Nrf2下游抗氧化酶的表达等能力。体内研究结果显示，PHPB能够明显改善APP/PS1-AD小鼠在跳台实验中表现出的认知功能障碍，提升其脑组织中CAT、GPx、GSH等抗氧化物活力，降低脑组织中Aβ沉积及胶质细胞活化，并通过AKT/GSK3β途径上调Nrf2及其下游HO-1、NQO-1等蛋白的表达，此外PHPB对APP/PS1-AD小鼠脑组织中异常的线粒体代谢亦能起到一定恢复作用。然而上述变化在APP/PS1-Nrf2 KO小鼠中无法被观察到，提示PHPB对APP/PS1-AD小鼠的认知和病理改善可能和Nrf2密切相关。同时，体外结果进一步显示，PHPB能够剂量依赖性地改善SK-N-SH APPwt细胞模型的存活及LDH漏出率，降低ROS及MDA等氧化产物，提升Nrf2下游蛋白的表达，并且以上作用均可被Nrf2抑制剂ML385阻断。本项目的实施揭示了PHPB通过调控Nrf2信号通路抑制氧化应激改善AD症状的分子机制，为AD药物的开发提供了新的有效靶点和治疗策略。