蛋氨酸亚砜还原酶A负性调控ROS/MAPKs/NF-kB信号通路在小胶质细胞炎性活化中的作用及机制

Microglial activation results in the production of reactive oxygen species (ROS), which induces oxidative injury on neurons. Recently, it has been revealed that microglial ROS may act as a signal and positively regulate microglia function via activation of inflammatory signal transduction pathways. As the main scavengers of ROS in the body, endogenous antioxidant defense systems may serve as potential "brakes" of microglia activation. Methioine residues on proteins serve as a key antioxidant defense that protects protein oxidative damage and regulates redox-dependent siganl transduction. Methionine sulfoxide reductase A (MsrA) is a key regulator of methioine antioxidant defense. We firstly found that the expression of MsrA in microglia and its potential negative regulation on MAPKs/NF-kB pathway. However, until now, little is known about its role in microglia activation. In this term, we investigated the role of MsrA-catalytic methioine antioxidant defense in microglia activation and its mechanisms using cell biology approaches, molecular biology approaches and bioanalytical chemistry approaches. Our findings may supply experimental data for development of novel drugs that target MsrA system.

小胶质源性活性氧（ROS）是引起脑内氧化应激损伤的重要病理因素，近年来发现其还可作为"信使分子"，通过激活炎性信号通路，正反馈性的调控小胶质细胞本身的活化状态。体内负责清除ROS的各种内源性防御系统可能在小胶质细胞异常活化时扮演"刹车"的角色。蛋氨酸抗氧化防御系统是一种重要的蛋白质自我保护机制，参与调节氧化-还原依赖的信号转导过程。蛋氨酸亚砜还原酶A（MsrA）是体内调控蛋氨酸抗氧化机制的关键酶，申请者所在团队的前期研究发现MsrA表达在小胶质细胞中，且可负向调控炎性条件下MAPKs/NF-kB通路的激活。目前MsrA在小胶质细胞异常活化中的作用尚不清楚。本项目拟综合利用细胞生物学、分子生物学、生物分析化学等技术，探索MsrA酶促抗氧化系统在小胶质细胞炎性激活中的作用，并试图从关键蛋白氨基酸残基氧化-还原修饰的角度阐明其机制，为以MsrA为靶点开发相关新药提供实验依据。

氧化应激是小胶质细胞激活的早期事件之一。本研究阐述了一种抗氧化还原酶——蛋氨酸亚砜还原酶A在小胶质细胞炎性激活中的作用及其机制。蛋氨酸亚砜还原酶A在小胶质细胞中表达，并在LPS、亚硫酸盐等刺激下表达增加。通过shRNA下调蛋氨酸亚砜还原酶A表达恶化了小胶质细胞炎性激活，基因重组蛋白上调蛋氨酸亚砜还原酶A功能减弱了小胶质细胞炎性激活。进一步证实蛋氨酸亚砜还原酶A通过Cys72、Tyr103和Glu115残基和活性氧形成鎓盐中间体，促进活性氧和蛋氨酸残基的结合，清除活性氧，抑制活性氧引起下游信号通路如MAPKs/NF-κB 信号通路的激活，进而抑制小胶质细胞的激活。我们通过计算机虚拟筛选研究了蛋氨酸亚砜还原酶A的潜在底物，发现来自海洋生物的化合物二甲基硫醚可以选择性的和蛋氨酸亚砜还原酶A结合，在蛋氨酸亚砜还原酶A催化下，通过Cys72、Tyr103和Glu115残基和活性氧形成鎓盐中间体，快速和活性氧反应，清除活性氧，进而抑制活性氧引起下游信号通路如MAPKs/NF-κB 信号通路的激活，抑制小胶质细胞的激活。本研究项目首次阐明蛋氨酸亚砜还原酶A在小胶质细胞相关炎性激活中的作用，并为二甲基硫醚在细胞保护和神经炎症调节中的应用提供实验依据。