大豆皂甙调控TLR4信号通路关键靶点的发现及其抗慢性炎症机理

Non-communicable chronic diseases (NCDs) are the primary causes of death of the whole world and also the major threats to the people’s health in China. Chronic inflammation is one of the key factors that cause NCDs. Many phytochemicals such as soyasaponins have strong anti-inflammatory activities. The appropriate application of such phytochemicals may be very effective in preventing NCDs. Studies from us and others have recently shown that soyasaponins can antagonize inflammation through regulation of the TLR4 signaling pathway. However, the primary targets and the associated mechanisms have not been established yet. To address these issues, we propose to investigate the effects of soyasaponins (A1, A2 and I) on expression, phosphorylation, recruitment, and dimerization of components of the TLR4 signaling pathway into lipid rafts by using in vitro (macrophage) and in vivo (mouse) inflammation models. A series of sophisticated technologies including fluorescence resonance energy transfer (FRET), immunoprecipiation, immunoblotting, and cell flowcytometry will be employed. Furthermore, the effects of soyasaponins on clustering, lipid composition of lipid rafts and production of reactive oxygen species will be investigated. Completion of this project will shed new lights on the primary targets and molecular mechanisms of soyasaponins in suppression of inflammation by modulating the TLR4 signaling pathway, and potentially provide new revenues to prevent chronic inflammation-mediated NCDs. Besides, it may provide clues for developing pharmaceutical inflammation inhibitors from soyasaponins.

慢性非传染性疾病（NCDs）是当今全球致死的主要原因，也是严重危害我国人民健康的重要问题。慢性炎症是引起NCDs的关键因素之一，很多植物化学物如大豆皂甙有很强的抗炎活性，其合理使用对于有效防治NCDs具有重要意义。我们和国内外其他研究小组的结果显示大豆皂甙可能经调节TLR4信号通路发挥抗炎活性，但其调控的关键靶点目前国内外未见报道。因此，我们拟运用巨噬细胞和小鼠体内慢性炎症模型，结合荧光共振能量转移、免疫共沉淀、蛋白印迹和流式细胞分选等技术研究大豆皂甙（A1、A2和I）对TLR4通路信号分子表达与磷酸化、募集进入脂筏及二聚化作用的影响；并进一步分析它们对脂筏成簇、脂筏内脂质构成及活性氧自由基的调节作用。旨在弄清大豆皂甙调控TLR4信号通路的关键靶点，阐明其抗慢性炎症的分子机理，为从营养学角度合理利用大豆皂甙防治慢性炎症介导的NCDs提供理论依据，也为药学上开发大豆皂甙类炎症抑制剂提供线索。

慢性非传染性疾病（NCDs）是当前危害我国人民健康的一个主要问题，慢性炎症是引起NCDs的关键因素之一。膳食来源的植物化学物如大豆皂甙具有抗炎活性，弄清其作用机理对于合理使用它们预防NCDs具有重要意义。本项目采用荧光标记激光共聚焦、蔗糖梯度超速离心、免疫共沉淀、荧光共振能量转移、蛋白印迹、荧光定量PCR等手段结合体外、体内炎症模型，研究发现：（1）在脂多糖和棕榈酸刺激的RAW264.7小鼠巨噬细胞体外炎症模型中，大豆皂甙（SS-A1、SS-A2和SS-I）可抑制TLR4及其配体分子MyD88和TRIF在脂筏区的募集和二聚体复合物（TLR4/MyD88和TLR4/TRIF）的形成、以及下游NF-κB的激活和促炎因子（iNOS与COX-2）的表达，这一活性与大豆皂甙阻断NADPH氧化酶亚基gp91phox和p47phox的相互作用、抑制NADPH氧化酶活性、清除ROS以及抑制脂筏成簇有关。（2）在脂多糖刺激的ICR小鼠和高脂饲料诱导的C57BL/6J肥胖小鼠体内炎症模型中，大豆皂甙（SS-A1、SS-A2和SS-I）可不同程度的降低促炎细胞因子（TNF-а、IL-1β和IL-6）、炎症介质（NO和PGE2）和炎症酶（iNOS和COX-2）含量或表达以及提高抗氧化酶（SOD）水平。（3）体内外实验表明，大豆皂甙可调控TLR4通路信号分子MD-2、TLR4、MyD88、TIRAP、TRAM、TRIF和TRAF6的表达以及IRAK4和IRAK1的磷酸化，关键靶点是其可抑制MyD88的分子表达。这些结果表明，大豆皂甙具有体内外抗慢性炎症的活性，其分子机理主要与其调控TLR4炎症信号通路有关，大豆皂甙调控TLR4炎症信号通路的关键靶点是其可抑制MyD88的分子表达并减少TLR4、MyD88和TRIF在脂筏中的募集和二聚化相互作用。本项目研究结果为促进大豆皂甙在慢性炎症介导的NCDs预防中的合理利用提供了理论依据，也为药学上开发大豆皂甙类炎症抑制剂提供了一些线索。