双苄基异喹啉类生物碱抑制小胶质细胞和巨噬细胞过度活化的作用机制研究

Hyperactivation of microglia and macrophage has been considered to be associated with several inflammatory diseases. Liensinine and other bisbenzylisoquinoline alkaloids (BBI) are inhibitors of calcium. They can inhibit the hyperactivation of microglia and macrophage. Studies have demonstrated that by binding to calmodulin (CaM), BBI may inhibit the CaM from binding to calcium channels and calcium influx in the cells. CaM and calcium are essential for the synthesis of NO by inducible NO synthase (iNOS). Thus, it is a key point that whether BBI could inhibit the hyperactivation of these two cells by binding to CaM. In this project, the binding of BBI to CaM and the effects of BBI on the level of intracellular free calcium and NO production in LPS-activated microglia or macrophage will be studied. According to the structure-activity relationship，three to five BBI will be selected for further studies. Then, the antagonism of over expressed CaM on the effects of BBI and the change of signal transduction-related proteins (such as CaM, NF-κB, and CaMKII) caused by BBI will be investigated, by which the mechanism of BBI on hyperactivation of microglia and macrophage as well as the ralationship between the inhibition of activation and CaM binding can be illuminated. This study will be helpful for rapidly screening drug candidates from BBI that can inhibit the hyperactivation of these two cells. In addition, this study may help lay a theoretical foundation for chemical modifications of BBI.

小胶质细胞和巨噬细胞过度活化会导致多种炎症相关疾病的发生。莲心碱等双苄基异喹啉类生物碱（BBI）是钙抑制剂，并具有抑制上述细胞过度活化的作用。已有研究表明BBI可能通过与钙调蛋白（CaM）结合而阻止CaM与钙离子通道结合，抑制钙离子内流；CaM和钙离子为诱导型一氧化氮合酶（iNOS）催化生成NO所必须。因此BBI是否通过与CaM结合而抑制两种细胞过度活化是迫切需要解决的关键科学问题。本项目拟考察BBI与CaM的结合，及对LPS活化的上述细胞钙离子浓度和NO生成的抑制作用，经构效关系分析优选3-5种BBI，考察CaM过表达对BBI作用的拮抗能力，BBI作用时细胞内CaM、iNOS、NF-κB、CaMKII等信号通路相关蛋白的变化，阐明BBI抑制两种细胞过度活化与结合CaM能力之间的关系及作用机制。本研究将为从BBI中快速筛选抑制上述细胞过度活化的候选药物及BBI的化学修饰改造提供重要理论依据

小胶质细胞和巨噬细胞过度活化会导致多种炎症相关疾病的发生。双苄基异喹啉类生物碱（BBI）是钙抑制剂，并具有抑制上述细胞过度活化的作用。已有研究表明BBI可能与钙调蛋白（CaM）结合，抑制钙离子内流；CaM和钙离子为诱导型一氧化氮合酶（iNOS）催化生成NO所必须。因此BBI是否通过与CaM结合而抑制两种细胞过度活化是迫切需要解决的关键科学问题。本项目研究结果表明：（1）多种BBI结构中苯环上酚羟基的甲基化可使其抑制小胶质细胞和巨噬细胞活化的活性增强；头尾相连的结构使分子扭曲度提高，利于与靶点结合，有助于活性的提高；1位的手性碳原子构型引起的分子空间构型和构象对活性有较大影响；BBI的抗小胶质细胞和巨噬细胞活化作用与其钙调蛋白结合能力具有一定的正相关性。（2）甲基莲心碱、莲心碱和异莲心碱可通过与CaM结合、抑制CaM的表达和活性、以及抑制[Ca2+]i水平，从而抑制过度激活的巨噬细胞和小胶质细胞内Ca2+与CaM结合，进而抑制细胞内IκBα磷酸化和降解，抑制NF-κB的活化，进一步抑制iNOS及炎症因子的转录，抑制随后的细胞NO、TNF-α、IL-6、IL-1β以及IL-8的过度释放；3种BBI还可通过抗氧化抗自由基作用以及抑制活化的两种细胞过度迁移，进而发挥抑制巨噬细胞和小胶质细胞过度激活作用。（3）莲子心生物碱对Aβ损伤的神经元细胞具有的保护作用，并初步证明莲心总碱可改善阿尔茨海默病小鼠的学习和记忆能力。本研究为从BBI中快速筛选抑制小胶质细胞和巨噬细胞过度活化的候选药物和BBI的化学修饰改造提供重要理论依据；上述研究成果对神经退行性疾病，尤其对阿尔茨海默病的防治及药物的研发具有重要意义。