石斛抗糖尿病性白内障活性成分与靶蛋白之间分子识别研究

Molicular identification of small-molecule acitive component and its target proten belongs to the leading herbal research nowadays. The results of previous research of our project have revealed that dendrophnol and syringic acid were the anti-diabetic cataract effective components of Herba Dentrobii, and aldose reductase (AR) and inducible nitric oxide synthase (iNOS) were the target protein. The in-vitro and in-vivo experimental animal studies as well as the phase 0 clinical trial have showed significantly therapeutic effect of drugs of the acitive component from Herba Dentrobii in treating diabetic cataract. In this study, we will perform real-time monitoring of the intracellular transmembrane absorption, transportation and anchoring of AR and iNOS by fluorescence labeling of dendrophnol and syringic acid and by using total internal reflection fluorescence microscopy (TIRFM) on the basis of previous achievements for molecule-docking technique and dynamics model, and will construct bimolecular fluorescence expression system by double-gene cotransfection of AR-iNOS carrier gene. We will also detect the secondary structure, functional groups, chemical bonds, three-dimension structure, bonding modes, and acting force of dendrophnol and syringic acid in identifying AR and iNOS in the living cells by using single-molecule dynamic imaging techniques such as AFM, LSCM, FRET, micro-Raman spectroscopy, emission spectrum, fluorescence spectrum, by which we will further explore the pharmacological mechanism of Herba Dentrobii in treating diabetic cataract. Furthermore, we will investigate the synergistic action of AR and iNOS by using bimolecular fluorescence complementation technique.

时空合一的活细胞单分子成像探究生命活动的奥秘是现代生命科学的前沿与热点；研究中药活性小分子与靶蛋白间分子识别，对于揭示其药理本质具有重要意义。本项目前期研究发现:1.石斛抗糖尿病性白内障（DC）活性成分为石斛酚和丁香酸；靶蛋白为醛糖还原酶(AR)和诱导型一氧化氮合酶(iNOS)；2.制成抗DC药物，动物体内外及0期临床试验均表现显著疗效。本项目拟通过全内反射荧光显微术、LSCM、基因沉默实时观察石斛酚和丁香酸靶向锚定AR和iNOS的过程；双基因共转染构建双分子荧光表达体系，利用AFM、LSCM、荧光共振能量转移(FRET)、拉曼光谱、荧光光谱等活细胞单分子动态成像技术，原位、实时、动态观察检测石斛酚、丁香酸识别AR、iNOS的过程及其二级结构、官能团、化学键、构象、作用力等；基因突变构建FRET双分子突变系统，利用双分子荧光互补、FRET技术检测AR、iNOS协同作用的结构域和作用模式。

该项目前期研究发现石斛抗唐糖尿病性白内障的活性成分为石斛和丁香酸，靶蛋白为醛糖还原酶（AR）和诱导型一氧化氮合酶（iNOS），但石斛酚、丁香酸如何在活细胞原位环境中识别靶蛋白的机制不清楚。本项目利用时空合一的单分子活细胞荧光动态成像技术体系，系统地研究了研究石斛酚和丁香酸识别AR、iNOS分子机制。首先采用化学全合成的方法对石斛酚和丁香酸进行荧光标记，应用DNA重组构建绿色荧光标记的AR、红色荧光标记的iNOS的AR-GFP、iNOS-DsRed晶状体上皮细胞系。荧光光谱显示石斛酚、丁香酸均通过氢键和范德华力与AR结合，结合常数随温度上升而下降；同步荧光光谱显示石斛酚使AR侧链氨基酸残基酪氨酸、色氨酸残基荧光强，疏水性增加；拉曼光谱显示石斛酚使AR的α-螺旋、β折叠、苯丙氨酸、色氨酸和酪氨酸的特征峰发生改变；原子力显微镜显示石斛酚导致AR凝聚。拉曼光谱显示丁香酸使AR的α-螺旋、苯丙氨酸、色氨酸及酪氨酸残基特征谱改变；原子力显微镜显示丁香酸使AR分子轻微聚集、节点增多、高度增加。荧光光谱显示石斛酚与iNOS通过氢键与范德华力结合，其结合常数随着温度的上升而下降；同步荧光光谱显示石斛酚使iNOS侧链色氨酸和酪氨酸荧光强度增加，且色氨酸发射波长轻微蓝移，说明色氨酸所处极性改变；拉曼光谱显示石斛酚使iNOS的α-螺旋、苯丙氨酸、色氨酸及酪氨酸特征谱线强度发生改变，说明这些结构在细胞中的含量发生改变，或环境发生了变化；原子力显微镜显示石斛酚导致iNOS大量聚集、高度显著增加；能量共振转移联合激光共聚焦显微显示，在高糖环境下晶状体上皮细胞内AR与iNOS存在相互作用，石斛酚能增强其相互作用。上述结果表明，石斛酚、丁香酸能选择性识别AR、iNOS形成复合物，并通过对其构象和氨基酸侧链的影响发挥其抑制AR和iNOS活性的作用。本项目从动态分子水平上诠析了石斛酚、丁香酸与AR、iNOS识别、转运过程，深刻揭示了石斛抗糖尿病性白内障的药理本质，为其新药创制提供了更深入的科学依据。