靶酶催化电子转移阻抑及靶细胞捕集法快速筛选藏药短管兔耳草降尿酸物质基础研究

It is very important to screen active compounds based on target enzyme (xanthine oxidase) and target cell (renal tubular epithelial cell) for lowering uric acid levels from traditional Chinese herbal medicines with effect of antigout, as these active components could be used for development of new drugs with higher safety than that of western antigout drugs. The active constituents from Chinese herbal medicines used for treatment of gout are mainly focused on target enzyme, and the activities of these compounds are still evaluated with ultraviolet method based on the products of the target enzyme catalytic reaction, which can easily result in false results and need a large amount of samples, while the active constituents on target cell have not been studied. Hence, these phenomenons seriously limited the development of new medicines with higher safety based on Chinese herbal medicines. Our previous results proved the Tibetan medicine of lagotis brevituba Maxim could effective reduce the uric acid level through target enzyme and target cell. Hence, it is very valuable to study the effective materials for treatment of gout..Here, our group build a novel method named target enzyme catalytic electron transfer blocking based on the electron transfer signal of enzyme catalytic reaction for screening active constituents on target enzyme, and apply target cell capture combined with LC-MS, RT-qPCR and Western blot methods to build a new way for screening the active constituents of Tibetan medicine of lagotis brevituba Maxim on target cell. These strategies can not only avoid the shortcomings of present used methods but also break through the bottleneck of screening active constituents on target cell..This work is very attractive as it can not only provide us with effective substance for development of safe and effective drugs for treatment of gout from lagotis brevituba Maxim, but also, it can provide us a new method and new strategy to discover active compounds from the whole traditional herbal medicines used for treatment of gout.

基于调控尿酸生成的靶酶与尿酸排泄的靶细胞筛选中草药降尿酸物质基础对于开发抗痛风新药保障痛风的安全防治具有重要意义。目前中草药靶酶降尿酸物质基础均根据酶催化反应产物进行筛选，往往操作复杂所需样品量大易引起假性结果；靶细胞降尿酸物质基础仍停留在提取物研究阶段，严重束缚新药开发。课题组前期研究表明藏药短管兔耳草基于靶酶与靶细胞降尿酸效果显著相对安全，物质基础有重要研究价值。本项目根据酶催化反应过程中的电子转移信号及纳米材料信号放大功能创建靶酶催化电子转移阻抑法筛选靶酶降尿酸物质基础，采用靶细胞捕集结合LC-MS、RT-qPCR、Western blot技术开辟靶细胞降尿酸物质基础筛选新途径，快速阐明短管兔耳草降尿酸物质基础。本研究可避免当前靶酶降尿酸物质基础筛选不足并推进靶细胞降尿酸物质基础深入筛选，为开发新药保障痛风的安全防治奠定物质基础，并为其它中草药降尿酸物质基础的筛选提供新方法与新思路。

项目以XOD为靶酶捕集并应用UPLC-Q-TOF/MS技术鉴定了藏药短管兔耳草中16个与XOD作用成分。以HK-2肾上皮细胞为靶细胞捕集并应用UPLC-Q-TOF/MS技术鉴定了藏药短管兔耳草中８个与靶细胞作用成分。在此基础上，项目采用导向分离与系统分离相结合方式，分离、制备并鉴定了26个藏药短管兔耳草化学成分。在可操作条件下，应用靶酶催化电子转移阻抑方法筛选了这些成分的XOD抑制活性，结果发现毛蕊花糖苷、松果菊苷、兔耳草苷H、6'-O-(4-甲氧基-(Z)-肉桂酰基)-α/β-D-吡喃葡萄糖苷、木犀草素、邻苯二甲酸二丁酯7个XOD抑制活性成分。应用Western Blotting技术筛选了化合物对URAT1、GLUT9及OAT1的表达调控，发现兔耳草苷B、兔耳草苷C、6-O-(Z)-肉桂酰基-α/β-D-吡喃葡萄糖苷、syringaresinol-4'-O-β-D-monoglucoside、globularidin、baldaccioside、10-O-(3,4-二甲氧基-(Z)-肉桂酰基)-梓醇、10-O-(3,4-二甲氧基-(E)-肉桂酰基)-梓醇及邻苯二甲酸二丁酯可显著下调URAT1的表达水平，3,4-二甲氧基苯甲醛与邻苯二甲酸二丁酯可显著下调GLUT9的蛋白表达水平，但化合物对OAT1的表达调控无显著性差异。分子对接显示这些短管兔耳草降尿酸活性成分可通过氢键、范德华力、pi-pi作用和疏水作用等方式分别与XOD、URAT1及GLUT9结合。这些项目研究成果既为藏药短管兔耳草安全有效的降尿酸类抗痛风新药开发奠定了物质与理论基础，也为其它降尿酸中草药的开发利用提供了新的方法和思路。