翻白草通过LKB1/AMPK-mTOR信号通路抑制大肠炎癌转化的物质基础及作用机制研究

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in western countries, and its incidence is increasing rapidly in Asia. flammatory responses play decision roles at different stages of tumor development, including initiation,promotion,malignant conversion,invasion,and metastasis. The therapy targeting inflammation is one of the stratagy to fight colorectal cancer. LKB1/AMPK/mTOR is involved in the development of CRC.Potentilla discolor Bunge, a folk Chinese medicine, has a long history of clinical application and reliable therapeutic efficacy on diarrhea and malaria. Our previous researches have proved P. discolorcontains a serials of rare triterpenoids, which significantly inhibited the colorectal tumorigenesis in mice. This project is aimed to elucidate the anti-colorectal cancer active compounds in P. discolorby the new technologies of phytochemistry, biochemical analysis and pharmacology. At the same time, considering the drug metabolism has great influence on its pharmacological action, the pharmacokinetic profiles of P. discolorwill also be investigated. Furthermore, as our previous studies have shown triterpenoids in P. discolor could regulate the LKB1/AMPK/mTOR pathway, it raise a question whether P. discolor exerts an anti-colorectal cancer effect by modulating the signaling pathway. Therefore, its possible molecular mechanisms will be also discussed in vivo and in vitro. These studies will provide scientific bases for the new drug development.

在恶性肿瘤中，大肠癌的发病率位居前列，而肠道炎症是其重要发病因素之一。基于“炎-癌”链的治疗策略已成为大肠癌防治的热点。LKB1/AMPK-mTOR信号通路是连接“炎-癌”的纽带，为大肠癌防治提供了新的靶标。翻白草用于湿热泻痢、便血等病症已有悠久历史，而这与慢性肠炎的病症相吻合。我们研究发现，翻白草中存在一系列稀有三萜，具有显著抗大肠肿瘤活性。本项目拟在前期研究的基础上，通过化学生物学、分析化学、药理学等多学科理念和手段，开展翻白草中预防大肠炎癌转化有效成分的探寻，动态跟踪体内过程对活性成分和药效的重要影响；对活性成分群，针对 LKB1/AMPK/mTOR这一主线，阐明翻白草预防大肠炎癌转化的作用机制，进而阐释其用于湿热泻痢的科学内涵，实现在传承的基础上对中医药理论和实践的创新；同时拓展翻白草用于防治大肠癌的新用途，为抗肿瘤创新中药研发提供科学依据。

翻白草，为蔷薇科委陵菜属植物翻白草Potentilla discolor Bunge．的干燥全草，具有清热解毒、止血止痢、消肿之功效。在本项研究中，我们对翻白草抗大肠炎癌药效物质基础及活性机制进行了相关研究，主要包括以下三方面，（1）活性三萜成分的分离提取：利用现代分离技术（中压色谱柱，HPLC 制备色谱，硅胶，凝胶等）对发现的目标成分进行分离，基于体外抗大肠肿瘤活性筛选，获得了Maslinic acid（MA）、Corosolic acid（CRA）等高活性化合物，为后续的体内分析及活性机制研究提供了基础；（2）通过原位胃吸收和原位单次灌注技术，对CRA的大鼠胃肠道吸收进行了系统研究；建立UHPLC-QTOF-MS方法开展了活性三萜化合物CRA体内代谢研究，并评价了CRA体内代谢物药理活性，结果显示CRA具有良好的胃和小肠吸收，其在体内可通过甲基羧化，羟化，葡萄糖醛酸化和乙酰化代谢为不同代谢产物，P450同工酶CYP1A2，CYP3A4，葡萄糖醛酸转移酶和乙酰转移酶可能参与体内生物转化，含CRA/代谢物血浆显示出良好的体外抗肿瘤及糖尿病活性；（3）在活性三萜化合物MA和CRA的抗肿瘤活性机制研究中，通过体外MTT、集落克隆形成、流式细胞分析及Transwell实验等方法；结合在体裸鼠移植瘤、AOM/DSS小鼠模型活性验证，证实了MA可通过调控caspase-3及EMT相关蛋白表达来诱导大肠癌细胞凋亡、抑制大肠癌细胞迁移，其体内外抗肿瘤活性与AMPK-mTOR信号通路的调控作用密切相关；而CRA抑制多发性肠腺瘤（ApcMin/+）小鼠肠道肿瘤发展，可能与调节肠道炎症反应及菌群结构有关。以上研究将为翻白草活性三萜成分的抗大肠炎癌作用机制阐明、创新中药研发、指导临床合理用药提供重要的科学依据。