混合型高分辨质谱联用解析CADs代谢谱及对恶性肿瘤协同干预作用机制

Caffeic Acid Derivatives（CADs）were widely spread in food such as fruits, vegetables and honey, etc. Epidemiological surveys showed that regular consumption of CADs could prevent the occurrence of malignant tumor. But a series of follow-up study found that the CADs' bioactivities in vivo and in vitro studies had bigger difference. It's necessary to carry out correlation analysis between multi-CADs metabolic expression and their anti-tumor activities. This project was to establish CADs metabolic uHPLC-ESI-MS-MS fingerprinting and expression method to find CADs metabolic components, metabolic fingerprint region and the growth trend based on metabonomics method and hyphenated technology, a ultra-high performance liquid chromatography (uHPLC) -quadrupole-electrostatic field orbit trap high resolution tandem mass spectrum, chemical metrology analysis coupling organism model. It was used to evaluate the joint protection of CADs metabolic fingerprint region on DNA strand breakage to detect possible CADs antitumor active ingredient based on uHPLC-DPPH-DAD/ESI-MS-MS-uCL hyphenated detection. The influence of CADs and its metabolic marker on malignant tumor metabolic differentially expressed was deep to prove possible metabolic and anticancer pathway. The cytology and tumor-burdened bone in vivo animal experiment was employed to observe synergistic intervention action and molecular mechanism of CADs metabolic markers on anticancer. A new principle and new method was proposed for further research on malignant tumor of dietary intervention.

咖啡酸衍生物（CADs）广泛存在于果蔬及蜂蜜等食品中。流行病学调查表明，经常食用CADs可预防恶性肿瘤发生。但跟踪研究发现，CADs生物活性的体内、体外实验结果有较大差异，多种CADs代谢表达与抗肿瘤活性的关联性尚不清楚。本项目基于代谢组学方法和超高效液相色谱-四极杆-静电场轨道阱高分辨混合质谱手段，探索CADs代谢uHPLC-ESI-MS-MS指纹图谱构建及表征方法，以发现CADs代谢组分和代谢指纹区；基于uHPLC-DPPH-DAD/ESI-MS-MS-uCL联用技术评价CADs代谢指纹区对DNA链断裂的协同抑制作用，以发现CADs可能的抗肿瘤活性成分；解析CADs/代谢标志物对恶性肿瘤代谢差异表达的影响，以探明CADs及指纹区抗癌作用途径；开展细胞学和荷瘤动物实验，以观察CADs代谢标志物对恶性肿瘤的协同干预效应及机制。为恶性肿瘤膳食干预研究提供卫生分析新原理、新方法。

流行病学调查和科学研究表明，植物性膳食有利于慢性病及恶性肿瘤防控，但其作用机制尚不清楚。本项目以膳食中广泛存在的植物化学物CADs（咖啡酸衍生物，Caffeic Acid Derivatives，CADs）为研究对象，利用高分辨质谱Q Exactive UPLC-MS-MS、GC-MS-MS并结合16S rRNA宏基因组分析研究典型CADs代谢变化谱及对恶性肿瘤干预作用机制。结果表明：CADs多聚体在体内分解为单体化合物，且裂解途径相似；咖啡酸（CaA）单体作为CADs骨架结构，是其典型的代谢标志物；除甲基取代反应外，CADs体内代谢主要为二相代谢硫酸化和葡糖苷化反应，与肝脏中磺酸转移酶(SULTs)或葡萄糖醛酸转移酶(UGTs)有关。代谢单体CaA可通过miR-148a介导TGFβ-SMAD2信号通路的阻断抑制肝癌肿瘤干细胞特性。CADs单聚体绿原酸（CGA）和四聚体丹酚酸（Sal B）单独或协同干预可通过上调实验动物超氧化物歧化酶、谷胱甘肽过氧化物酶、血红素加氧酶及核转录因子Nrf2而明显减少致癌物PCB126导致的氧化应激损伤，CGA和Sal B具有协同作用。基于慢性不可预知应激（CUMS）代谢轮廓及代谢组学与行为学关联分析，发现代谢组学方法能够用来评价和预测CUMS损伤程度；结合炎症因子分析，CADs对CUMS损伤以及预防有良好干预效果。肝癌细胞和实验动物诱发性肝癌非靶标代谢组学研究发现了CaA和ChA扭转肝癌代谢的具体差异标志物，CaA和ChA通过谷氨酰胺转运通路发生作用；CaA、ChA和多聚体SalB可减少肝损伤、炎症和肝癌发生发展进展，其机制与肝代谢、肠道菌群与炎症反应有关。基于代谢组学，本研究揭示了CADs对恶性肿瘤（肝癌）发生、发展的协同干预作用及其与肠道菌群相关性，对重新解释和评估CADs健康促进效果及慢性病营养干预具有重要理论意义和潜在应用价值。