菊苣酸及其代谢产物对体内抗氧化防御酶调节与作用机制研究

Research that about uncovering the effects of dietary ployphenols on preventing oxidative stress through regulating antioxidants defence enzymes attracted much attention recently. Chicoric acid (CA), a caffeic acid derivative, originates from chicory and exists bioactivities such as inhibiting insulin resistance and enhancing immune system. Previous research of candidates indicated that CA was readily metabolized in liver microsome system in vitro. The metabolites of CA also had significant antioxidant effect. However, the cross-talk between in vivo metabolism of CA and its in vivo antioxidant effects are not elucidated clearly. This project is aimed at uncovering the bioavailability, CA metabolites and itself tissue distribution, and HAS-binding activity of CA in vivo by employing metabonomics technologies such as UPLC-Q-TOF-MS/MS. Furthermore, this project will examine the effects of CA on the expression of antioxidants defence enzymes such as HO-1 and NQO1 in liver and brain through in vitro and in vivo experiments. Meanwhile the regulatory mechanism of CA and its metabolites on the antioxidative signaling pathway Keap1/Nrf2/ARE will be determined through technologies including immunohistochemistry, siRNA, molecular docking, and electrophoretic mobility shift assays (EMSA). The results are planned to explore the metabolic and regulatory mechanism of CA on antioxidants defence enzymes. It is expected to provide new theoretical foundation for the studies of biological metabolic regularity and in vivo nutritional function of CA and other dietary ployphenols.

近年来，以调控抗氧化防御酶为靶向，开展膳食多酚体内抗氧化机制的研究已经成为新的热点。菊苣酸是菊苣菜中的一种具有抗氧化、增强免疫等生物活性的膳食多酚。申请者前期研究发现菊苣酸在体外代谢体系中易发生代谢且代谢物具有较强抗氧化活性，然而，其体内的抗氧化作用与其自身代谢之间的相关性和潜在规律尚不明确。本项目旨在通过UPLC-Q-TOF-MS/MS等代谢组学方法研究菊苣酸在体内的生物利用率、代谢产物、原型化合物及代谢产物的组织分布和蛋白结合特性；在此基础上通过体内外实验评价菊苣酸对肝脏和脑内的HO-1、NQO1等抗氧化防御酶表达的影响，并利用RNA沉默、分子模拟、凝胶迁移等技术研究菊苣酸及其代谢物对抗氧化防御信号通路Keap1/Nrf2/ARE的调控作用，据此揭示菊苣酸对抗氧化防御酶调控作用的物质基础与分子机制。研究结果预期为菊苣酸及其他膳食多酚生物代谢规律与体内营养功能机制的探索提供新的理论依据。

菊苣酸（Cichoric acid, CA）是菊苣和紫锥菊中极为重要的多酚化合物，具有抗氧化、抗炎和增强免疫等生物活性，现已作为食品抗氧化剂或免疫增强剂被广泛使用。本项目围绕菊苣酸体内代谢转化特征、体内外抗氧化机制以及二者之间相关性展开研究，获得研究结果如下：（1）建立生物组织中CA高灵敏度的HPLC-MS/MS定量测定方法，阐明CA代谢动力学、组织分布、蛋白结合等生物学特性；通过UPLC/Q-TOF-MS手段鉴定大鼠（灌胃,100 mg/kg）血浆、尿液和粪便中CA代谢物，分别在血浆、尿液和粪便中检测出6、12和11个代谢物，其中I相代谢物7个、II相代谢物12个，CA在大鼠体内主要经过水解、还原、甲基化、磺化、葡萄糖醛酸化、乙酰化、异构化和脱氧等代谢途径发生生物转化。（2）明确了菊苣酸甲基化代谢物的生物活性，采用体外肝匀浆法结合制备液相色谱分离制得CA单甲基化和双甲基化代谢物，细胞实验表明，CA及其甲基化代谢产物能够显著抑制H2O2导致的HepG2细胞活力的降低；CA及其甲基化代谢物缓解H2O2诱导的HepG2细胞线粒体膜电势的降低，提升细胞内GSH含量至正常水平；CA及其甲基化代谢物显著降低CAT、MAD的水平和升高细胞内GSH含量，甲基化程度增加，生物活性增强。（3）采用D-半乳糖构建氧化应激动物模型以及H2O2诱导SH-SY5Y细胞模型，解释CA调控抗氧化防御酶作用及分子机制，CA对D-半乳糖引起的小鼠学习、工作及空间探索能力的衰退具有显著的改善作用，CA显著提高小鼠大脑皮质和肝脏中Nrf2通路相关蛋白的表达，且能显著提高小鼠海马中HO-1和NQO1的mRNA水平，CA具有激活Keap1-Nrf2-ARE 信号通路、促进抗氧化防御酶的表达并维持细胞内氧化还原平衡作用。（4）为提高菊苣酸的稳定性和生物吸收率，采用热处理法构建乳铁蛋白-菊苣酸纳米复合物，乳铁蛋白对CA抗氧化具有协同作用且提高了CA体内稳定性及生物可及性。通过本项目实施，基本明确了CA体内分子生物学特性，初步揭示了CA调控抗氧化防御酶作用的分子机制，为CA在营养功能食品中的合理利用提供理论依据。在此项目资助下，共发表SCI 论文8篇，申报国家专利1项。