基于环腺苷酸/环鸟苷酸（cAMP/cGMP）信号通路的茶多酚抗氧化作用机理解析

Tea polyphenols has been widely consumed as food antioxidant with potential antitumor and antidiabetogenic properties, but its mechanism of action inside the cell remains a mystery. Based on our previous study that tea polyphenols inhibited cyclic AMP (cAMP) and cyclic GMP (cGMP) -degrading phosphodiesterases and elevated cAMP levels in HepG2 cell,we hypothesize that the cAMP/cGMP signaling pathway is involved in the antioxidative mechanism of tea polyphenols. The overall aim of the proposal is to verify the above hypothesis. The specific aims include: 1) to establish the antioxidative model of tea polyphenols in vitro as well as in HepG2/C2C12 cell, 2) to clarify the relationship between the antioxidant ability of tea polyphenols, cell viability and cAMP/cGMP level, 3) to investigate the effect of tea polyphenols on the metabolism of cAMP/cGMP as well as the corresponding mechanism by crystal structures of PDE and its complex with tea polyphenols, 4) to understanding the molecular mechanism of cellular regulation underlying cAMP/cGMP signaling and gene expression by TF activation profiling plate array (TFAPPA),especially that responses to oxidative stress, 5) to determine whether tea polyphenols activates the CamKKβ-AMPK pathway responds to the cAMP/cGMP action in the presence of Ca2+chelator, Ca2+ release channel of IP3 receptor and CamKKβ inhibitors.The studies on the regulation of the cAMP/cGMP signaling pathway of tea polyphenols will elucidate the biochemical pathway of its antioxidant activity, which will provide supplement insight into its antioxidative mechanism.

茶多酚作为可抗肿瘤、抗糖尿病等的天然食品抗氧化剂已得到日益关注，但其在靶细胞内的作用路径尚需进一步明确。本项目基于前期研究中发现的茶多酚可抑制环核苷酸磷酸二酯酶（PDEs）、提高HepG2胞内cAMP含量的现象，拟从构建茶多酚抗氧化模型入手，确定茶多酚调控HepG2/C2C12细胞氧化胁迫的有效cAMP/cGMP浓度；通过制备茶多酚与PDEs共结晶并解析其三维结构，确定茶多酚对胞内cAMP/cGMP的代谢调控机制；通过TFAPPA比较茶多酚对48种转录因子，尤其是16种氧化胁迫相关转录因子表达的影响，并利用Ca2+螯合剂、Ca2+通道抑制剂以及CamKK抑制剂揭示cAMP/cGMP通路与CamKKβ-AMPK信号通路的互作效应，揭示茶多酚通过cAMP/cGMP调控胞内氧化胁迫的机理。本项目可望阐明cAMP/cGMP信号通路调控茶多酚抗氧化作用的机制，丰富茶多酚作为食品抗氧化剂的现有理论。

茶多酚作为可抗肿瘤、抗糖尿病等的天然食品抗氧化剂已得到日益关注，但其在靶细胞内的作用路径尚需进一步明确。本项目通过构建茶多酚体外和细胞水平的抗氧化模型以及细胞死亡模型，并确定茶多酚调控细胞氧化胁迫和生活力的有效 cAMP/cGMP 浓度；联合分子对接及酶活测试解析了茶多酚与 PDEs 共结晶的三维结构，明确茶多酚对动物细胞胞内AMP/cGP 的代谢调控机制；获得并解析出酵母PDE的晶体结构及其水解cAMP的作用机制，基于环核苷酸信号通路茶多酚类物质调控糖尿病及神经元再生的机制以及调控拮抗酵母、病原菌细胞及果实抗氧化及其作用机制解析。本项目研究成果为茶多酚成为食品致病微生物防腐剂增效剂、作为降糖及健脑益智的功能性食品添加剂以及作为提高拮抗酵母生防效力的增效剂以的产业化应用提供研究思路和理论依据。