Nrf2与Smad3在肝纤维化-肝癌发展进程中的交互作用机制及黄芪甲苷的干预

Oxidative stress and inflammatory response are important factors facilitating the progression of hepatic fibrosis-carcinoma. Activated nuclear factor erythroid 2-related factor 2 (Nrf2) exerts anti-inflammatory and antioxidant activities resulting in against liver injury, and activated Nrf2 has also anti-hepatofibrosis effect involved in modulating transforming growth factor beta (TGF-β)/Smad signal pathway; however recently, it is emerging that continuous activation of Nrf2 maybe facilitates hepatocarcinogenesis-progress. In view of these findings that the shift of Smad3 C-terminal and linker region phosphorylated respectively (pSmad3C/pSmad3L) facilitates the progression of hepatic fibrosis-carcinoma, astragaloside IV (AS-IV) has anti-inflammatory and antioxidant activities. We speculate that cross-talking between Nrf2 and Smad3 maybe occur in the progression of hepatic fibrosis-carcinoma, which is a possible molecular mechanism of AS-IV's anti-hepatic fibrosis-carcinoma. Therefore, in vitro and in vivo models of hepatic fibrosis-carcinoma with up-regulated or down-regulated expression of Nrf2 and/or variant phosphorylation of Smad3 at C-terminal and linker region (pSmad3C/pSmad3L plasmid transfection and/or mutant gene knock-in) will be used in the project; the expression and activation of Nrf2, Smad3 and will be detected by immunohistochemistry and/or immunofluorescence, immunoprecipitation and/or immunoblotting, quantitative real-time polymerase chain reaction, luciferase-reporter gene assays and so on. These studies purpose to uncover the cross-talking mechanisms between Nrf2 and Smad3 and the intervention of AS-IV, which is helpful to clarify the possible mechanisms of hepatocarcinogenesis-progress and provide a theoretical foundation for clinical application of AS-IV.

氧化应激与炎症反应是促进肝纤维化-肝癌进程的重要因素。活化的核因子E2相关因子2（Nrf2）具有抗炎抗氧化作用而抗肝损伤，且其抗肝纤维化与TGF-β/Smad信号通路相关；近年发现Nrf2持续活化却促进肝癌的发生发展。鉴于Smad3的C末端向连接区磷酸化转换（pSmad3C→pSmad3L）亦促进肝纤维化-肝癌进程，黄芪甲苷（AS-IV）具有抗炎抗氧化作用。推测：肝纤维化-肝癌进程中可能存在Nrf2与Smad3交互作用，AS-IV可能通过干预该交互作用而延缓该进程。故拟采用Nrf2表达上调/下调、pSmad3C/pSmad3L质粒或点突变模式动物等体内外肝纤维化-肝癌模型，IHC/IF、IP/IB、qRT-PCR、荧光素酶报告基因等技术检测Nrf2、Smad3表达及活化情况，探讨Nrf2与Smad3的交互作用机制及AS-IV的干预，阐明肝癌发生发展机制并为AS-IV临床应用提供理论依据。

氧化应激与炎症反应是促进肝纤维化-癌进程的重要因素。Smad3磷酸化亚型作用相反且相互拮抗，pSmad3L介导的促癌信号可向肿瘤抑制pSmad3C转换。此外，Nrf2对肿瘤具有“阴阳”双向调节作用。黄芪甲苷（AS-IV）具有抗炎抗氧化作用。本课题通过构建Smad3C磷酸化位点与Nrf2突变体内外模型，明确肝纤维化-癌进程中Nrf2与Smad3的交互作用及AS-IV的干预机制。结果表明：①AS-IV可抑制TGF-1诱导HSC-T6、HepG2和HuH-7细胞增殖和迁移，同时抑制两种肝癌细胞侵袭，其机制与促进pSmad3L/C转换，抑制pSmad2C/L介导的肝纤维化信号，同时活化Nrf2/HO-1信号有关。 ②AS-IV可通过调控pSmad3C/L及Nrf2/HO-1信号通路抑制DEN/CCl4/C2H5OH诱导C57BL/6J小鼠肝纤维化-肝癌发生发展。③选择性过表达Nrf2可促进pSmad3L/PAI-1/c-Myc促癌信号→pSmad3C/p21抑癌信号转换增强AS-IV抑制HepG2细胞增殖、迁移和侵袭；而选择性抑制Nrf2则作用相反。 ④选择性上调pSmad3C可激活Nrf2/HO-1信号轴增强AS-IV抑制HepG2细胞增殖、迁移和侵袭；而选择性上调pSmad3L则作用相反。⑤Nrf2基因敲除抑制AS-IV抗小鼠肝纤维化-肝癌过程中pSmad3C/p21信号通路的上调与pSmad3L/PAI-1信号通路的下调。⑥Smad3基因C末端磷酸化位点突变可抑制pSmad3C/p21介导的抑癌信号上调与pSmad3L/PAI-1/c-Myc介导的促癌信号下调；并抑制Nrf2/HO-1信号通路激活减弱AS-IV在肝纤维化-肝癌小鼠中的抗肝癌形成作用。结论：Nrf2与pSmad3C/pSmad3L在肝纤维化-肝癌进展中存在交互作用，AS-IV促进pSmad3L→pSmad3C转换并激活Nrf2信号发挥抗肝纤维化-肝癌作用，此研究为肝癌防治提供了新的有效靶点（pSmad3C/pSmad3L与Nrf2交互）及候选药物（AS-IV）。本研究达到预期目标，已发表SCI论文6篇，待发表英文论文3篇，多次参加国际国内学术会议交流，提交会议摘要11篇，课题组多人受本课题资助获得奖励和荣誉，培养了博士和硕士生。