Jegal Kyung Hwan, Ko Hae Li, Park Sang Mi, Byun Sung Hui, Kang Keon Wook, Cho Il Je, Kim Sang Chan
Department of Herbal Formulation, MRC-GHF, College of Korean Medicine, Daegu Haany University, Gyeongsan, Gyeongsangbuk-do, 38610, Republic of Korea.
College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul, 08826, Republic of Korea.
Apoptosis. 2016 May;21(5):642-56. doi: 10.1007/s10495-016-1233-6.
Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) has many pharmacological activities including anti-inflammation, anti-oxidant and anti-cancer effects. Autophagy is the basic cellular machinery involving the digestion of damaged cellular components. In the present study, we investigated the protection effects of eupatilin against arachidonic acid (AA) and iron-induced oxidative stress in HepG2 cells and tried to elucidate the molecular mechanisms responsible. Eupatilin increased cell viability against AA + iron in a concentration-dependent manner and prevented mitochondrial dysfunction and reactive oxygen species (ROS) production. In addition, AA + iron increased the levels of pro-apoptotic proteins and these changes were prevented by eupatilin. Eupatilin also induced autophagy, as evidenced by the accumulation of microtubule-associated protein 1 light chain3-II and the detection of autophagic vacuoles. Furthermore, the protective effects of eupatilin on mitochondrial dysfunction and ROS production were significantly abolished by autophagy inhibitors. Eupatilin also increased the mRNA level of sestrin-2 and its promoter-driven reporter gene activity, which resulted in the up-regulation of sestrin-2 protein. Finally, gene silencing using sestrin-2 siRNA and the ectopic expression of recombinant adenoviral sestrin-2 indicated that sestrin-2 induction by eupatilin was required for autophagy-mediated cytoprotection against AA + iron. Our results suggest that eupatilin activates sestrin-2-dependent autophagy, thereby preventing oxidative stress induced by AA + iron.
灯盏乙素（5,7 - 二羟基 - 3,4,6 - 三甲氧基黄酮）具有多种药理活性，包括抗炎、抗氧化和抗癌作用。自噬是涉及消化受损细胞成分的基本细胞机制。在本研究中，我们研究了灯盏乙素对花生四烯酸（AA）和铁诱导的HepG2细胞氧化应激的保护作用，并试图阐明其分子机制。灯盏乙素以浓度依赖的方式提高细胞对AA + 铁的活力，并预防线粒体功能障碍和活性氧（ROS）的产生。此外，AA + 铁增加了促凋亡蛋白的水平，而灯盏乙素可阻止这些变化。灯盏乙素还诱导自噬，微管相关蛋白1轻链3-II的积累和自噬泡的检测证明了这一点。此外，自噬抑制剂显著消除了灯盏乙素对线粒体功能障碍和ROS产生的保护作用。灯盏乙素还增加了 sestrin-2的mRNA水平及其启动子驱动的报告基因活性，从而导致sestrin-2蛋白上调。最后，使用sestrin-2 siRNA进行基因沉默以及重组腺病毒sestrin-2的异位表达表明，灯盏乙素诱导sestrin-2对于自噬介导的针对AA + 铁的细胞保护是必需的。我们的结果表明，灯盏乙素激活依赖于sestrin-2的自噬，从而预防AA + 铁诱导的氧化应激。
