Department of Toxicology, School of Public Health, Peking University, Beijing 100191, China.
Department of Toxicology, School of Public Health, Peking University, Beijing 100191, China; Key Laboratory of State Administration of Traditional Chinese Medicine for Compatibility Toxicology, Beijing 100191, China; Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, China.
Phytomedicine. 2019 Nov;64:152929. doi: 10.1016/j.phymed.2019.152929. Epub 2019 Apr 16.
Euphorbia factor L1 (EFL1), a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L. (Euphorbiaceae), has been reported for many decades to induce gastric irritation, but the underlying mechanisms remain unclear.
The objective of this study was to investigate EFL1-induced cytotoxicity and the potential mechanisms of action on the human normal gastric epithelial cell GES-1.
GES-1 cells were treated with EFL1 (12.5-200 μM) for different time intervals, and cell survival, LDH release, intracellular reactive oxygen species (ROS), malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity were detected. Mitochondrial membrane potential (MMP) assay, DAPI staining, DNA fragment assay, and annexin V-FITC/PI staining were performed. The interaction between EFL1 and Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K, AKT, and mTOR proteins was simulated by molecular docking. The mRNA and protein expression of apoptosis and autophagy factors were detected by RT-qPCR and Western blotting.
EFL1 decreased survival, increased LDH leakage, and induced abnormal production of ROS, MDA and SOD in GES-1 cells. Mitochondria-mediated apoptosis was characterized by decreased MMP, condensed nuclei, fragmented DNA, and increased apoptosis rate. EFL1 interacted with proteins via hydrogen bonding. The mRNA, total or phosphorylated protein expression of Bcl-2, mitochondrial cytochrome c, PI3K, AKT, mTOR and p62 were downregulated; in contrast, those of cytoplasmic cytochrome c, cleaved caspase-9, cleaved caspase-3, LC3-ll and Beclin-1 were upregulated.
These findings indicated that EFL1 decreased the survival of GES-1 cells through EFL1-induced oxidative stress, activation of the mitochondria-mediated apoptosis as well as autophagy via inhibition of the PI3K/AKT/mTOR pathway.
大狼毒萜醇 L1（EFL1）是一种来自大戟科大戟属植物狼毒（Euphorbia lathyris L.）的拉坦类二萜，几十年来一直被报道具有胃刺激性，但作用机制尚不清楚。
本研究旨在探讨 EFL1 诱导的人正常胃上皮细胞 GES-1 细胞毒性及其潜在作用机制。
用不同浓度 EFL1（12.5-200 μM）处理 GES-1 细胞不同时间，检测细胞存活率、LDH 释放、细胞内活性氧（ROS）、丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。进行线粒体膜电位（MMP）测定、DAPI 染色、DNA 片段测定和 Annexin V-FITC/PI 染色。通过分子对接模拟 EFL1 与 Bcl-2、细胞色素 c、caspase-9、caspase-3、PI3K、AKT 和 mTOR 蛋白的相互作用。通过 RT-qPCR 和 Western blot 检测凋亡和自噬相关因子的 mRNA 和蛋白表达。
EFL1 降低了 GES-1 细胞的存活率，增加了 LDH 的漏出，并诱导了 ROS、MDA 和 SOD 的异常产生。线粒体介导的凋亡特征为 MMP 降低、核固缩、DNA 片段化和凋亡率增加。EFL1 通过氢键与蛋白质相互作用。Bcl-2、线粒体细胞色素 c、PI3K、AKT、mTOR 和 p62 的 mRNA、总蛋白或磷酸化蛋白表达下调；相反，细胞质细胞色素 c、裂解 caspase-9、裂解 caspase-3、LC3-ll 和 Beclin-1 的表达上调。
这些发现表明，EFL1 通过诱导氧化应激、激活线粒体介导的凋亡以及通过抑制 PI3K/AKT/mTOR 通路激活自噬，降低 GES-1 细胞的存活率。
