背景:大黄因子L1(EFL1),一种来自药用草本植物一品红(Euphorbiaceae)的二萜,几十年来一直有报道会引起胃刺激,但潜在的机制仍不清楚。
目的:本研究的目的是研究EFL1诱导的细胞毒性及其对人正常胃上皮细胞GES-1的潜在作用机制。
方法:用EFL1(12.5-200μM)处理GES-1细胞不同的时间间隔,和细胞存活,LDH释放,细胞内活性氧(ROS),丙二醛(MDA)含量,检测超氧化物歧化酶(SOD)活性。线粒体膜电位(MMP)测定,DAPI染色,DNA片段分析,进行膜联蛋白V-FITC/PI染色。EFL1与Bcl-2、细胞色素c、caspase-9,caspase-3,PI3K,AKT,通过分子对接模拟mTOR蛋白。RT-qPCR和Westernblotting检测细胞凋亡和自噬因子的mRNA和蛋白表达。
结果:EFL1降低了生存率,LDH泄漏增加,并诱导ROS的异常产生,MDA和SOD在GES-1细胞中的表达。线粒体介导的细胞凋亡的特征是MMP降低,凝聚核,片段化的DNA,细胞凋亡率增加。EFL1通过氢键与蛋白质相互作用。mRNA,Bcl-2、线粒体细胞色素c、PI3K,AKT,mTOR和p62下调;相比之下,细胞质细胞色素c,切割的半胱天冬酶-9、切割的半胱天冬酶-3、LC3-11和Beclin-1上调。
结论:这些发现表明EFL1通过EFL1诱导的氧化应激降低了GES-1细胞的存活,通过抑制PI3K/AKT/mTOR通路激活线粒体介导的凋亡和自噬。
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.