诸葛菜碱Ⅰ对H2O2所致HepG2细胞氧化损伤的保护作用

Protective Effect of Orychophragmuspine I Against Oxidative Damage in HepG2 Cells Induced by Hydrogen Peroxide

摘要:

本文采用柱色谱技术对诸葛菜种子的提取物进行分离纯化，采用波谱技术结合化学方法进行结构鉴定，首次获得新化合物，命名为诸葛菜碱Ⅰ，并通过建立H2O2诱导的细胞损伤模型，对诸葛菜碱Ⅰ保护H2O2所致HepG2细胞氧化损伤的作用及其机制进行了研究。于H2O2刺激前，加入不同浓度的诸葛菜碱Ⅰ预处理HepG2细胞12 h，然后以400 μmol/L H2O2损伤细胞2 h建立模型。MTT法检测细胞存活率，微板法检测细胞培养液中LDH活性和细胞MDA含量及SOD、GSH-PX的活性，Western Blot检测细胞内Nrf 2、HO-1蛋白表达，以评价诸葛菜碱Ⅰ对H2O2所致HepG2细胞氧化损伤的保护作用。结果表明，与模型组相比，诸葛菜碱I给药组（53.5、107、214 μmol/L）预处理12 h后，增加了细胞存活率（p<0.01），显著降低LDH向细胞外液的释放（p<0.01），显著降低细胞内MDA含量（p<0.05，p<0.01），显著提高细胞内SOD、GSH-PX的活性（p<0.05，p<0.01），增高了Nrf2蛋白水平。因此，诸葛菜碱Ⅰ对H2O2所致HepG2细胞氧化损伤具有保护作用。

Abstract:

Orychophragmus violaceus seed extract was isolated and purified by silica gel chromatography, and the structures were identified by spectral analysis and chemical methods. A new compound was identified for the first time and named as Orychophragmuspine I. The protective effect and mechanism of Orychophragmuspine I on hydrogen peroxide (H2O2)-induced oxidative damage in HepG2 cells was investigated by establishing a model of H2O2-induced cell damage. Before hydrogen peroxide stimulation, HepG2 cells were pretreated with different concentrations of Orychophragmuspine I for 12 h, and then cells were treated with 400 μmol/L H2O2 for two hours. The survival rate of cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Lactate dehydrogenase (LDH) activity in the medium, cellular malondialdehyde (MDA) content, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were measured using a microplate method, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expression was measured by western blot. The results showed that, compared with the model group, 12 h of Orychophragmuspine I treatment (53.5, 107, and 214 μmol/L) increased the cell survival rate (p<0.01), significantly reduced the release of LDH to the extracellular fluid (p<0.01), decreased MDA content in cells (p<0.05, p<0.01), and increased the intracellular activities of SOD and GSH-PX (p<0.05, p<0.01) and the level of Nrf2 protein. Therefore, Orychophragmuspine I has a protective effect against H2O2-induced oxidative damage in HepG2 cells.