BACKGROUND 4,5-di-O-caffeoylquinic acid methyl ester (4,5-CQME) is a caffeoylquinic acid (CQA) isolated from Lonicera japonica Thunb., a traditional Chinese medicine. To date, the biological activity of 4,5-CQME has not been fully investigated. PURPOSE The aim of the current study was to explore the anti-oxidative activity and the underlying mechanism of 4,5-CQME. METHODS MTT assay was used to evaluate the cytoprotective effect of 4,5-CQME. DCFH-DA was used as a fluorescence probe to detect intracellular ROS. The mitochondrial membrane potential was detected using the fluorescent probe JC-1. MDA and GSH levels were measured using MDA and GSH commercial kits, respectively. Apoptosis assay was performed using the Annexin V-FITC/PI method. The functional mechanism of 4,5-CQME was investigated by analyzing relative signaling pathways through immunofluorescent staining, quantitative PCR and western blot analysis. RESULTS HepG2 cells were incubated with different concentrations of 4,5-CQME for 12 h before exposure to 500 μM H2O2 for 3 h. 4,5-CQME attenuated H2O2-induced oxidative damage and had a higher cytoprotective effect than 3-caffeoylquinic acid, 3-caffeoylquinic acid methyl ester, or 4,5-di-O-caffeoylquinic acid. 4,5-CQME also reduced ROS and MDA levels and rescued GSH depletion. Western blots demonstrated that 4,5-CQME decreased Bax/Bcl-2 and Bak levels. A mechanistic study confirmed that 4,5-CQME significantly suppressed H2O2-induced MAPKs phosphorylation but had little effect on MAPKs phosphorylation under normal conditions. By contrast, 4,5-CQME induced AKT phosphorylation in the presence or absence of H2O2. 4,5-CQME also regulated the Keap1/Nrf2 signaling pathway and enhanced both the mRNA and protein expressions of HO-1 and NQO1. The anti-oxidative effect of 4,5-CQME was greatly abolished by co-incubation with the Nrf2 inhibitor ML385 or PI3K inhibitor wortmannin. CONCLUSIONS Taken together, these results showed that 4,5-CQME offered significant protection against H2O2-induced oxidative stress, and its effect was in part due to the modulation of the Keap1/Nrf2 pathway.

中文翻译：

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从忍冬忍冬中分离得到的4，5-二-O-咖啡酰奎尼酸甲酯。靶向Keap1 / Nrf2通路以减轻H2O2诱导的HepG2细胞肝氧化损伤。

背景技术4，5-二-O-咖啡酰奎尼酸甲酯（4,5-CQME）是从中药忍冬属植物忍冬中分离出来的咖啡酰奎尼酸（CQA）。迄今为止，尚未完全研究4,5-CQME的生物活性。目的本研究的目的是探讨4,5-CQME的抗氧化活性及其潜在机理。方法采用MTT法评价4，5-CQME的细胞保护作用。DCFH-DA用作荧光探针以检测细胞内ROS。使用荧光探针JC-1检测线粒体膜电位。分别使用MDA和GSH商业试剂盒测量MDA和GSH水平。使用膜联蛋白V-FITC / PI方法进行凋亡测定。4，功能机制 5-CQME通过免疫荧光染色，定量PCR和Western印迹分析来分析相对信号传导途径。结果HepG2细胞与不同浓度的4,5-CQME孵育12小时，然后暴露于500μMH2O2 3小时。4,3-CQME减弱了H2O2引起的氧化损伤，并具有比3-咖啡酰奎尼酸，3-咖啡酰奎尼酸甲酯或4,5-二-O-咖啡酰奎尼酸更高的细胞保护作用。4,5-CQME还降低了ROS和MDA水平，并挽救了GSH消耗。蛋白质印迹表明4,5-CQME降低了Bax / Bcl-2和Bak水平。一项机理研究证实，在正常条件下，4,5-CQME显着抑制H2O2诱导的MAPKs磷酸化，但对MAPKs磷酸化的影响很小。相比之下，4 在存在或不存在H2O2的情况下，5-CQME诱导的AKT磷酸化。4,5-CQME还调节Keap1 / Nrf2信号通路，并增强HO-1和NQO1的mRNA和蛋白表达。与Nrf2抑制剂ML385或PI3K抑制剂渥曼青霉素共同孵育可大大消除4,5-CQME的抗氧化作用。结论综上所述，这些结果表明4，5-CQME对H 2 O 2诱导的氧化应激提供了显着的保护，并且其作用部分是由于Keap1 / Nrf2途径的调节。