4,5-di-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. targets the Keap1/Nrf2 pathway to attenuate H2O2-induced liver oxidative damage in HepG2 cells.,Phytomedicine

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4,5-di-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. targets the Keap1/Nrf2 pathway to attenuate H2O2-induced liver oxidative damage in HepG2 cells.
Phytomedicine ( IF 6.7 ) Pub Date : 2020-04-23 , DOI: 10.1016/j.phymed.2020.153219
Lingyun Xiao ₁ , Shu Liang ₂ , Lanlan Ge ₁ , Haoqiang Wan ₃ , Weigang Wu ₂ , Jia Fei ₄ , Shipin Wu ₂ , Boping Zhou ₂ , Xiaobin Zeng ₅

Affiliation

Centre Lab of Longhua Branch and Department of Infectious Disease, 2nd Clinical Medical College (Shenzhen People's Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China; Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, Guangdong Province, China.
Centre Lab of Longhua Branch and Department of Infectious Disease, 2nd Clinical Medical College (Shenzhen People's Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China.
Centre Lab of Longhua Branch and Department of Infectious Disease, 2nd Clinical Medical College (Shenzhen People's Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China; Department of Pathology (Longhua Branch), 2nd Clinical Medical College (Shenzhen People's Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China.
Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, Guangdong Province, China.
Centre Lab of Longhua Branch and Department of Infectious Disease, 2nd Clinical Medical College (Shenzhen People's Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China; Department of Pathology (Longhua Branch), 2nd Clinical Medical College (Shenzhen People's Hospital) of Jinan University, Shenzhen 518020, Guangdong Province, China; Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Medicine School of Shenzhen University, Shenzhen 518037, Guangdong Province, China.

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：

从 Lonicera japonica Thunb 中分离出 4,5-二-O-咖啡酰奎尼酸甲酯。靶向 Keap1/Nrf2 通路，减轻 H2O2 诱导的 HepG2 细胞肝脏氧化损伤。

背景技术4,5-二-O-咖啡酰奎尼酸甲酯(4,5-CQME)是从中药忍冬中分离得到的咖啡酰奎尼酸(CQA)。迄今为止，4,5-CQME 的生物活性尚未得到充分研究。目的本研究的目的是探索4,5-CQME的抗氧化活性及其潜在机制。方法采用MTT法评价4,5-CQME的细胞保护作用。 DCFH-DA用作荧光探针来检测细胞内ROS。使用荧光探针JC-1检测线粒体膜电位。 MDA 和 GSH 水平分别使用 MDA 和 GSH 商业试剂盒测量。使用Annexin V-FITC/PI方法进行细胞凋亡测定。通过免疫荧光染色、定量PCR和蛋白质印迹分析相关信号通路来研究4,5-CQME的功能机制。结果 HepG2 细胞与不同浓度的 4,5-CQME 孵育 12 小时，然后暴露于 500 μM H2O2 3 小时。 4,5-CQME 可减轻 H2O2 诱导的氧化损伤，并且比 3-咖啡酰奎尼酸、3-咖啡酰奎尼酸甲酯或 4,5-二-O-咖啡酰奎尼酸具有更高的细胞保护作用。 4,5-CQME 还可以降低 ROS 和 MDA 水平并挽救 GSH 消耗。蛋白质印迹表明 4,5-CQME 降低 Bax/Bcl-2 和 Bak 水平。机制研究证实，4,5-CQME 显着抑制 H2O2 诱导的 MAPKs 磷酸化，但在正常条件下对 MAPKs 磷酸化影响不大。相比之下，无论有或没有 H2O2，4,5-CQME 都会诱导 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。 4,5-CQME 的抗氧化作用通过与 Nrf2 抑制剂 ML385 或 PI3K 抑制剂渥曼青霉素共孵育而被大大消除。结论综上所述，这些结果表明 4,5-CQME 对 H2O2 诱导的氧化应激具有显着的保护作用，其作用部分归因于 Keap1/Nrf2 通路的调节。

更新日期：2020-04-23

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