Protective Effect of the Anthocyanidin Extract of Hippophae rhamnoides L. on H1299 Cell Injury Induced by Hydrogen Peroxide and Effect of Nrf2/HO-1 Pathway
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摘要: 目的：研究沙棘花青素提取物（The anthocyanidin extract of Hippophae rhamnoides L.，HRAE）对双氧水诱导H1299细胞氧化损伤的保护作用及其机制。方法：利用双氧水诱导H1299细胞氧化损伤模型，采用MTT法检测HRAE对细胞活力的影响，采用荧光探针DCFH-DA检测细胞内活性氧（Reactive oxygen species，ROS）的含量；采用试剂盒分别测定超氧化物歧化酶（Superoxide dismutase，SOD）、过氧化氢酶（Catalase，CAT）、谷胱甘肽过氧化物酶（Glutathion peroxidase，GSH-Px）及丙二醛（Malondialdehyde，MDA）的含量；采用Western Blot法检测血红素氧合酶-1（Heme oxygenase-1，HO-1）、醌氧化还原酶-1（NAD(P)H quinine oxidoreductase 1，NQO1）、Kelch样环氧氯丙烷相关蛋白1（Kelch like ECH-associated protein 1，Keap1）和核转录因子E2相关因子（Nuclear factor E2-related factor 2，Nrf2）的蛋白质水平；采用相关数据库和软件对沙棘花青素提取物的关键活性成分表儿茶素和儿茶素与氧化应激关键靶点蛋白Nrf2进行分子对接验证。结果：MTT实验表明，在0~800 μg/mL浓度范围内HRAE对细胞活力无显著性抑制作用（P>0.05）；与模型组相比，给药组中MDA水平下降，SOD、CAT和GSH-Px等活性均有不同程度升高（P<0.05或P<0.01）；与模型组相比，给药组ROS的含量显著降低（P<0.05）；Western Blot显示HRAE可显著（P<0.05）调节HO-1、NQO1、KEAP和Nrf2的蛋白质水平（P<0.05）；分子对接结果表明表儿茶素和儿茶素与氧化应激关键蛋白Nrf2具有良好的自由结合活性。结论：沙棘花青素提取物以浓度依赖降低双氧水导致的H1299细胞氧化损伤，其机制可能与促进Nrf2/HO-1信号通路的激活有关。
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关键词:
- 沙棘花青素提取物 /
- H1299细胞 /
- 氧化应激 /
- Nrf2/HO-1通路 /
- 活性氧
Abstract: Objective: The present study was to explore the protective effects of the anthocyanidin extract of Hippophae rhamnoides L. (HRAE) against hydrogen peroxide (H2O2) induced by oxidative damage in H1299 cells and its mechanism. Methods: The H2O2-induced oxidative damage in H1299 cells was established. MTT assay was used to detect the cell viability of HRAE. Then the content of reactive oxygen species (ROS) was measured by fluorescence probe DCFH-DA. The contents of superoxide dismutase (SOD), catalase (CAT), glutathion peroxidase (GSH-Px), and malondialdehyde (MDA) were determined respectively by kit. Also the expression of proteins heme oxygenase-1 (HO-1), NAD(P)H quinine oxidoreductase 1 (NQO1), Kelch-like epichlorohydrin-associated protein 1 (Keap1) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were tested. At last, the research collaboratory for structural bioinformatics (RCSB) protein data bank and AutoDock software were used to verify the molecular docking between epicatechin and Nrf2, the key target protein of oxidative stress. Results: MTT assay showed that HRAE had no effect on cell viability in the range of 0~800μg/mL (P>0.05). Compared with model group, MDA level was decreased, while SOD, CAT and GSH-Px protein expression levels increased significantly in drug administration group (P<0.05 or P<0.01). HRAE significantly reduced the ROS levels in oxidative injured cells (P<0.05). Western Blot showed that HRAE significantly activated the protein levels of HO-1, KEAP and Nrf2 (P<0.05). Molecular docking results showed that epicatechin and catechin had good free binding activity with Nrf2 protein. Conclusion: HRAE could reduce the oxidative damage of H1299 cells induced by H2O2 in a concentration dependent manner, and its mechanism may be related to promoting the activation of Nrf2/HO-1 signaling pathway. -
图 1 双氧水对H1299细胞活力的影响
Figure 1. Effect of hydrogen peroxide on H1299 cell viability
注：***表示与空白组对比差异极其显著（P<0.001）。
图 2 沙棘花青素提取物对H1299细胞活力的影响
Figure 2. Effect of anthocyanins from Sea-buckthorn on H1299 cell viability
注：*表示与空白组对比差异显著（P<0.05）；**表示与空白组对比差异极显著（P<0.01）。
图 3 HRAE对双氧水诱导的H1299细胞保护作用
Figure 3. Protective effect of HRAE on H1299 cells induced by hydrogen peroxide
注：***表示与空白组对比差异极其显著（P<0.001）；#表示与模型组对比差异显著（P<0.05）；##表示与模型组对比差异极显著（P<0.01）。
图 4 HREA对H2O2诱导H1299细胞MDA、SOD、GSH-Px和CAT水平的影响
Figure 4. Effects of HREA on MDA, SOD, GSH-Px and CAT in H2O2-exposed H1299 cells
注：*表示与空白组对比差异显著（P<0.05）；***表示与空白组对比差异极其显著（P<0.001）；#表示与模型组对比差异显著（P<0.05）；##表示与模型组对比差异极显著（P<0.01）；###表示与模型组对比差异极其显著（P<0.001）。
图 5 HREA对H2O2诱导H1299细胞活性氧水平的影响（100×）
Figure 5. Effect of HREA on ROS expressions in H2O2-exposed H1299 cells (100×)
图 6 HREA对H2O2诱导H1299细胞Nrf2、HO-1、PPARγ、p44/42 MAPK和KEAP1蛋白的影响
Figure 6. Effects of HREA on Nrf2, HO-1, PPARγ, p44/42 MAPK and KEAP1 protein expressions in H2O2-exposed H1299 cells
注：***表示与空白组对比差异极其显著（P<0.001）；#表示与模型组对比差异显著（P<0.05）；###表示与模型组对比差异极其显著（P<0.001）；C表示空白组；M表示模型组；低、中、高分别表示沙棘花青素提取物50 μg·mL?1组、100 μg·mL?1组、200 µg·mL−1组。
图 7 沙棘花青素提取物与受体蛋白的对接模式图和2D展示图
Figure 7. Docked pattern and 2D display of anthocyanin extracts from seabuckthorn and receptor proteins
A：表儿茶素和Nrf2；B：儿茶素和Nrf2；C：表儿茶素和Nrf2分子对接2D展示图；D：儿茶素和Nrf2分子对接2D展示图。
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