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Introduction
Results and Discussion
首先从PubChem数据库中共得到33 个金丝桃苷和129 个槲皮素的靶蛋白（图1A和B）。线的粗细与靶点之间的关系强度有关（线条越粗，靶点与靶点之间的相互作用越强）。GO-BP结果表明，金丝桃苷和槲皮素在细胞凋亡过程中都起着负调节作用，并且可以调节炎症反应（图1C和D）。GO-CC结果表明，金丝桃苷和槲皮素主要在细胞核、细胞核质及细胞质中发挥作用。GO-MF结果显示，金丝桃苷和槲皮素主要通过与蛋白结合发挥作用（图1G、H）。KEGG通路富集结果显示，金丝桃苷和槲皮素均可作用于活性氧途径(图1I、J）。“金丝桃苷-靶点-通路”和“槲皮素-靶点-通路”显示，抗氧化相关基因主要参与Nrf2、MAPK和NF-κB信号通路（图1K和L）。
图1 利用网络药理学方法预测了与金丝桃苷/槲皮素相互作用的可能靶点和途径
研究发现槲皮素对氧化损伤的保护作用可能优于金丝桃苷。因为75 μmol/L槲皮素组的ROS水平显著低于75 μmol/L金丝桃苷组（图2A和D）。ROS水平升高会引发线粒体和细胞功能障碍、代谢紊乱和凋亡。该研究中，槲皮素在维持线粒体膜电位和防止ROS引起的损伤后的凋亡方面比金丝桃苷更有效（图2B、C和E），并且槲皮素在提高抗氧化酶GSH-Px、SOD和CAT的活性与降低MDA水平上显著优于金丝桃苷（图3）。
图2 细胞内ROS水平与线粒体膜电位
图3 细胞内抗氧化酶活力与MDA水平
图4 细胞内Nrf2、NQO1、HO-1、GCLC、GCLM、SOD1和CAT 的mRNA表达
图5 细胞核中Nrf2的蛋白表达水平与细胞内的ROS水平
图6 N2线虫寿命（A）；线虫体内脂褐素与ROS水平（B）；skn-1突变体线虫寿命（C）；skn-1在细胞质与细胞核中的比例（D）小鼠肠道菌群多样性和组成分析
Conclusion
研究结果证实，金丝桃苷和槲皮素都可以通过启动和上调Nrf2信号通路来减轻细胞的氧化损伤，并且在相同摩尔浓度下，槲皮素比金丝桃苷具有更好的抗氧化作用。从营养角度来看，槲皮素可能是一种比金丝桃苷更好的预防细胞氧化损伤的功能性药物。
Reduction of the oxidative damage to H2O2-induced HepG2 cells via the Nrf2 signalling pathway by plant flavonoids Quercetin and Hyperoside
Meijing Zhanga,1, Gaoshuai Zhanga,1, Xiangxing Menga, Xinxin Wanga, Jiao Xieb, Shaoshu Wangc, Biao Wangc, Jilite Wangd, Suwen Liue,*, Qun Huangb,*, Xu Yangf,*, Jing Lia,*, Hao Wanga,*
a State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology (TUST), Tianjin 300457, China
b Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang 550025, China
c College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology (TUST), Tianjin 300457, China
d Department of Agriculture, Hetao College, Bayannur 015000, China
e College of Food Science & Technology, Hebei Normal University of Science and Technology, Qinhuangdao 066004, China
f National Center of Supervision and Inspection for Processed Food Quality, Tianjin Institute for Food Safety Inspection Technology, Tianjin 300457, China
1 These authors contributed equally to this work and should be considered co-first authors.
*Corresponding author.
Abstract
Hyperoside and quercetin are similar in molecular structures. In this study, the antioxidant regulatory targets of hyperoside and quercetin are mainly in the nuclear factor (erythroid-2-derived)-related factor 2 (Nrf2) pathway predicted by network pharmacology. And the antioxidant effect and mechanism of hyperoside and quercetin were measured and compared in H2O2-induced HepG2 cells and Caenorhabditis elegans. The findings indicated that quercetin was more effective than hyperoside in reducing oxidative damage, which was proved by improved cell viability, decreased reactive oxygen species (ROS) production, decreased cellular apoptosis, and alleviated mitochondrial damage. In addition, quercetin was more efficient than hyperoside in enhancing the expression of Nrf2-associated mRNAs, increasing the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), and reducing the cellular malondialdehyde (MDA) content. Quercetin was superior to hyperoside in prolonging the lifespan of worms, decreasing the accumulation of lipofuscin, inhibiting ROS production, and increasing the proportion of skn-1 in the nucleus. With the Nrf2 inhibitor ML385, we verified that quercetin and hyperoside primarily protected the cells against oxidative damage via the Nrf2 signalling pathway. Furthermore, molecular docking and dynamics simulations demonstrated that the quercetin- Kelch-like ECH-associated protein 1 (Keap1) complex was more stable than the hyperoside-Keap1 complex. The stable structure of the complex might hinder the binding of Nrf2 and Keap1 to release Nrf2 and facilitate its entry into the nucleus to play an antioxidant role. Overall, quercetin had a better antioxidant than hyperoside.
ZHANG M J, ZHANG G S, MENG X X, et al. Reduction of the oxidative damage to H2O2-induced HepG2 cells via the Nrf2 signalling pathway by plant flavonoids Quercetin and Hyperoside[J]. Food Science and Human Wellness, 2024, 13(4): 1864-1876. DOI:10.26599/FSHW.2022.9250155.
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