Academy for Advanced Interdisciplinary Studies, Peking University, No. 5 Yiheyuan Road, Haidian District, Beijing, 100871, China.
Key Laboratory of Particle & Radiation Imaging, Ministry of Education, Department of Engineering Physics, Tsinghua University, No. 30 Shuangqing Road, Haidian District, Beijing, 100084, China.
Biotechnol Lett. 2020 Nov;42(11):2453-2466. doi: 10.1007/s10529-020-02982-2. Epub 2020 Aug 11.
The aim of this study is to evaluate the cytoprotection and potential molecular mechanisms of cyanidin-3-glucoside (C3G) on hydrogen peroxide (HO)-induced oxidative damage in HepG2 cells.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to examine the viability of HepG2 cells exposure to HO or C3G. Meanwhile, the antioxidant properties of C3G were measured by determining the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and the malondialdehyde (MDA) levels. Flow cytometry was employed to determine HepG2 cells apoptosis, and HepG2 cells were stained with Hoechst 33342 to observe cell morphology. 2',7'-dichlorofluorescin diacetate (DCFH-DA) was used to evaluate the production of intracellular reactive oxygen species (ROS). Finally, the expression of apoptosis-related protein was monitored through western blot analysis.
HepG2 cells induced with HO presented a remarkable decrease in cell viability that was suppressed when HepG2 cells were interfered with C3G (2.5-10 μM). C3G interference memorably and dose-dependently inhibited HO-induced intracellular ROS and MDA overproduction, while C3G treatment markedly increased HO-induced the activities of intracellular SOD, GSH-Px and CAT. Eventually, the relative proteins expression levels of p53, cleaved caspase-9/3, cytochrome c, Fas-L, Fas, FADD and caspase-8 were substantially up-regulated in HO-triggered HepG2 cells, and Bax/Bcl-2 ratio and the relative protein expression levels of PARP were dramatically down-regulated. However, the expression levels of these relative proteins were reversed in C3G-interfered HepG2 cells.
C3G could protect HepG2 cells from oxidative damage, and the effects that were mediated by the mitochondrial apoptotic pathways and the external pathways.
本研究旨在评估矢车菊素-3-葡萄糖苷（C3G）对过氧化氢（HO）诱导的 HepG2 细胞氧化损伤的细胞保护作用及其潜在的分子机制。
采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法检测 HO 或 C3G 作用后 HepG2 细胞活力。同时，通过测定超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（CAT）活性和丙二醛（MDA）水平来评估 C3G 的抗氧化特性。采用流式细胞术检测 HepG2 细胞凋亡，并用 Hoechst 33342 染色观察细胞形态。用 2',7'-二氯荧光素二乙酸酯（DCFH-DA）评估细胞内活性氧（ROS）的产生。最后，通过 Western blot 分析监测凋亡相关蛋白的表达。
HO 诱导的 HepG2 细胞活力显著下降，而当 HepG2 细胞用 C3G（2.5-10 μM）干预时，这种下降受到抑制。C3G 干预显著且呈剂量依赖性地抑制 HO 诱导的细胞内 ROS 和 MDA 过度产生，而 C3G 处理显著增加了 HO 诱导的细胞内 SOD、GSH-Px 和 CAT 的活性。最终，HO 诱导的 HepG2 细胞中 p53、裂解的 caspase-9/3、细胞色素 c、Fas-L、Fas、FADD 和 caspase-8 的相对蛋白表达水平显著上调，Bax/Bcl-2 比值和 PARP 的相对蛋白表达水平显著下调。然而，在 C3G 干预的 HepG2 细胞中，这些相对蛋白的表达水平发生了逆转。
C3G 可保护 HepG2 细胞免受氧化损伤，其作用机制涉及线粒体凋亡途径和外部途径。
