Department of Cardiology, The Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine.
Int Heart J. 2022;63(5):904-914. doi: 10.1536/ihj.21-585.
Forsythiaside, one of the main bioactive components of Chinese medicine Lian Qiao, exerts antioxidant, anti-bacterial, and anti-inflammatory effects. To date, the mechanism of Forsythiaside in cardiomyocyte injury remains unclear. However, the antioxidant effects of Forsythiaside on cardiac cells are currently unknown. This study investigated the effect and mechanism of Forsythiaside on oxidative stress in H9c2 cardiomyocytes. H9c2 cells were treated with HO and Forsythiaside and then transfected with small-interfering RNA against nuclear factor erythroid 2-related factor 2 (siNrf2). Cell viability, apoptosis, accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential were measured using methyl thiazolyl tetrazolium (MTT), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123, respectively. The levels of oxidative stress-related markers were determined using their respective detection kits. Furthermore, the levels of apoptosis- and Nrf2 pathway-related molecules were determined via Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Forsythiaside had no obvious toxicity on H9c2 cells. HO suppressed the viability, and reduced the levels of mitochondrial membrane potential, B-cell lymphoma-2 (Bcl-2), glutathione peroxidase (GSH-Px) and catalase (CAT) and superoxide dismutase (SOD), while promoted apoptosis, ROS accumulation, and elevated the levels of cleaved caspase 3, BCL2-Associated X (Bax) and malondialdehyde (MDA) in H9c2 cells. Contrarily, Forsythiaside reversed the aforementioned effects. HO advanced the levels of cytoplasm Nrf2, heme oxygenase-1 (HO-1), and nucleus Nrf2 in H9c2 cells, whereas Forsythiaside enhanced these effects. SiNrf2 reversed the functions of HO or Forsythiaside in cell viability, MDA, SOD, GSH-Px, CAT, Nrf2, and HO-1 in H9c2 cells, whereas Forsythiaside reversed the aforementioned effects of siNrf2. In sum, Forsythiaside protected H9c2 cells from oxidative stress and apoptosis induced by HO by activating the Nrf2/HO-1 pathway.
连翘中的主要生物活性成分之一连翘苷具有抗氧化、抗菌和抗炎作用。迄今为止，连翘苷在心肌细胞损伤中的作用机制尚不清楚。然而，连翘苷对心肌细胞的抗氧化作用目前尚不清楚。本研究探讨了连翘苷对 H9c2 心肌细胞氧化应激的作用及其机制。用 H2O2 和连翘苷处理 H9c2 细胞，然后用核因子红细胞 2 相关因子 2（Nrf2）的小干扰 RNA（siNrf2）转染。用噻唑蓝（MTT）比色法、末端脱氧核苷酸转移酶（TdT）介导的 dUTP 缺口末端标记（TUNEL）法、荧光探针 2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）和罗丹明 123 分别测量细胞活力、细胞凋亡、活性氧（ROS）积累和线粒体膜电位。使用各自的检测试剂盒测定与氧化应激相关的标记物的水平。此外，通过 Western blot 和定量逆转录聚合酶链反应（qRT-PCR）测定凋亡和 Nrf2 通路相关分子的水平。连翘苷对 H9c2 细胞无明显毒性。H2O2 抑制 H9c2 细胞活力，降低线粒体膜电位、B 细胞淋巴瘤-2（Bcl-2）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）以及超氧化物歧化酶（SOD）水平，促进细胞凋亡，增加 ROS 积累，并提高 H9c2 细胞中半胱天冬酶 3 裂解（cleaved caspase 3）、BCL2 相关 X（Bax）和丙二醛（MDA）的水平。相反，连翘苷逆转了上述作用。H2O2 增加了 H9c2 细胞胞浆 Nrf2、血红素加氧酶-1（HO-1）和核 Nrf2 的水平，而连翘苷增强了这些作用。用 siNrf2 转染 H9c2 细胞后，siNrf2 逆转了 H2O2 或连翘苷对细胞活力、MDA、SOD、GSH-Px、CAT、Nrf2 和 HO-1 的影响，而连翘苷逆转了 siNrf2 的上述作用。总之，连翘苷通过激活 Nrf2/HO-1 通路，保护 H9c2 细胞免受 H2O2 诱导的氧化应激和细胞凋亡。
