Chen Yongchang, Wu Yan, Dong Juan, Zhang Chuanming, Tang Jia
Department of Pulmonary Disease, Yangzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Yangzhou, 225000, Jiangsu, China.
Department of Traditional Chinese Medicine Culture Publicity, Yangzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Yangzhou, 225000, Jiangsu, China.
Cell Biochem Biophys. 2025 Jun;83(2):2499-2510. doi: 10.1007/s12013-024-01659-1. Epub 2025 Jan 3.
Chronic obstructive pulmonary disease (COPD) stands as a major contributor to mortality worldwide, with cigarette smoke being a primary causative factor. Acacetin has been reported to possess lung protective effects. However, the precise role and mechanism of Acacetin in COPD remains elusive. In this study, human bronchial epithelial cell line HBE135-E6E7 was treated with Acacetin under cigarette smoke extract (CSE) conditions. Cellular viability was assessed using CCK-8 and LDH kits. Reactive oxygen species (ROS) generation was tested with DCFH-DA staining. JC-1 staining was employed to examine the mitochondrial membrane potential (MMP). Additionally, hydroxynonenal (4-HNE) level was tested using immunofluorescence staining and mitochondrial lipid peroxidation was evaluated using MitoPeDPP staining. MitoSOX staining was used to detect mitochondrial (mito)-ROS. Fe level was measured using FerroOrange staining and the expression of ferroptosis-related proteins was detected with western blot. Besides, the binding between Acacetin and NRF2 was analyzed by molecular docking. The sequent NRF2 overexpression or knockdown was used to explore the regulation of Acacetin on NRF2/SLC7A11/GPX4 signaling. Results indicated that CSE significantly reduced the viability, augmented ROS generation and decreased MMP in HBE135-E6E7 cells, which were blocked by Acacetin addition. Moreover, Acacetin inhibited lipid peroxidation and ferroptosis in CSE-treated HBE135-E6E7 cells. Specifically, Acacetin targeted NRF2 and activated the NRF2/SLC7A11/GPX4 signaling in CSE-induced HBE135-E6E7 cells. Furthermore, NRF2 deficiency or ML-385 treatment notably restored the influences of Acacetin on oxidative stress and ferroptosis in HBE135-E6E7 cells challenged with CSE. In conclusion, Acacetin alleviated CSE-induced injury in HBE135-E6E7 cells by activating The NRF2/SLC7A11/GPX4 signaling to inhibit ferroptosis.
慢性阻塞性肺疾病（COPD）是全球死亡率的主要促成因素，香烟烟雾是主要致病因素。据报道，芹菜素具有肺保护作用。然而，芹菜素在COPD中的具体作用和机制仍不清楚。在本研究中，人支气管上皮细胞系HBE135-E6E7在香烟烟雾提取物（CSE）条件下用芹菜素处理。使用CCK-8和LDH试剂盒评估细胞活力。用DCFH-DA染色检测活性氧（ROS）的产生。采用JC-1染色检测线粒体膜电位（MMP）。此外，使用免疫荧光染色检测羟基壬烯醛（4-HNE）水平，使用MitoPeDPP染色评估线粒体脂质过氧化。用MitoSOX染色检测线粒体（mito）-ROS。使用FerroOrange染色测量铁水平，并用蛋白质印迹法检测铁死亡相关蛋白的表达。此外，通过分子对接分析芹菜素与NRF2之间的结合。随后进行NRF2过表达或敲低，以探讨芹菜素对NRF2/SLC7A11/GPX4信号通路的调节作用。结果表明，CSE显著降低了HBE135-E6E7细胞的活力，增加了ROS的产生并降低了MMP，而芹菜素可阻断这些作用。此外，芹菜素抑制了CSE处理的HBE135-E6E7细胞中的脂质过氧化和铁死亡。具体而言，芹菜素靶向NRF2并激活CSE诱导的HBE135-E6E7细胞中的NRF2/SLC7A11/GPX4信号通路。此外，NRF2缺陷或ML-385处理显著恢复了芹菜素对CSE攻击的HBE135-E6E7细胞中氧化应激和铁死亡的影响。总之，芹菜素通过激活NRF2/SLC7A11/GPX4信号通路抑制铁死亡，减轻了CSE诱导的HBE135-E6E7细胞损伤。
