Wang Yanqing, Zhao Xi, Chen Ban, Chen Shaoman, Liang Yongbai, Chen Dongfeng, Li Xican
Department of Anatomy, School of Basic Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
Molecules. 2024 Dec 6;29(23):5764. doi: 10.3390/molecules29235764.
In this study, homoisoflavone methylophiopogonanone A (MOA) was investigated for its inhibitory effect on ferroptosis of H9c2 cells using a set of cellular assays, such as BODIPY-probed and HDCFDA-probed flow cytometry analyses, cell counting kit-8 analysis (CCK-8), and lactate dehydrogenase (LDH) release analysis. All these cellular assays adopted Fer-1 as the positive control. Subsequently, MOA and Fer-1 were subjected to two antioxidant assays, i.e., 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO)-scavenging and 2,2'-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid radical (ABTS)-scavenging. Finally, MOA, along with Fer-1, were systematically analyzed for molecular docking and dynamics simulations using a set of software tools. The experimental results revealed that MOA could inhibit ferroptosis of H9c2 cells but did not effectively scavenge PTIO and ABTS free radicals. Two molecular simulation methods or algorithms suggested that MOA possessed similar binding affinity and binding free energy (∆G) to Fer-1. Visual analyses indicated various hydrophobic interactions between MOA and one of the seven enzymes, including superoxide dismutase (SOD), dihydroorotate dehydrogenase (DHODH), ferroportin1 (FPN), ferroptosis suppressor protein 1 (FSP1), glutathione peroxidase 4 (GPX4), nicotinamide adenine dinucleotide phosphate (NADPH), and solute carrier family 7 member 11 (SLC7A11). Based on these experimental and molecular simulation results, it is concluded that MOA, a homoisoflavonoid with -di-OHs, can inhibit ferroptosis in H9c2 cells. Its inhibitory effect is mainly attributed to the regulation of enzymes rather than direct free radical scavenging. The regulation of enzymes primarily depends on hydrophobic interactions rather than H-bond formation. During the process, flexibility around position 9 allows MOA to adjust to the enzyme binding site. All these findings provide foundational information for developing MOA and its derivatives as potential drugs for myocardial diseases.
在本研究中，使用一系列细胞实验，如BODIPY探针法和HDCFDA探针法流式细胞术分析、细胞计数试剂盒-8分析（CCK-8）以及乳酸脱氢酶（LDH）释放分析，研究了高异黄酮甲基麦冬黄酮A（MOA）对H9c2细胞铁死亡的抑制作用。所有这些细胞实验均采用Fer-1作为阳性对照。随后，对MOA和Fer-1进行了两种抗氧化实验，即2-苯基-4,4,5,5-四甲基咪唑啉-1-氧基3-氧化物自由基（PTIO）清除实验和2,2'-偶氮二（3-乙基苯并噻唑啉-6-磺酸）自由基（ABTS）清除实验。最后，使用一组软件工具对MOA以及Fer-1进行了分子对接和动力学模拟的系统分析。实验结果表明，MOA可以抑制H9c2细胞的铁死亡，但不能有效清除PTIO和ABTS自由基。两种分子模拟方法或算法表明，MOA与Fer-1具有相似的结合亲和力和结合自由能（∆G）。可视化分析表明，MOA与七种酶之一之间存在各种疏水相互作用，这七种酶包括超氧化物歧化酶（SOD）、二氢乳清酸脱氢酶（DHODH）、铁转运蛋白1（FPN）、铁死亡抑制蛋白1（FSP1）、谷胱甘肽过氧化物酶4（GPX4）、烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和溶质载体家族7成员11（SLC7A11）。基于这些实验和分子模拟结果，得出结论：具有二羟基的高异黄酮MOA可以抑制H9c2细胞的铁死亡。其抑制作用主要归因于对酶的调节，而非直接清除自由基。对酶的调节主要取决于疏水相互作用，而非氢键形成。在此过程中，9位周围的灵活性使MOA能够适应酶结合位点。所有这些发现为开发MOA及其衍生物作为心肌疾病的潜在药物提供了基础信息。
