Abstract
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 H(2)DCFDA-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(bind)) 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 meta-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.