Kuwanon A from Morus alba L. Alleviates H2O2-Induced Oxidative Damage in HaCaT Keratinocytes by Inhibiting Ferroptosis and Enhancing Antioxidant Capacity

The root bark of Morus alba L. is commonly used as a natural antioxidant; however, its active constituents and underlying molecular mechanisms remain unclear. In this study, a bioactivity-guided isolation approach was employed to identify antioxidant substances from the root bark of Morus alba L. and to investigate their protective effects against oxidative damage in HaCaT cells. Using techniques such as silica gel column chromatography and semi-preparative HPLC, combined with NMR and HR-ESI-MS analysis, 22 compounds were isolated and identified from the dichloromethane extract of Morus alba L. root bark, including Diels–Alder adducts, flavonoids, and benzofurans. Among them, compounds 1 and 2 are new compounds, while compounds 12 and 16 were isolated from this plant for the first time. Bioactivity screening revealed that Kuwanon A (compound 17) exhibited significant cytoprotective effects in an H2O2-induced HaCaT cell injury model, effectively scavenging intracellular reactive oxygen species (ROS), restoring mitochondrial function, and enhancing the activities of antioxidant enzymes such as SOD and GSH. Further studies indicated that H2O2 induced ferroptosis in HaCaT cells, characterized by abnormal Fe2+ levels, lipid peroxidation, and elevated levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Kuwanon A significantly ameliorated these pathological changes. Consistently, ELISA and Astral DIA quantitative proteomics analyses demonstrated that Kuwanon A specifically upregulates the expression of the sulfurtransferase NFS1, thereby promoting the expression of the core antioxidant enzyme GPX4 and the iron storage protein ferritin-H, collectively inhibiting ferroptosis. This study elucidates that Kuwanon A is a key active component responsible for the antioxidant and anti-inflammatory effects of Morus alba L. root bark, and its mechanism is closely associated with regulating the NFS1-mediated ferroptosis defense pathway.