Inonotus obliquus Crude Melanin Ameliorates DSS-Induced Colitis with Modulation of Gut Microbiota and Neutrophil pro-NETotic Activation

Background: Inonotus obliquus, a fungus known for its edible and medicinal properties, has traditionally been used as a herbal tea to relieve gastrointestinal disorders. Melanin, a major active constituent of this fungus, exhibits antioxidant, anti-inflammatory, and immune-modulating effects. This study aimed to investigate the therapeutic potential and mechanisms of Inonotus obliquus crude melanin (IOM) in treating dextran sulfate sodium (DSS)-induced colitis in mice. Methods: The study assessed colonic inflammation, mucosal damage, and intestinal barrier integrity. It also measured the levels of proinflammatory cytokines and oxidative stress markers. Gut microbiota composition was analyzed using 16S rRNA gene sequencing following IOM treatment. Additionally, label-free quantitative proteomic analysis was performed to explore the underlying mechanisms. Results: IOM administration significantly ameliorated colitis symptoms, strengthened the intestinal barrier, and reduced inflammation and oxidative stress in a dose-dependent manner. Furthermore, IOM modulated gut microbiota composition by increasing the relative abundance of Lactobacillus and Muribaculaceae, while reducing that of Bacteroides, Escherichia-Shigella, and Romboutsia. Proteomic analyses revealed that IOM treatment regulated the neutrophil pro-NETotic signaling pathway, which was further verified by immunohistochemistry or Western blot analysis of key pro-NETotic markers (e.g., PAD4, CitH3, MPO). Importantly, the relative abundances of these bacterial taxa were significantly correlated with the ulcerative colitis (UC) progression and neutrophil pro-NETotic activation. Conclusions: IOM mitigates DSS-induced colitis alongside the modulation of gut microbiota and the suppression of neutrophil pro-NETotic activation, suggesting its potential as a functional food ingredient for the management of UC.