Integrative Multi-Omics Analysis Reveals the Immunoregulatory Effects of Sepia Ink on ADHD-like Phenotypes

Attention-Deficit/Hyperactivity Disorder (ADHD), affecting 5-10% of children globally, faces treatment limitations due to adverse effects and uncertain long-term risks of current pharmacotherapies. This study investigated the therapeutic potential of sepia ink (SI), a marine-derived natural complex from cuttlefish, in a scopolamine-induced ADHD-like mouse model. The chemical constituents of SI were characterized via Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). The behavioral assessments, histopathological examinations, flow cytometry, and complete blood counts were utilized to evaluate its effects on ADHD-like phenotypes, neuroinflammation, and immune function. Integrated transcriptomic, plasma metabolomic, and 16S rRNA sequencing were used to explore the underlying mechanisms. SI significantly alleviated hyperactivity and improved spatial learning and memory deficits. It reduced hippocampal neuronal damage, attenuated neuroinflammation, and reversed scopolamine-induced immunosuppression in spleen and thymus. SI also restored the balance of immune cell subsets in both mesenteric lymph nodes and spleen, and the peripheral blood cell counts. Multi-omics analyses suggested that the beneficial effects of SI were associated with reduced neuroinflammation, rebalanced systemic immune responses, partial correction of lipid metabolic disturbances, and restoration of gut microbiota homeostasis. Collectively, our findings indicate that SI effectively mitigates the in vivo ADHD-like impairments by coordinating immune, metabolic, and gut microbiota-related processes, thereby supporting its potential as a marine-derived therapeutic candidate for further ADHD treatment.