Xiaoyaosan and isoliquiritigenin remodel depression-associated chromatin 3D structure and phase separation of NF-κB p65

Xiaoyaosan (XYS) is a classical traditional prescription with well-documented antidepressant effects. However, the active components and molecular mechanisms to alleviate depression remain unclear. In this study, using chronic social defeat stress (CSDS) and lipopolysaccharide (LPS)-induced depression models, we found that XYS and its key ingredient isoliquiritigenin (ILG) not only significantly improved depressive-like behaviors in mice but also exerted dual regulatory effects at the cellular level, promoting hippocampal neural stem cell proliferation and differentiation, while inhibiting microglia-mediated neuroinflammation. Using Hi-C technology, we discovered that XYS and ILG reversed the abnormal chromatin 3D structure in hippocampal microglia of depression models. Integrated analyses of transcriptomics, molecular docking, and 3D genomics identified the transcription factor Nfkb1 as a target of XYS and ILG. Co-culture experiments with microglia and neural stem cells further confirmed that high expression of Nfkb1 promotes inflammation and inhibits neuronal growth. In depressed mice, NF-κB1 expression was elevated and further formed phase separated condensates, which was effectively suppressed by XYS and ILG. This study reveals how XYS and its active component ILG function mechanistically to exert therapeutic effects on depression, including restoring chromatin 3D structure, and normalizing pathological phase separation of NF-κB1, and reversing depressive phenotypes. Collectively, these findings provide a solid experimental foundation for the development of natural antidepressants. • Identification of the key bioactive substance, Isoliquiritigenin(ILG), responsible for Xiaoyaosan(XYS)’s antidepressant effects. • XYS and ILG reverse abnormal chromatin 3D structure in microglia of depression mouse models. • Multi-omics analysis and experimental validation identifies NF-κB1 as a key target of ILG. • It was discovered and confirmed that NF-κB1 undergoes pathological phase separation in depression, and XYS/ILG can specifically reverse this process.