Neuroimmunological Mechanisms of Xiao-Yao-San Against Chronic Stress-Induced Colorectal Cancer: A Bioinformatics and Single-Cell Sequencing Study

Objective: This study aimed to integrate bioinformatics, network pharmacology, and single-cell sequencing to explore the potential neuro-immune mechanisms by which the traditional Chinese medicine formula Xiao-Yao-San (XYS) ameliorates chronic stress-induced colorectal cancer (CRC) progression. Methods: Transcriptomic and survival data of CRC patients were obtained from public databases (TCGA, GEO). Combined with literature review, 130 neurotransmitter-related receptor genes (NRGs) were screened, and their expression and prognostic value in CRC were analyzed. Based on 51 XYS components previously identified by UHPLC/Q-TOF-MS, network pharmacology was employed to predict their potential targets (1,228 in total). These targets were intersected with differentially expressed NRGs. Molecular docking was further performed to evaluate the binding affinity between XYS components and receptors. A mouse model of chronic unpredictable mild stress (CUMS) combined with orthotopic CRC transplantation was established. Neurotransmitter levels were measured by mass spectrometry, and 10x Genomics single-cell transcriptomic sequencing was applied to analyze changes in the tumor immune microenvironment. Results: Bioinformatics analysis revealed that 100 NRGs were significantly dysregulated in CRC tissues. After intersecting with XYS targets, 14 potential NRGs were identified, among which seven (ADRA2C, DRD1, GABRA2, HTR1B, HTR1D, HTR2A, HTR2B) showed significant correlation with patient prognosis. Molecular docking demonstrated favorable binding activity between XYS components (such as 8-Debenzoylpaeoniflorin and Sucrose) and the hub NRGs. In vivo experiments confirmed that XYS reversed CUMS-induced abnormalities in tumor and plasma levels of norepinephrine, dopamine, and serotonin. Single-cell sequencing further indicated that XYS reduced the CUMS-induced increase in myeloid-derived suppressor cell (MDSC) proportion and modulated the expression of key NRGs, including HTR2A in macrophages and HTR1B in T cells. KEGG enrichment analysis suggested that neuroactive ligand-receptor interaction and immune-related pathways were significantly regulated upon XYS intervention. Conclusion: XYS modulates neurotransmitter homeostasis and the tumor immune microenvironment in CRC under chronic stress through a multi-component and multi-target manner, which is closely associated with the regulation of key NRGs such as HTR1B and DRD1. This study provides neuro-immunological evidence for the clinical application of XYS in CRC patients with emotional disorders.