Depression has become the leading cause of disability worldwide and a growing public health problem in China. In addition, intestinal flora may be associated with depression. This study investigated the effect of the decoction Xiaoyaosan (XYS) against depressive behavior through the regulation of intestinal flora. Fifty-two healthy male Sprague-Dawley rats were randomly divided into four groups (i.e., control, model, XYS, and fluoxetine). The latter three groups were subjected to 21 days of chronic restraint stress to produce the stress depression model. Rats in the XYS and fluoxetine groups received intragastric administration of XYS and fluoxetine, respectively. The behavioral changes of the rats were observed after 21 days. Stool specimens were sequenced using the 16S rDNA high-throughput method to detect the structure and changes in intestinal flora. There was no difference observed in alpha diversity among the groups. At the phylum level, XYS regulated the abundance of Bacteroidetes, Proteobacteria, Firmicutes, Chloroflexi, and Planctomycetes. At the genus level, XYS reduced the abundance of the Prevotellaceae_Ga6A1_group, Prevotellaceae_UCG-001, and Desulfovibrio. On the contrary, it increased the abundance of the Ruminococcaceae family to improve depression-like behavior. The mechanism involved in this process may be related to short-chain fatty acids, lipopolysaccharides, and intestinal inflammation.

Depression is the neurological manifestation most commonly associated with gastrointestinal diseases. The release of inflammatory cytokines mediated by TLR4/NLRP3 inflammasome signaling-induced immunoinflammatory activation may represent a common pathogenic process underlying the development of gastrointestinal diseases and depression. Clinical studies have indicated that Xiaoyaosan (XYS) can relieve depressive behavior by improving gastrointestinal symptoms. We previously demonstrated that XYS can reduce colonic inflammation in a rat model of chronic unpredictable mild stress; however, the precise anti-inflammatory mechanisms involved remain unclear. Here, we investigated whether XYS can ameliorate depressive behavior through regulating the TLR4/NLRP3 inflammasome signaling pathway, thereby inhibiting immunoinflammatory activation and reducing colonic proinflammatory cytokine levels. Fifty-two healthy male SpragueDawley rats were randomly divided into four groups (control, model, XYS, and fluoxetine). The latter three groups were subjected to 21 days of chronic restraint stress to generate a model of stress-induced depression. XYS and fluoxetine were administered intragastrically. Behavioral changes in the rats were assessed after 21 days. Serum and colon samples were collected, and the relative levels of the inflammation indicators IL-6, IL-1β, and TNF-α were determined by ELISA. Pathological changes in colon tissue were assessed by hematoxylin and eosin staining. The levels of TLR4, MyD88, NF-κB-p65, TAK1, IRAK1, and TRAF6 were detected by immunohistochemistry, while the gene and protein expression levels of TLR4, MyD88, NF-κB-p65, TAK1, IRAK1, TRAF6, NLRP3, ASC, and caspase-1 were detected by quantitative polymerase chain reaction (qPCR) and Western blotting. The results indicated that XYS could improve the depressive-like behavior and the weight loss of rats with stress-induced depression. Furthermore, depressed rats treated with XYS exhibited decreased expression levels of TLR4, MyD88, NF-κB-p65, TAK1, IRAK1, TRAF6, NLRP3, ASC, and caspase-1 in colonic tissue; reduced colon and serum concentrations of the inflammatory factors IL-6, IL-1β, and TNF-α; and lowered levels of colonic inflammation.

Chronic spontaneous urticaria (CSU) pathogenesis involves mast cell degranulation induced by the inositol 1,4,5-trisphosphate/diacylglycerol (IP3/DAG) pathway, but the condition lacks specific biomarkers. This study was performed to investigate long non-coding RNA (lncRNA) expression profiles, identify those associated with IP3/DAG pathway, and assess their diagnostic and prognostic value for CSU. Ten samples were selected from CSU and control groups, and microarray was performed to screen differentially expressed (DE) lncRNAs and mRNAs. Bioinformatic and co-expression network analyses were used to identify lncRNAs associated with IP3/DAG pathway. Quantitative real-time polymerase chain reaction was used to validate lncRNA expression levels. Combined with disease characteristics and serum indices detected with enzyme-linked immunosorbent assays, Spearman analysis and logistic regression were applied to analyze lncRNA-associated disease risk. Receiver operating characteristic (ROC) curves and 2-year follow-up data were applied to evaluate lncRNA diagnostic and prognostic value. A total of 678 up- and 573 downregulated DE lncRNAs and 609 up- and 176 downregulated DE mRNAs were identified. Seven lncRNAs (upregulated T264761, T280622, ENST00000587970, T224062, ENST00000562459, and his-1_RNA_dna; downregulated ENST00000417930) were associated with the IP3/DAG pathway. D-dimer and histamine levels were significantly different between the two groups. Correlation analysis showed that his-1_RNA_dna positively correlated with the frequency of symptom appearance, while his-1_RNA_dna, ENST00000417930, T264761, and T280622 negatively correlated with the maximum wheal diameter. Regression analysis showed T264761 was associated with CSU risk. ROC analysis showed that the specificity of T264761 was 90%, with an area under the curve of 0.666. In follow-up, the rate of well-controlled disease in the low T264761 expression group was 82.61%. This study established lncRNA and mRNA expression profiles in CSU and identified lncRNAs associated with IP3/DAG pathway, which is mechanistically involved in this disease. T264761 may be a novel biomarker for CSU, but further study is needed to confirm its specific mechanism.

With the continued rise in the incidence and prevalence of depression globally, treatment options involving traditional Chinese medicine (TCM) are receiving increasing attention. Xiaoyaosan, which is commonly used to treat depression, has been used in clinical practice for more than 1000 years in China. Our previous research has confirmed that Xiaoyaosan (XYS), an herbal TCM prescription, can improve depressive-like behavior by regulating the gut microbes closely related to immune-inflammatory processes, providing a potential basis for the antidepressant actions of XYS. The aim of this research is to investigate the antidepressant mechanism of XYSspecifically, we examined the downregulation of the PI3K/Akt/mTOR signaling pathway, restoration of Th17/Tregs, and reduction of inflammatory responseusing network pharmacology, transcriptomics, 4D proteomics, molecular docking, molecular dynamics simulation, and animal experiments. The chemical components of XYS were obtained using the PubChem database and HERB. The ADMET lab 3.0 drug absorption, distribution, metabolism, excretion, and toxicity prediction tool was used to screen the potential ingredients of the compound. The Swiss Target Prediction and SEA TCM ingredient target prediction platforms were used to predict the targets of potential active ingredients. The STRING database was used for protein-protein interaction (PPI) network analysis of the target. A chronic immobilization stress (CIS) rodent model of depression was established using male Sprague-Dawley rats (n = 75). Tests for depressive-like behaviors included the sucrose preference test, open field test, and forced swim test. Transcriptomics and 4D-DIA proteomics were employed to explore gene and protein expression changes in the colon tissue. Molecular docking and molecular dynamics simulations verify the binding of XYS's components and core targets. Rat colons were processed for Western blot detection of PI3K, p-PI3K, mTOR, p-mTOR, Akt, p-Akt, and Th17/Treg-related receptors (RORγ and Foxp3) and for RT-PCR detection of PI3K, mTOR, Akt, RORγt, and Foxp3. Flow cytometry was used to detect Th17 and Treg cell subsets in the colon, and ELISA was applied to detect IL-1 β, TNF-α, IL-17, IL-21, IL-22, IL-10, and TGF-β levels. The core components and core targets were screened by network pharmacology, which combined with molecular dynamics simulation, molecular docking, 4D proteomics, and transcriptomics sequencing revealed that the PI3k/Akt/mTOR signaling pathway is an important pathway underlying the antidepressant effects of XYS. XYS administration ameliorated depressive-like behavior and reduced weight loss in rats with CIS. Western blotting and RT-qPCR experiments showed that compared with the CIS model group, the expression of p-PI3K, p-mTOR, p-Akt, and RORγt was reduced, while that of Foxp3 was increased, in the H-XYS group. Moreover, flow cytometry suggested restored Th17/Treg balance in the colon, while ELISA indicated reduced TNF-α, IL-21, IL-22, IL-17, and IL-1β but increased IL-10 and TGF-β levels in the colons of rats in the H-XYS group. Network pharmacology, combined with 4D proteomics, transcriptomics, molecular docking, molecular dynamics simulation, and in vivo experiments, was performed to explore the antidepressant mechanism of XYS. XYS downregulated the PI3K/Akt/mTOR signaling pathway to restore Th17/Treg balance, reduce the release of proinflammatory factors, and ameliorate depressive behaviors in a CIS rat model.

Introduction: Studies exploring the molecules and potential mechanisms linking air pollution exposure to depression remain scarce. This study aimed to elucidate the potential regulatory molecules and possible mechanisms associated with air pollutants and depression, thereby providing scientific clues for disease prevention and treatment. Methods: Seven air pollutants (SO ₂ , NO, benzene, CO, O ₃ , NO ₂ , and toluene) were included. A multilevel research framework was employed. Network toxicology and the Gene Expression Omnibus (GEO) database were used to identify potential targets and pathways of air pollutants; intersection genes were used to construct machine learning diagnostic models, and the optimal model was selected for validation. Mendelian randomization (MR) was applied to assess the genetic causal association between depression and relevant genes. Single-cell virtual perturbation was performed for network perturbation analysis to dissect potential molecular mechanisms. Molecular docking and molecular dynamics simulations were used to evaluate the binding affinity and complex stability between specific pollutants and depression-related genes. Finally, in vitro cell experiments were conducted to preliminarily validate the computational findings. Results: By intersecting air pollution-related targets with the Gene Expression Omnibus depression dataset, nine candidate target genes were identified. Ensemble machine learning analysis suggested that nitric oxide synthase 2 (NOS2) showed expression in depression. Mendelian randomization analysis suggested that elevated NOS2 expression may be associated with depression, supporting a possible genetic causal association between the two. Single-cell virtual perturbation suggested that perturbing NOS2 in microglia could affect inflammation- and oxidative stress-related pathways. Molecular docking predicted that all seven pollutants might bind to NOS2, but the predicted binding energies varied, with benzene and toluene showing relatively higher binding affinity. Molecular dynamics simulations further evaluated the stability of the benzene–and toluene–NOS2 complexes, suggesting that toluene binding to NOS2 was more stable. In vitro cell experiments preliminarily suggested that toluene exposure could induce inflammation and oxidative stress in BV2 cells, and changes in NOS2 expression may be involved in this process. However, these results cannot yet prove that NOS2 is a causal mediator of depression, and further functional experiments are needed for validation. Discussion: Integrating computational prediction, genetic association, network perturbation, and cellular validation, NOS2 may be a candidate molecular node linking air pollution exposure to depression, and its potential mechanism may involve neuroinflammation and oxidative stress. This study provides new hypotheses and theoretical clues for the possible molecular mechanisms linking air pollutants and depression, but the underlying mechanisms still require further experimental investigation.