Integrated network pharmacology, mass cytometry and multi-omics analysis the effect of Jingfang granule on intestinal immune disorder in mice with cold-dampness syndrome

The pathogenesis of cold-dampness syndrome (CDS) is closely related to intestinal inflammation and immune disorders induced by cold-dampness pathogen. CDS is the root cause of a variety of chronic inflammatory and immune diseases. Jingfang granule (JF) was widely used to treat a variety of diseases closely related to CDS. JF is well known for its clinical effect of dispelling cold and eliminating dampness, but the pharmacological effect and mechanism of JF on the improvement of CDS are still unclear. This study aimed to explore the efficacy and mechanism of JF in improving CDS from the perspective of intestinal immunity. In this study, mass spectrometry (CyTOF), metabolomics, network pharmacology, proteomics and molecular biology experiments were performed to investigate the therapeutic effects and underlying mechanisms of JF on intestinal inflammation and immune disorders in CDS mice. These results showed that JF could improve the clinical symptoms and increase the thymus index of CDS mice. Most strikingly, JF ameliorated intestinal inflammation and immune disorders in CDS mice, as indicated by increased frequency of TH1, CD8 + Tem, CD8 + TEFF, gdT and iNK cells and decreased frequency of Naive B cells, M1-macrophages, DCs and eosinophils. Metabolomics results showed that JF reversed the content of docosahexaenoic acid, arachidonic acid, linoleic acid, inosine and hypoxanthine in CDS mice. Correlation analysis showed that these metabolites were strongly correlated with a variety of intestinal immune cells, indicating that there was a certain regulatory effect between them. Then, 271 JF targets, 316 metabolite targets and 18374 disease targets were integrated to obtain 75 common targets and 138 pathways (such as PI3K/ AKT and MAPK pathway, etc). Furthermore, molecular docking, proteomics and western blotting demonstrated that PI3K/AKT signaling pathway might be the key molecular mechanism by which JF regulated intestinal immune disorders in CDS mice. These results suggested that JF may act on the PI3K/AKT pathways to further regulate the levels of metabolites to exert intestinal immunomodulatory effects. In summary, we confirmed the beneficial effects of JF on intestinal immune disorders in CDS mice.