Galactose-rich polysaccharides from Ganoderma lucidum have emerged as promising immunomodulatory agents, yet their structural characteristics and mechanisms of action remain poorly understood. Here, we isolated and characterized a novel galactose-rich polysaccharide (GLP-1b, 16.79 kDa) from Ganoderma lucidum. The analysis revealed a unique structure comprising an α-1,6-linked galactan backbone with C-2/C-3 acetylation, and complex branching with diverse terminal residues including T-α-L-Fuc-(-4-OAc), T-α-D-Man, and T-α-D-Glc. In vitro studies demonstrated that GLP-1b enhanced macrophage function through TLR4-dependent activation of NF-κB and MAPK signaling pathways, resulting in increased NO production and pro-inflammatory cytokine secretion. In a cyclophosphamide-induced immunosuppression mouse model, GLP-1 administration significantly restored immune homeostasis by normalizing CD4+/CD8+ T cell ratios, elevating CD19+ B cell percentages, enhancing immunoglobulin production, and maintaining intestinal barrier integrity. Analyses revealed concurrent changes in immune parameters, gut microbiota composition (including Lactobacillus and Alistipes populations), and metabolic profiles Metabolomics identified key changes in immune-supportive metabolites, including phospholipids and TCA cycle intermediates, as well as modulation of bile acid pathways. These findings demonstrate that GLP-1b exhibits immunomodulatory activities both in vitro and in vivo, with immune effects that coincide with changes in gut microbiota and host metabolism. These observations provide insights for developing polysaccharide-based immunotherapeutics.
