Immunomodulatory mechanism of Radix Bupleuri polysaccharide on RAW264.7 cells and cyclophosphamide-induced immunosuppressed mice

Radix Bupleuri polysaccharides (RBP) are heteropolysaccharides with a molecular weight distribution ranging from 1.10 to 121.21 kDa, extracted from the traditional Chinese medicinal herb Radix Bupleuri . Using DEAE-cellulose column chromatography, one neutral polysaccharide (RBP-1) and two acidic polysaccharides (RBP-2 and RBP-3) were isolated. These polysaccharides exhibited the capability to activate RAW264.7 macrophages, with the acidic fractions demonstrating greater effects than the neutral ones. RNA sequencing revealed that differentially expressed genes (DEGs) were significantly enriched in MAPK and NF-κB signaling pathways following RBP-3 treatment. Mechanistic studies further demonstrated that RBP-3 activates these pathways through toll-like receptors TLR2 and TLR4. Additionally, RBP effectively alleviated immunosuppression by modulating gut microbiota and restoring the integrity of the intestinal mucosal barrier in cyclophosphamide (CTX)-induced immunosuppressed mice. RBP influenced the generation of colonic short-chain fatty acids (SCFAs), regulated CD4+ T cell differentiation, and enhanced intestinal immune function. These findings highlight RBP potential as a natural immunomodulator and establish a theoretical foundation for the formulation of immune-supportive botanical products derived from Radix Bupleuri .