Panoramic characterization and structure-activity relationship of astragalus polysaccharides: Modulating M2-to-M1 repolarization via the TLR4-NF-κB pathway.

Over 30 Astragalus polysaccharides have been reported, yet most studies focus on individual structures with inconsistent findings, which hinders structure-activity relationship (SAR) analysis and the association between efficacy and active substances. Here, Astragalus polysaccharides were characterized at three levels: crude polysaccharides (ARCP), neutral (ARN) and acidic (ARA) fractions, and purified homogeneous polysaccharides (ARNPs and ARAPs). Their M2-to-M1 repolarization effects, mechanisms, and SAR were evaluated. ARN and ARA accounted for 94% of ARCP in an approximately 1:1 ratio, with ARN primarily comprising low-molecular-weight (5.1-110.9 kDa) polysaccharides and ARA enriched in high-molecular-weight components (17.3-470.5 kDa). Four low-branched α-1,4-glucans were obtained from ARN, whereas two pectins from ARA were rich in homogalacturonan and differed in degree of methyl esterification (DM: 72% vs 23%). Both neutral (ARNPs) and acidic (ARAPs) purified polysaccharides significantly promoted M2-to-M1 repolarization. SAR revealed that medium-molecular-weight ARNP-2 (14.7 kDa) showed the strongest activity among ARNPs, while the C-6 carboxyl group was essential for ARAP activity, with low-DM ARAP-2 being more potent. Mechanistically, both activated the TLR4-NF-κB pathway, generating tumor-suppressive M1 macrophages. This study provides a panoramic structural profile of Astragalus polysaccharides, elucidates their activity, mechanisms in M2-to-M1 re-polarization and SAR, and offers a scientific basis for their further development.