Binding of the polysaccharide from Acanthopanax giraldii Harms to toll-like receptor 4 activates macrophages

Abstract Ethnopharmacological relevance The traditional Chinese medicine, Acanthopanax giraldii Harms, is commonly used to treat arthralgia due to wind, cold and dampness, as well as weakness in the feet and knees. Its other reported effects include eliminating flatulence, strengthening muscles and bones, and delaying aging. The polysaccharides in A. giraldii Harms are the major bioactive substances that confer the herb's antioxidant properties as well as anticancer and antiviral effects. Aims of the study To elucidate the underlying mechanism and signaling cascade involved in the homogeneous A. giraldii Harms polysaccharide II (AHP–II)–mediated immunomodulation of mice macrophages. Materials and methods The phagocytosis of neutral red and the production of nitric oxide, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), were measured to determine AHP–II–induced macrophage activation. Confocal microscopy and flow cytometry were used to confirm the binding of AHP-II to macrophages. The involvement of Toll-like receptor (TLR) 4 in AHP–II–induced macrophage activation was demonstrated using antibody blocking and macrophages from C3H/HeJ TLR4-mutant mice. Western blotting was used to map AHP–II–induced downstream signaling pathways. Results AHP-II increased the phagocytosis of macrophages and the release of nitric oxide, IL-6 and TNF-α cytokines. Direct, saturable and reversible binding of AHP-II to macrophages was observed, while it can be inhibited by the anti-TLR4 antibody. In addition, the presence of the anti-TLR4 antibody inhibited AHP–II–induced macrophage IL-6 and TNF-α production in the peritoneal macrophages of C3H/HeJ mice. Moreover, AHP–II–TLR4-stimulated macrophages activate the downstream intracellular ERK and JNK/nuclear factor (NF)-κB signaling pathways. In addition, the AHP–II–mediated regulation of IL-6 and TNF-α production from macrophages was greatly affected by specific ERK, JNK and NF-κB inhibitors. Conclusion Our study elucidated the immunomodulatory mechanism of AHP-II in macrophage activation and identified TLR4 as the main receptor coordinating AHP-II binding. Our findings suggest AHP-II may be used as a novel immunopotentiator for medical purposes.