Glycyrrhiza uralensis polysaccharide GiP-B1 activates macrophage RAW264.7 via TLR4/MyD88/NF – κ B signaling pathway
The innate immune system is an important defense line for the body to resist pathogen invasion, and Toll like receptors (TLRs), as well as antigen-presenting cells such as macrophages and dendritic cells expressing TLRs, are important components of the innate immune system. Research on the involvement of TLRs in macrophage immune recognition, regulation of signal transduction, and initiation of immune response has shown that all members of the TLR family, except TLR3, can recognize ligands such as lipopolysaccharides (LPS), bind to myeloid differentiation factor 88 (MyD88), recruit relevant kinases, and activate the kinase inhibitor of nuclear factor kappa B (IKK), which phosphorylates the inhibitor of NF – κ B (I κ B) that binds to NF – κ B. And dissociate, activate NF – κ B into the nucleus, initiate or inhibit the transcription of target genes, thereby regulating the innate immune response.
Polysaccharides, as an effective class of biological response regulators, activate macrophage immune responses through mechanisms related to TLRs/NF – κ B signaling pathway transduction. Research has found that many plant polysaccharides can specifically bind to receptors such as TLR2/4 on the surface of macrophages, activate the NF – κ B signaling pathway, regulate the transcription of cytokines and the expression of co stimulatory molecules in phagocytic cells, and initiate downstream cascades to activate innate and acquired immunity.
Glycyrrhiza inflata Bat. is a characteristic medicinal plant resource in Xinjiang. Its dried roots and rhizomes have been included in the Chinese Pharmacopoeia as medicinal herbs. Its crude polysaccharide GiP and purified components have been confirmed by our research group to promote the proliferation of macrophages and lymphocytes. The purified component GiP-B1, which has a high content, is an acidic polysaccharide with a poly Rha GalA main chain, with a relative molecular weight greater than 2.0 × 106Da and a uronic acid content of 16.8%. It can significantly promote the proliferation and phagocytosis of RAW264.7 macrophages, increase the secretion of IL-1 β and TNF – α by macrophages, and promote the generation of NO and NOS. The research team further used monoclonal antibody anti-TLR4 to intervene in macrophages and found that the secretion of TNF – α, IL-1 β, 1L-6, and NO induced by GiP-B1, as well as the expression of iNOS, 1L-1 β, TNF – α, and 1L-6 mRNA, were inhibited to varying degrees, suggesting that GiP-B1 activation of macrophages may be related to TLR4 and its related signaling pathways. In order to investigate the relationship between GiP-B1 activated macrophages and the TLR4/NF – κ B signaling pathway, this study transfected RAW264.7 macrophages with TLR4 interfering RNA (TLR4 siRNA) in vitro to explore whether GiP-B1 intervention can activate macrophages through the TLR4 mediated NF – κ B signaling pathway, in order to elucidate the molecular mechanism of GiP-B1 activated macrophages.

Macrophages are important executors of innate immunity in the body. As specialized tissue phagocytic cells, they can recognize conserved pathogen associated molecular patterns (PAMPs) on the surface of pathogenic microorganisms through pattern recognition receptors (PRRs) such as TLRs expressed on their surface. Through downstream signaling pathways, they regulate the expression of various immune response genes to clear pathogens. Research has shown that many natural plant polysaccharides can serve as ligands for TLR4, facilitating macrophage signaling through both MyD88 dependent and non dependent pathways, promoting NF – κ B entry into the nucleus and binding to specific recognition sequences on DNA promoters, mediating the expression of cytokines such as IL-1, IL-6, TNF – α, etc. These cytokines also act as stimulators of NF – κ B, further activating NF – κ B to cause sustained inflammatory responses and activate adaptive immune responses.
As a source plant of traditional Chinese medicine licorice, the main medicinal substances of swollen fruit licorice include not only chalcones with anticancer activity, but also polysaccharides with antioxidant, alpha glucosidase inhibitory activity, and immune regulatory activity. In the preliminary research of our research group, it was found that the crude polysaccharide GiP and its purified components of Glycyrrhiza uralensis have the effect of promoting macrophage and lymphocyte proliferation. Among them, the purified component GiP-B1 with higher content has a significant promoting effect on the proliferation, phagocytosis, and cytokine secretion of RAW264.7 macrophages. The preliminary research group used anti-TLR4 to treat cells and found that its mechanism of action is related to the activation of the TLR4/NF – κ B signaling pathway after sample intervention. This article uses siRNA technology to silence the TLR4 gene through TLR4 siRNA, and finds that GiP-B1 promotes the proliferation of RAW264.7 macrophages and inhibits the secretion of cytokines TNF – α, IL-1 β, and IL-6. This indicates that TLR4 is involved in GiP-B1’s effect on RAW264.7 macrophage proliferation and cytokine secretion, that is, GiP-B1 activation of macrophages is related to TLR4, which is consistent with the results of previous antibody blockade experiments. In addition, based on the types of cytokines secreted by macrophages activated by GiP-B1 through TLR4, such as TNF – α, IL-1 β, IL-6, etc., it suggests that GiP-B1 activation of macrophages can induce polarization towards M1 type. M1 type macrophages not only promote inflammatory responses, but also have antigen presentation abilities, which can activate adaptive immune responses of T cells, induce Th1 cell immune responses, and exert host immune clearance functions against pathogens and tumor cells. Previous studies have also found that GiP-B1 has certain in vitro anti-tumor effects. Whether its mechanism of action is related to GiP-B1 promoting macrophage polarization and whether it can promote the body’s anti-tumor immune function by regulating macrophages in the tumor microenvironment still needs further in-depth research.
In order to further demonstrate the relationship between GiP-B1 activation of macrophages and the TLR4/NF – κ B pathway, this experiment also studied the mRNA and protein expression changes of upstream regulatory genes TLR4, dependent molecule MyD88, downstream receptor genes NF κ Bp65, I κ B α, etc. in macrophages regulated by GiP-B1 under TLR4 siRNA intervention. The results showed that due to the silencing of the TLR4 gene, the expression level of TLR4 mRNA decreased, the content of free NF – κ Bp65 in the nucleus significantly decreased, and the activation of the NF – κ B pathway also decreased accordingly; GiP-B1 not only increases the expression of TLR4 gene in macrophages under normal conditions, upregulates the mRNA and protein expression of TLR4 signaling dependent molecule MyD88 and downstream genes NF – κ Bp65 and p-I κ B α, but also upregulates the mRNA and protein expression of TLR4 and MyD88, NF – κ Bp65, and p-I κ B α caused by RNA interference, further proving that GiP-B1 can act as one of the TLR4 ligands, activate the TLR4/NF – κ B signaling pathway dependent on MyD88, activate nuclear transcription factor p65 in the NF – κ B family, leading to phosphorylation degradation of I κ B and dissociation from NF – κ B, and promote NF – κ B. Transferring from the cytoplasm to the nucleus, binding to specific DNA sequences, regulating the transcription of inflammatory cytokines, cell proliferation (anti apoptosis) and other genes, participating in inflammatory responses, immune responses, etc. GiP-B1 activates macrophages through the TLR4/MyD88/NF – κ B signaling pathway, which is almost identical to the pathway of action of polysaccharides in the aforementioned studies.
In summary, GiP-B1 can regulate the immune function of macrophages RAW264.7 through the TLR4/NF – κ B signaling pathway, and its regulatory effect is mainly achieved through the MyD88 dependent pathway. This experiment provides experimental evidence for the activation of macrophages by Glycyrrhiza uralensis polysaccharides, which helps to elucidate the mechanism of action of Glycyrrhiza uralensis polysaccharides in inflammatory response, immune response, and other aspects.