The Immunomodulatory Effects Of Glycyrrhizin And Its Mechanisms Against Salmonella Infection In Mice

Glycyrrhizin (GL), a major biologically active constituent of licorice root accounting for the sweet taste, is a potent immune modulator with the ability to stimulate innate immune response. The aim of this research was to investigate the effects of GL on host immnue responses against Salmonella enterica serovar Typhimurium (ST) infection in mice, and further explore the impacts on murine DC maturation and macrophages polarization. Additionally, we further studied the immunopotentiating effects of GL on chicken macrophages. The main research content and results are listed as follows:1 Effect of GL on mice immune functions and its ability to anti-Salmonella infectionWe first determined the in vitro antibacterial activity of GL against ST using radial diffusion assay and bacterial growth kinetics, and found that 50-200 μg/mL GL affected neither the growth nor the proliferation of ST. Meanwhile, RT-PCR analysis showed that 200 μg/mL GL had no effect on the expression of key virulence genes InvA, SopD, SipB, HilA and ssrB within 4 h, indicating that GL could not inhibit ST in vitro.We further analyzed the effect of GL on mice immune functions. The data showed that: (a) 80 mg/kg GL had no effects on the growth, intestinal mucosa and liver structure of mice; (b) GL significantly enhanced the production of slgA in ileum, NO in intestinal mucosa, IFN-γ, IL-12, IL-6 and IL-10 in intestinal mucosa, serum, and spleen, and upregulated the gene expression of IFN-y and IL-12 in liver; (c) GL significantly increased the spleen index of mice; (d) GL increased the relative abundance of Lactobacillus, Desulfovibrio, Helicobacter, and Bilophila, while reduced Akkermansia, Bacteroides, and Anaerostipes in cecum. These data indicated that 80 mg/kg is safe for mice, which could improve gut microbiota and immune functions of mice.We also investigated the effect of GL on ST-infected mice, the results showed:(a) GL could improve the weight loss of mice caused by ST, and significantly reduce the intestinal mucosa damage; (b) GL significantly reduced ST colonization in ileum, colon and the translocation to liver and spleen; (c) GL decreased ST-induced IFN-y, TNF-a, and IL-6 production in ileum, serum, and spleen, and downregulated the expression of IFN-γ, TNF-α, and IL-6 in liver; (d) GL could increase IL-10 secretion in colon, serum, and spleen of ST-infected mice; (e) GL could ameliorate ST-induced liver and spleen damage, while decrease caspase-1 activity in liver, and suppress ST-induced apoptosis of liver cells; (f) GL reduced Akkermansia, Sutterella, Prevotella, and Coprococcus, while enriched Parabacteroides and Anaerotruncus in cecum of ST-infected mice. The results suggested that GL could suppress ST infection by improving gut microbiota and immune functions of mice.2 Effect of GL on phenotypic and functional maturation of murine BMDCsThe aim of this experiment was to investigate phenotypic and functional maturation of murine bone marrow-derived dendritic cells (BMDCs) using GL (25-200 μg/mL). These effects of GL on BMDCs were assessed with cytochemistry assay, flow cytometry (FCM), bio-assay, real-time PCR and enzyme linked immunosorbent assay (ELISA). We found that the safe concentration of GL for BMDCs ranges from 0 to 400 μg/mL. GL could induce phenotypic maturation as evidenced by an increased surface expression of CD40, CD80, CD83, CD86, and major histocompatibility complex-II (MHC-II). The functional tests showed that the activity of acidic phosphatase (ACP) inside BMDCs were downregulated after treatment with GL. Moreover, we found that GL increased the production of IL-12, IFN-y, TNF-a, IL-6, IL-10, and TGF-β in BMDCs, and upregulated the gene expression of TLR2, while downregulated TLR3 and TLR4, indicating that GL could activate TLR pathways. Finally, we found that NF-κB, ERK, and p38 MAPK were required for GL-induced IL-12 and IL-6 secretion, suggesting that NF-κB, ERK, and p38 MAPK pathways were involved in GL-induced BMDCs maturation.3 Effect of GL on macrophage polarization in murine BMDMsThe aim of this experiment was to investigate the effect of GL from licorice on macrophage polarization. Both phenotypic and functional activities of murine bone marrow-derived macrophages (BMDMs) treated by GL (100 ug/mL) were assessed with flow cytometry, real-time PCR, cytochemistry assay, western blotting and ELISA. Our results showed that GL obviously increased the cell surface expression of CD80, CD86 and MHC-II molecules. Meanwhile, GL upregulated the expression of CCR7 and the production of TNF-a, IL-12, IL-6, and NO (the markers of classically activated (M1) macrophages), whereas it downregulated the expression of MR, Yml, and Argl (the markers of alternatively activated (M2) macrophage). The functional tests showed that GL dramatically enhanced the uptake of FITC-dextran and E. coli K88 by BMDMs and markedly decreased the intracellular survival of E. coli K88 and S. typhimurium. Moreover, we demonstrated that JNK and NF-κB activation were required for GL-induced NO and M1-related cytokines production, while ERK 1/2 pathway exhibited a regulatory effect via induction of IL-10. Additionally, GL-treatment could also downregulate IL-4-induced Argl, FIZZ1, MR, Yml, PPAR-y, and KLF4 expression, while upregulate the expression of iNOS, TNF-a, IL-12p40, and IL-6. Together, these findings indicated that GL promoted polarization of M1 macrophages and enhanced its phagocytosis and bactericidal capacity. The results expanded our knowledge about the role of GL in macrophage polarization.4 Effect of GL on chicken HD11 macrophage cell lineSince macrophages play an essential role in controlling of Salmonella infection, the aim of this study was to evaluate the effect of GL from licorice on chicken macrophages (HD11). Our results showed that GL (100 μg/mL) increased the internalization of both FITC-dextran and ST by HD11 cells, and markedly decreased the intracellular survival of ST when compared to control group. Meanwhile, iNOS and NOX-1 expression were induced by GL and thereby enhanced the production of NO and H2O2 in HD11 cells. Gene expression analysis showed that GL also upregulated the expression of cell surface molecules (CD40, CD80, CD83, and CD197) and cytokines IFN-γ, IL-6, IL-10, which were associated with the activation of macrophages. These findings demonstrated that GL exhibits a potent immune modulatory effect to activate chicken macrophage and enhance its Salmonella-killing capacity.In conclusion,50-200 μg/mL GL could not suppress the growth and proliferation of ST in vitro, while it could enhance the ability of mice to resist Salmonella challenge by improving gut microbiota, inducing DC maturation and M1 macrophage polarization. Moreover, we demonstrated that GL could activate chichen HD11 macrophage cells with enhanced phagocytosis and Salmonella-killing capacity.