Study On Effects Of Echinacea Purpurea Extract On Immunity And Anti-Salmonella Infection Ability Of Mice And Its Mechanism

Echinacea purpurea is a safe, efficient herb with multiple biological activities, such as immunomodulation, anti-inflammation, anti-oxidation, anti-bacteria, anti-fung, anti-virus and anti-cancer, and attracts much attention widely in recent years. Macrophages and dendritic cells (DCs) are both crucial members of immune system. Macrophages possess multiple biological functions, including phagocytosis, secretion, tissue repair and antigen presentation, whereas DCs are professional antigen presenting cells and can secrete a variety of cytokines. The above two cells both play important roles in innate immunity (non-specific immunity) and adaptive immunity (specific immunity). Toll-like receptors (TLRs) are mainly expressed in macrophages and DCs. After recognizing their ligands, these receptors can not only induce the secretion of inflammatory cytokines and type I IFN to trigger innate immune responses, but also enhance the maturation of DCs and their antigen presenting capacity to trigger adaptive immune responses. Therefore, the purpose of this study is to investigate the effects of EE on body immune functions and anti-Salmonella infection ability of mice, and explore the effects of Echinacea purpurea extract (EE) on the immune functions of murine macrophages and DCs, and the role of TLR signaling pathway in these immunomodulatory activities. Finally, the mechanism that EE modulates celluar and body immunity and anti-Salmonella infection ability of mice will be clarified. The main research content and results are listed as follows:1Effect of EE on body immune functions and anti-Salmonella infection ability of miceA total of fifty5-week old C57BL/6female mice with similar body weight were randomly divided into5groups, including control group, EE group, ST (Salmonella enterica serovar Typhimurium) group, EE+ST group and GM (gentamycin)+ST group, with10mice per group. The mice in control group and EE group were gavaged with PBS and EE respectively for17days to investigate the effect of EE on body immune functions of mice; The mice in ST group, EE+ST group and GM+ST group were gavaged with PBS, EE and PBS respectively for17days and infected with5×108CFU ST also by gavaging on the15th day, to investigate the effect of EE on anti-Salmonella infection ability of mice. The results are listed as follows:Effect of EE on body immune functions of mice:(a) EE had no effects on the body weight and intestinal mucosa structure of mice, indicating that EE is safe for the body of mice;(b) EE could significantly increase the spleen index of mice, indicating that EE could enhance their body immune immunity;(c) EE could significantly stimulate sIgA secretion in the small intestine of mice, but had no effect on IgG secretion, indicating that EE could enhance the antibody secretion in mice to some extent;(d) EE could not noly significantly upregulate the expression of proinflammatory cytokines interleukin (IL)-6, IL-12, tumor necrosis factor (TNF)-a, interferon (IFN)-y and anti-inflammatory cytokine IL-10in the blood, ileum, colon, spleen, mesenteric lymph node (MLN) and liver of mice, but also increase NO production in their ileal and colonic mucosa, indicating that EE could enhance the non-specific immune functions of mice.Effect of EE on anti-Salmonella infection ability of mice:(a) EE could eliminate the weight loss of mice caused by Salmonella enterica serovar Typhimurium (ST) infection, and significantly reduce the intestinal mucosa damage, splenomegaly and the decrease of the ratio of splenic CD3+CD4+cells to CD3+CD8+cells in mice caused by ST infection, indicating that EE could mitigate the body and immune system damage of mice caused by ST;(b) EE could significantly reduce the neutrophil infiltration in the ileal and colonic mucosa of ST-infected mice, the ST colonization in their colon and the translocation of ST to their liver and spleen, indicating that EE could reduce ST infection extent in the intestinal mucosa of ST-infected mice;(c) EE could significantly stimulate IgG and sIgA secretion in mice, indicating that EE could enhance the antibody secretion in ST-infected mice;(d) EE could not noly significantly upregulate the expression of proinflammatory cytokines IL-6, IL-12, TNF-a, IFN-y and anti-inflammatory cytokine IL-10in the blood, spleen, MLN and liver of ST-infected mice, but also increase NO production in their ileal and colonic mucosa, indicating that EE could enhance the non-specific immune function of ST-infected mice.The above results suggested that EE could enhance the body immune functions of mice, and increase their anti-infection ability.2Effect of EE on immune functions of murine macrophagesIn this study, RAW264.7murine macrophage cell line and bone marrow-derived macrophages (BMDMs) were treated with200μg/mL and100μg/mL EE respectively to investigate the Effects of EE on the immune functions of both cells. The results are listed as follows:Effect of EE on immune functions of RAW264.7murine macrophage cell line:(a) The safe concentration of EE for RAW264.7cells range from0μg/mL to200μg/mL;(b) EE could significantly increase the activities of macrophage marker enzymes ACP and LDH, indicating that EE could activate RAW264.7cells;(c) EE could significantly enhance the phagocytic function of RAW264.7cells;(d) EE could significantly upregulate the gene expression and secretion of proinflammatory cytokines IL-1β,IL-6, IL-12, TNF-a, IFN-y, anti-inflammatory cytokines IL-10, transforming growth factor (TGF)-β1and antiviral cytokine IFN-β, indicating that EE could enhance cytokine secretion in RAW264.7cells;(e) EE could significantly induce iNOS gene expression and elevate its activity, and increase NO production, indicating that EE could enhance NO synthesis in RAW264.7cells.Effect of EE on immune functions of murine BMDMs:(a) The safe concentration of EE for murine BMDMs range from0μg/mL to100μg/mL;(b) EE could upregulate the expression of M1macrophage marker genes IL-6, TNF-a, IFN-y, iNOS and surface marker CD197, indicating that EE could induce the M1polarization of murine BMDMs;(c) EE could significantly increased the activities of macrophage marker enzymes ACP and LDH, and upregulate the surface expression of macrophage activation markers CD80, CD86and MHC-II, indicating that EE could activate murine BMDMs and enhance their antigen-presenting capacity;(d) EE could significantly enhance the phagocytic function of murine BMDMs;(e) EE could significantly upregulate the gene expression and secretion of proinflammatory cytokines IL-1β, IL-6, IL-12, TNF-a, IFN-γ, anti-inflammatory cytokines IL-10, TGF-β1and antiviral cytokine IFN-β, indicating that EE could enhance cytokine secretion in murine BMDMs;(f) EE could significantly induce iNOS expression and elevate its activity, and increase NO production, indicating that EE could enhance NO synthesis in murine BMDMs (g) EE could modulate the gene expression of TLRs and their adaptors MyD88, TRIF, and activate signaling molecules ERK, JNK, p38MAPK and NF-κB, to modulate cytokine secretion in murine BMDMs:TLR-ERK pathway mediated EE-induced IL-1β, TNF-a and TGF-β1gene expression and secretion, TLR-JNK pathway mediated EE-induced IL-1β, IL-6, IL-12, TNF-a, IFN-y, IL-10and TGF-β1gene expression and secretion, TLR-p38MAPK pathway mediated EE-induced IL-1β, IL-6, IL-12, IFN-y, TGF-β1and IFN-β gene expression and secretion, TLR-NF-κB pathway mediated EE-induced IL-6, IL-12, IFN-y, IL-10, TGF-β1and IFN-β gene expression and secretion; Meanwhile, TLR-JNK pathway also mediated EE-induced iNOS expression and NO production.The above results suggested that EE could not only activate murine macrophages and enhance their immune functions, but also modulate their cytokine secretion and NO synthesis via TLR signaling pathway.3Effect of EE on immune functions of murine DCsIn this study, murine bone marrow-derived DCs (BMDCs) were treated with400μg/mL EE to investigate the Effects of EE on their immune functions. The results are listed as follows:(a) The safe concentration of EE for murine BMDCs range from0μg/mL to400μg/mL;(b) EE could upregulate the surface expression of DC maturation markers CD40, CD80, CD83, CD86and MHC-II, indicating that EE could promote the maturation of murine BMDCs and enhance their antigen-presenting function;(c) EE could reduce the phagocytic function and ACP activaity of murine BMDCs, indicating that EE could downregulate their antigen uptake capacity;(d) EE could significantly upregulate the gene expression and secretion of proinflammatory cytokines IL-1β, IL-6, IL-12, TNF-a, IFN-y, anti-inflammatory cytokines IL-10, TGF-β1and antiviral cytokine IFN-P, indicating that EE could enhance cytokine secretion in murine BMDCs;(e) EE could modulate the gene expression of TLRs and their adaptors MyD88, TRIF, and activate signaling molecules ERK, JNK, p38MAPK and NF-κB, to modulate cytokine secretion in murine BMDCs:TLR-ERK pathway mediated EE-induced IL-1β, IL-6, TNF-a, IFN-y, IL-10and TGF-β1gene expression and secretion, TLR-JNK pathway mediated EE-induced IL-1β, IFN-y and IFN-P gene expression and secretion, TLR-p38MAPK pathway mediated EE-induced IL-1β, TNF-a, IL-10, TGF-β1IFN-β gene expression and secretion, TLR-NF-κB pathway mediated EE-induced IL-12, IFN-y, TGF-β1and IFN-β gene expression and secretion, indicating that EE could modulate cytokine secretion in murine BMDCs through TLR signaling pathways. The above results suggested that EE could not only promote the maturation of murine DCs and enhance their immune functions, but also modulate their cytokine secretion via TLR signaling pathway.In conclusion, EE could induce M1macrophage polarization and DC maturation to enhance the immune functions of both cells, and enhance the body immune functions of mice to increase their anti-Salmonella infection ability.