【作者】 徐歆；

【导师】 李卫芬；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 紫锥菊(Echinacea purpurea)是近年来备受关注的一种安全、高效多功能药用植物,其具有强大的免疫调节活性。巨噬细胞和树突状细胞(dendritic cell, DC)同为机体免疫系统的重要成员,前者具有吞噬、分泌、组织修复、抗原递呈等多种功能,后者为专职抗原提呈细胞并能分泌多种细胞因子,两者均在非特异性免疫(先天性免疫)和特异性免疫(获得性免疫)中有重要作用。Toll样受体(Toll-like receptor, TLR)主要表达于巨噬细胞和DC,该类受体在识别配体后,既能够通过激活炎症因子和I型IFN分泌,启动非特异性免疫反应,又可以通过促进DC成熟并递呈抗原,启动特异性免疫反应。因此,本研究拟先以小鼠为研究材料,探讨紫锥菊提取物(Echinacea purpurea extract, EE)对其机体免疫功能和抗沙门氏菌感染能力的影响,再以小鼠巨噬细胞和DC为研究对象,研究EE对两者免疫功能的影响以及TLR信号通路在此免疫调节活动中的作用,最终结合两方面的研究结果,阐明EE调控小鼠细胞和机体免疫功能以及抗沙门氏菌感染能力的机理。主要研究内容及结果如下：(一)EE对小鼠机体免疫功能和抗沙门氏菌感染能力的影响50只体重相近的C57BL/6雌性小鼠被随机分为5组,对照组、EE组、鼠伤寒沙门氏菌(Salmonella enterica serovar Typhimurium, ST)组、EE+ST组和庆大霉素(gentamycin, GM)+ST组,每组10只。对照组、EE组分别以PBS和EE (10mg)连续灌胃17天,研究EE对正常小鼠机体免疫功能的影响；ST组、EE+ST组和GM+ST组分别以PBS、EE (10mg)和PBS连续灌胃17天,并于第15天再灌胃5×10~8CFU ST感染小鼠,研究EE对小鼠机体抗沙门氏菌感染能力的影响。结果如下：EE对正常小鼠机体免疫功能的影响：(1)EE对正常小鼠生长和肠粘膜结构无显著影响,说明其对小鼠机体具有良好的安全性；(2)EE可显著提高小鼠机体免疫功能,提高其脾脏指数；(3)EE可在一定程度上可增强小鼠机体的抗体分泌功能,增加小肠分泌型IgA (secretory IgA, sIgA)分泌,但对IgG分泌没有影响；(4)EE可增强小鼠机体的非特异性免疫功能,显著上调小鼠血液、回肠、结肠、脾脏、肠系膜淋巴结(mesenteric lymph node, MLN)和肝脏中促炎细胞因子白介素(interleukin, IL)-6、IL-12、肿瘤坏死因子(Tumor necrosis factor, TNF)-α、干扰素(interferon, IFN)-γ和抑炎细胞因子IL-10的表达并显著增加其回肠和结肠的NO产生。EE对小鼠机体抗沙门氏菌感染能力的影响：(1)EE可缓解ST感染对小鼠机体和免疫系统的损伤,消除鼠伤寒沙门氏菌(Salmonella enterica serovar Typhimurium, ST)感染引起的小鼠体重下降,大幅减轻ST感染引起的肠粘膜损伤以及脾脏肿大和CD4+/CD8+值下降；(2)EE可降低肠粘膜的ST感染程度,显著减少回肠和结肠粘膜的中性粒细胞浸润以及ST在结肠的定植和ST向肝脏和脾脏中的移位；(3)EE可增强ST感染小鼠机体的抗体分泌功能,显著促进IgG和sIgA分泌；(4)EE可增强ST感染小鼠机体的非特异性免疫功能,显著上调血液、脾脏、MLN和肝脏中促炎细胞因子IL-6、IL-12、TNF-α、IFN-y和抑炎细胞因子1L-10的表达并显著增加其回肠和结肠的NO产生。以上结果提示,EE能够通过增强小鼠的机体免疫功能,提高其抗感染能力。(二)EE对小鼠巨噬细胞免疫功能的影响本研究分别以200μg/mL和100μg/mL EE处理RAW264.7小鼠巨噬细胞系和骨髓源巨噬细胞(bone marrow-derived macrophage, BMDM),研究EE对两者免疫功能的影响。结果如下：EE对RAW264.7小鼠巨噬细胞系免疫功能的影响：(1)EE对RAW264.7细胞的安全浓度为0-200μg/mL;(2) EE可激活RAW264.7细胞,显著提高酸性磷酸酶(acid phosphatase, ACP)和乳酸脱氢酶(Lactate dehydrogenase, LDH)活性；(3)EE可增强RAW264.7细胞的吞噬功能；(4)EE可增加RAW264.7细胞的细胞因子分泌,显著上调促炎因子IL-1β、IL-6、IL-12、TNF-α、IFN-γ抗炎因子IL-10、转化生长因子(transforming growth factor, TGF)-β1和抗病毒细胞因子IFN-p的基因表达和分泌；(5)EE可增强RAW264.7细胞的NO合成功能,显著提高诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的基因表达和活性以及NO产量。EE对小鼠BMDM免疫功能的影响：(1)EE对小鼠BMDM的安全浓度为0-100μg/mL;(2) EE可诱导小鼠BMDM发生M1型极化,显著上调M1型巨噬细胞标志基因IL-6、TNF-α、IFN-γ、iNOS和表面标志CD197的表达；(3)EE可活化小鼠BMDM,并增强其抗原递呈能力,显著提高ACP和LDH活性,上调巨噬细胞活化标志CD80、CD86和MHC-II的表面表达；(4)EE可增强小鼠BMDM的吞噬功能；(5)EE可增加小鼠BMDM的细胞因子分泌,显著上调促炎因子IL-1β、IL-6、IL-12、TNF-α、IFN-γ、抑炎因子IL-10、TGF-β1和抗病毒细胞因子IFN-p的基因表达和分泌；(6)EE可提高小鼠BMDM的NO合成功能,提高iNOS基因表达和活性及NO产量；(7)EE可调控TLR及其衔接分子MyD88、 TRIF的基因表达,并激活信号分子ERK、JNK、p38MAPK及NF-κB,以调节小鼠BMDM的细胞因子分泌和NO合成。其中TLR-ERK通路介导了EE诱导的IL-1β,TNF-a和TGF-β1基因表达和分泌,TLR-JNK通路介导了EE诱导的IL-1β、 IL-6、IL-12、TNF-α、IFN-γ、IL-10和TGF-β1基因表达和分泌,TLR-p38MAPK通路介导了EE诱导的IL-1β、IL-6、IL-12、IFN-γ、TGF-β1和IFN-β基因表达和分泌,TLR-NF-κB通路介导了IL-6、IL-12、IFN-γ、IL-10、TGF-β1和IFN-β基因表达和分泌,同时,TLR-JNK通路还介导了iNOS的表达以及NO产生。以上结果提示,EE不仅能够激活小鼠巨噬细胞并增强其免疫功能,而且可以通过TLR信号通路调控其细胞因子分泌和NO合成。(三)EE对小鼠DC免疫功能的影响本研究分别以400μg/mL EE处理小鼠骨髓源DC (bone marrow-derived DC, BMDC),研究EE对其免疫功能的影响。结果如下：(1)EE对小鼠BMDC的安全浓度为0-400μg/mL;(2) EE可促进小鼠BMDC成熟,并增强其抗原递呈功能,显著上调DC成熟标志CD40、CD80、CD83、CD86和MHC-II的表面表达；(3)EE降低小鼠BMDC抗原摄取能力,降低其吞噬功能和ACP活性；(4)EE可增加小鼠BMDC的细胞因子分泌,上调促炎因子IL-1β、IL-6、IL-12、TNF-α、 IFN-γ、抑炎因子IL-10、TGF-β1和抗病毒细胞因子IFN-β的基因表达和分泌；(5)EE可调控TLR及其衔接分子MyD88、TRIF的基因表达,并激活信号分子ERK、JNK、p38MAPK及NF-κB,以调节小鼠BMDM的细胞因子分泌。其中TLR-ERK通路介导了EE诱导的IL-1β、IL-6、TNF-α、IFN-γ、IL-10和TGF-β1基因表达与分泌,TLR-JNK通路介导了EE诱导的IL-1β、IFN-y和IFN-β基因表达与分泌,TLR-p38MAPK通路介导了EE诱导的IL-1β、TNF-α、IL-10、TGF-β1和IFN-β基因表达与分泌,TLR-NF-κB通路介导了EE诱导的IL-12、IFN-γ、TGF-β1和IFN-β基因表达与分泌。以上结果提示,EE不仅能够促进小鼠DC成熟并增强其免疫功能,而且可以通过TLR信号通路调控其细胞因子分泌。综上所述,EE能够激活小鼠巨噬细胞并诱导其发生M1型极化,促进小鼠DC成熟,进而增强两者免疫功能,并通过提高小鼠机体免疫功能,增加其抗沙门氏菌感染能力。更多还原

【Abstract】 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.更多还原