甘草酸苷对小鼠免疫功能的调节作用及其抗沙门氏菌感染的机理研究

发布时间：2018-01-12 13:14

  本文关键词：甘草酸苷对小鼠免疫功能的调节作用及其抗沙门氏菌感染的机理研究　出处：《浙江大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 甘草酸苷 小鼠 沙门氏菌 巨噬细胞 树突状细胞 极化 成熟

【摘要】：甘草酸苷(glycyrrhizin, GL)是甘草根最主要的活性成分和甜味来源,具有显著的免疫调节活性。本研究首先以小鼠为研究对象,通过体内实验探究GL对小鼠免疫功能及抗沙门氏菌感染能力的影响,随后利用体外细胞实验进一步探讨了GL对小鼠树突状细胞(dendritic cell, DC)功能成熟和巨噬细胞极化的影响,旨在阐明GL调控小鼠免疫及其抗沙门氏菌感染的作用机理；同时通过体外实验研究GL对鸡巨噬细胞免疫及清除胞内沙门氏菌能力的影响验证其免疫调节的作用效果,主要研究结果如下：(一)GL对小鼠免疫功能和抗沙门氏菌感染能力的影响GL在体外对鼠伤寒沙门氏菌(Salmonella enterica serovar Typhimurium, ST)生长和毒力基因表达的影响：50-200 μg/mL GL对ST均无抑菌活性,且200 μg/mL GL与ST共培养1 h、2 h和4h对ST毒力基因InvA、SopD、SipB、HilA和ssrB的表达无显著影响,提示GL在体外不能直接抑制ST的生长、增殖和主要毒力基因的表达。GL对正常小鼠免疫功能的影响：(1)口服80 mg/kg GL在21天内对小鼠生长及回肠、结肠和肝脏组织结构无显著影响;(2)GL可显著上调小鼠回肠中sIgA的分泌,促进肠粘膜NO的合成,并提高肠粘膜、血清和脾脏中IFN-γ、IL-12、IL-6和IL-10的含量,上调肝脏中IFN-γ和IL-12的基因表达；(3)GL可提高小鼠的脾脏指数；(4)GL可上调小鼠盲肠中乳杆菌属(Lactobacillus)、脱硫弧菌属(Desulfovibrio) 、螺杆菌属(Helicobacter)和嗜胆菌属(Bilophila)的相对丰度,降低Akkermansia、拟杆菌属(Bacteroides)和Anaerostipes的相对丰度。以上结果表明,口服80 mg/kg GL对小鼠具有良好的安全性,且可改善小鼠肠道的菌群结构,增强肠粘膜和机体免疫功能。GL对小鼠抗ST感染能力的影响：(1)GL预防给药可显著缓解ST感染(4×108CFU/只)引起的小鼠体重下降,改善回肠和结肠的粘膜损伤；(2)GL可显著降低小鼠回肠、结肠、脾脏和肝脏中ST的数量；(3)GL可显著降低ST感染引起的回肠、结肠、血清和脾脏中促炎性细胞因子IFN-γ、TNF-α和IL-6的含量,同时下调结肠中IL-12的分泌及肝脏中IFN-γ、TNF-α和IL-6的基因表达,而显著提高ST感染小鼠结肠、血清和脾脏中抑炎细胞因子IL-10的分泌；(4)GL可降低ST感染引起的脾脏和肝脏组织病变,显著下调肝脏caspase-1的活力,抑制ST引起的肝脏细胞凋亡；(5)GL可降低ST感染小鼠盲肠中Akkermansia、萨特氏菌属(Sutterella)、普氏菌属(Prevotella)和粪球菌属(Coprococcus)的相对丰度,提高Parabacteroides和Anaerotruncus的相对丰度。以上结果提示,GL可通过改善ST感染小鼠的肠道菌群结构、提高肠道和机体的免疫功能,从而抑制ST在肠道中的定植和向组织中的移位,改善ST感染引起的组织病变和细胞凋亡,使小鼠免受沙门氏菌感染。(二)GL对小鼠骨髓来源树突状细胞成熟的影响本研究分别以25200 μg/mL GL处理小鼠骨髓来源树突状细胞(bone marrow-derived DC, BMDC),研究GL对其表型和功能成熟的影响。结果发现：(1)GL对小鼠BMDC的安全浓度范围为0-400 μg/mL; (2) 25-200 μg/mL GL以浓度依赖的方式显著降低BMDC胞内ACP的活力；(3)GL可显著上调BMDC中成熟标志分子CD40、 CD80、CD83、CD86和MHC-Ⅱ的表面表达及细胞因子IL-12、IFN-γ、TNF-α、IL-6、 IL-10和TGF-β的基因表达和分泌；(4)GL可在12 h内显著上调BMDC中TLR2的基因表达,降低TLR3和TLR4的基因表达；(5)GL可在60 min内激活BMDC中NF-κB、ERK和p38 MAPK信号分子,并介导IL-12和IL-6的分泌。以上结果提示,GL能够通过TLR激活NF-κB、ERK和p38 MAPK信号通路,促进BMDC成熟,提高其抗原递呈能力和免疫功能。(三)GL对小鼠骨髓来源巨噬细胞极化的影响本研究以100 μg/mL GL处理小鼠骨髓来源巨噬细胞(bone marrow-derived macrophage, BMDM),研究GL对其极化、功能及信号通路的影响。结果发现：(1)GL对小鼠BMDM的安全浓度范围为0-100μg/mL;(2)GL可显著上调BMDM中CD80、CD86和MHC-II的表面表达,表明GL能够激活小鼠BMDM,增强其抗原递呈能力；(3)GL可显著上调BMDM中M1型巨噬细胞特征分子CCR7、TNF-α、IL-12和IL-6的表面表达或分泌,降低M2型巨噬细胞特征分子Ym1和MR的基因表达,提示GL能够诱导BMDM向M1型极化；(4)GL可在24 h内显著上调BMDM中iNOS的基因表达,下调Argl的表达,并诱导iNOS的蛋白表达和NO的合成,表明GL能够促进BMDM中NO的合成,提商细胞的抗感染能力；(5)GL可增强BMDM吞噬FITC-Dextran和E.coli K88的能力,并显著降低ST和E.coli K88在细胞内的存活,表明GL能够增强BMDM的吞噬、杀菌能力；(6)GL在60 min内不能激活BMDM中STAT1信号分子,可激活NF-κB、ERK、JNK和p38 MAPK信号分子,其中NF-κB和JNK介导了BMDM中M1型特征分子NO和IL-6的产生,而ERK介导了IL-10的分泌,表明NF-κB和JNK信号通路参与了GL诱导的M1型巨噬细胞极化,而ERK通路通过调节抗炎细胞因子IL-10的分泌对上述过程进行调控；(7)GL可显著下调IL-4诱导的M2型巨噬细胞特征分子Arg1、FIZZ1、MR和Ym1的基因表达,提示GL能够抑制IL-4诱导的M2型巨噬细胞的产生；(8)GL可显著上调IL-4诱导的M2型巨噬细胞表达M1型特征分子iNOS、TNF-α、IL-12p40和IL-6,提示GL可促进IL-4诱导的M2型巨噬细胞向M1型转化；(9)GL可显著下调IL-4诱导的M2型极化相关信号分子PPAR-γ和KLF4的基因表达,提示GL可能通过降低PPAR-γ和KLF4的表达,抑制IL-4诱导的M2型巨噬细胞极化。(四)GL对鸡HD11巨噬细胞系免疫功能的影响本研究以100μg/mL GL处理鸡HD11巨噬细胞系,研究GL对其免疫功能的激活情况。结果如下：(1)GL对鸡HD11巨噬细胞系的安全浓度范围为0-400 μg/mL; (2) GL可显著增强HD11细胞吞噬FITC-Dextran和ST的能力,显著降低胞内ST数量；(3)GL在12h内可显著上调HD11细胞表面分子CD40、CD80、CD83和CD197及细胞因子IFN-γ、IL-6和IL-10的基因表达；(4)GL可显著提高细胞中iNOS和NOX-1的基因表达以及NO和H202的产生水平。上述结果表明,GL能够激活鸡HD11巨噬细胞系,提商其吞噬、杀菌和抗原递呈能力。综上所述,50-200μg/mL GL不具备直接杀死和抑制ST生长的能力,其主要通过激活小鼠DC和巨噬细胞,促进DC功能的成熟和巨噬细胞的M1型极化,改善肠道菌群结构和提高免疫功能等途径增强小鼠抗ST感染的能力。同时本研究还发现GL能够激活鸡HD11巨噬细胞系,提高其吞噬和杀灭胞内ST的能力。
[Abstract]:Glycyrrhizin (glycyrrhizin, GL) is the main active ingredient of licorice root and source of sweetness, has significant immunomodulatory activity. The mice as the research object, through in vivo experiments explore effects of GL on immune function in mice and the ability to resist the infection of Salmonella, followed by in vitro experiments to further explore the GL on mouse dendritic cells (dendritic cell DC) function maturation and macrophage polarization, to clarify the mechanism of GL regulation of mouse immune and anti Salmonella infection; at the same time through the in vitro effect of the GL to verify the immune regulation of chicken macrophage and removing cell Neishamenshi bacteria, the main results are as follows: (a) the influence of GL on immune function in mice and the ability to resist the infection of Salmonella GL in vitro on Salmonella typhimurium (Salmonella enterica serovar Typhimurium, ST) affects the growth and virulence gene expression: 50-200 g/mL GL had no antibacterial activity on ST, and 200 g/mL GL were co cultured with ST 1 h, 2 h and 4H of InvA ST, SopD SipB, virulence gene, no significant effect on the expression of HilA and ssrB, suggesting that GL in vitro could not be directly inhibit the growth of ST, the expression of.GL and proliferation of major virulence genes on the immune function of normal mice: (1) 80 mg/kg oral GL in 21 days on the growth of ileum and mice, no significant effect of colon and liver tissue structure; (2) GL can significantly increase the sIgA secretion of mouse ileum in the intestine, promote the synthesis of mucosal NO, and improve the intestinal mucosa, serum and spleen IFN- gamma, IL-12, content of IL-6 and IL-10, IFN- expression and up regulation of IL-12 gene in the liver; (3) GL can increase the spleen index; (4) GL can be up-regulated in caecum of mice belonging to the genus Lactobacillus (Lactobacillus), Desulfovibrio genus (Desulfovibrio ), Helicobacter (Helicobacter) and bile bacteria (Bilophila) relative abundance, reduce Akkermansia, Bacteroides (Bacteroides) and the relative abundance of Anaerostipes. These results indicate that oral administration of 80 mg/kg GL is safe for mice, bacteria can improve small intestinal structure and the enhancement effect the intestinal mucosa and immune function of.GL mouse anti ST infection ability: (1) GL prophylaxis can significantly relieve the infection of ST (4 * 108CFU/) caused a decrease of the body weight of mice, improve the ileum and colon mucosa injury; (2) GL can significantly reduce the number of mouse ileum, colon, liver and spleen ST; (3) GL can significantly reduce the ST infection of the ileum, colon, proinflammatory cytokine IFN- serum and spleen, the content of TNF- alpha and IL-6, and down-regulation of IL-12 in colon and liver secretion in IFN-, expression of TNF- and IL-6 genes, and significantly improve the sense of ST The infected mice colon, serum and spleen and secretion of inflammatory cytokines IL-10; (4) GL can reduce the spleen and liver tissue lesions caused by ST infection, significantly reduced liver caspase-1 activity, liver cell apoptosis inhibition induced by ST; (5) GL can reduce the ST infection of Akkermansia mice in the cecum, Sateshi sp. (Sutterella), P. Copri (Prevotella) and coprococcus (Coprococcus) the relative abundance, increase the relative abundance of Parabacteroides and Anaerotruncus. These results suggest that GL can improve the intestinal microflora of mice infected with ST, improve the intestinal immune function and the body, thereby inhibiting ST in intestinal colonization and shift in the organization, improve the ST infection caused by tissue lesions and apoptosis, the mice against Salmonella infection. (two) the influence of GL on murine bone marrow derived dendritic cells mature in this study were based on 25200 g/mL GL 鐞嗗皬榧犻�ㄩ珦鏉ユ簮鏍戠獊鐘剁粏鑳�(bone marrow-derived DC, BMDC),鐮旂┒GL瀵瑰叾琛ㄥ瀷鍜屽姛鑳芥垚鐔熺殑褰卞搷.缁撴灉鍙戠幇锛，

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