本文關(guān)鍵詞：甘草酸苷對小鼠免疫功能的調(diào)節(jié)作用及其抗沙門氏菌感染的機(jī)理研究　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甘草酸苷 小鼠 沙門氏菌 巨噬細(xì)胞 樹突狀細(xì)胞 極化 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來源,具有顯著的免疫調(diào)節(jié)活性。本研究首先以小鼠為研究對象,通過體內(nèi)實驗探究GL對小鼠免疫功能及抗沙門氏菌感染能力的影響,隨后利用體外細(xì)胞實驗進(jìn)一步探討了GL對小鼠樹突狀細(xì)胞(dendritic cell, DC)功能成熟和巨噬細(xì)胞極化的影響,旨在闡明GL調(diào)控小鼠免疫及其抗沙門氏菌感染的作用機(jī)理；同時通過體外實驗研究GL對雞巨噬細(xì)胞免疫及清除胞內(nèi)沙門氏菌能力的影響驗證其免疫調(diào)節(jié)的作用效果,主要研究結(jié)果如下：(一)GL對小鼠免疫功能和抗沙門氏菌感染能力的影響GL在體外對鼠傷寒沙門氏菌(Salmonella enterica serovar Typhimurium, ST)生長和毒力基因表達(dá)的影響：50-200 μg/mL GL對ST均無抑菌活性,且200 μg/mL GL與ST共培養(yǎng)1 h、2 h和4h對ST毒力基因InvA、SopD、SipB、HilA和ssrB的表達(dá)無顯著影響,提示GL在體外不能直接抑制ST的生長、增殖和主要毒力基因的表達(dá)。GL對正常小鼠免疫功能的影響：(1)口服80 mg/kg GL在21天內(nèi)對小鼠生長及回腸、結(jié)腸和肝臟組織結(jié)構(gòu)無顯著影響;(2)GL可顯著上調(diào)小鼠回腸中sIgA的分泌,促進(jìn)腸粘膜NO的合成,并提高腸粘膜、血清和脾臟中IFN-γ、IL-12、IL-6和IL-10的含量,上調(diào)肝臟中IFN-γ和IL-12的基因表達(dá)；(3)GL可提高小鼠的脾臟指數(shù)；(4)GL可上調(diào)小鼠盲腸中乳桿菌屬(Lactobacillus)、脫硫弧菌屬(Desulfovibrio) 、螺桿菌屬(Helicobacter)和嗜膽菌屬(Bilophila)的相對豐度,降低Akkermansia、擬桿菌屬(Bacteroides)和Anaerostipes的相對豐度。以上結(jié)果表明,口服80 mg/kg GL對小鼠具有良好的安全性,且可改善小鼠腸道的菌群結(jié)構(gòu),增強腸粘膜和機(jī)體免疫功能。GL對小鼠抗ST感染能力的影響：(1)GL預(yù)防給藥可顯著緩解ST感染(4×108CFU/只)引起的小鼠體重下降,改善回腸和結(jié)腸的粘膜損傷；(2)GL可顯著降低小鼠回腸、結(jié)腸、脾臟和肝臟中ST的數(shù)量；(3)GL可顯著降低ST感染引起的回腸、結(jié)腸、血清和脾臟中促炎性細(xì)胞因子IFN-γ、TNF-α和IL-6的含量,同時下調(diào)結(jié)腸中IL-12的分泌及肝臟中IFN-γ、TNF-α和IL-6的基因表達(dá),而顯著提高ST感染小鼠結(jié)腸、血清和脾臟中抑炎細(xì)胞因子IL-10的分泌；(4)GL可降低ST感染引起的脾臟和肝臟組織病變,顯著下調(diào)肝臟caspase-1的活力,抑制ST引起的肝臟細(xì)胞凋亡；(5)GL可降低ST感染小鼠盲腸中Akkermansia、薩特氏菌屬(Sutterella)、普氏菌屬(Prevotella)和糞球菌屬(Coprococcus)的相對豐度,提高Parabacteroides和Anaerotruncus的相對豐度。以上結(jié)果提示,GL可通過改善ST感染小鼠的腸道菌群結(jié)構(gòu)、提高腸道和機(jī)體的免疫功能,從而抑制ST在腸道中的定植和向組織中的移位,改善ST感染引起的組織病變和細(xì)胞凋亡,使小鼠免受沙門氏菌感染。(二)GL對小鼠骨髓來源樹突狀細(xì)胞成熟的影響本研究分別以25200 μg/mL GL處理小鼠骨髓來源樹突狀細(xì)胞(bone marrow-derived DC, BMDC),研究GL對其表型和功能成熟的影響。結(jié)果發(fā)現(xiàn)：(1)GL對小鼠BMDC的安全濃度范圍為0-400 μg/mL; (2) 25-200 μg/mL GL以濃度依賴的方式顯著降低BMDC胞內(nèi)ACP的活力；(3)GL可顯著上調(diào)BMDC中成熟標(biāo)志分子CD40、 CD80、CD83、CD86和MHC-Ⅱ的表面表達(dá)及細(xì)胞因子IL-12、IFN-γ、TNF-α、IL-6、 IL-10和TGF-β的基因表達(dá)和分泌；(4)GL可在12 h內(nèi)顯著上調(diào)BMDC中TLR2的基因表達(dá),降低TLR3和TLR4的基因表達(dá)；(5)GL可在60 min內(nèi)激活BMDC中NF-κB、ERK和p38 MAPK信號分子,并介導(dǎo)IL-12和IL-6的分泌。以上結(jié)果提示,GL能夠通過TLR激活NF-κB、ERK和p38 MAPK信號通路,促進(jìn)BMDC成熟,提高其抗原遞呈能力和免疫功能。(三)GL對小鼠骨髓來源巨噬細(xì)胞極化的影響本研究以100 μg/mL GL處理小鼠骨髓來源巨噬細(xì)胞(bone marrow-derived macrophage, BMDM),研究GL對其極化、功能及信號通路的影響。結(jié)果發(fā)現(xiàn)：(1)GL對小鼠BMDM的安全濃度范圍為0-100μg/mL;(2)GL可顯著上調(diào)BMDM中CD80、CD86和MHC-II的表面表達(dá),表明GL能夠激活小鼠BMDM,增強其抗原遞呈能力；(3)GL可顯著上調(diào)BMDM中M1型巨噬細(xì)胞特征分子CCR7、TNF-α、IL-12和IL-6的表面表達(dá)或分泌,降低M2型巨噬細(xì)胞特征分子Ym1和MR的基因表達(dá),提示GL能夠誘導(dǎo)BMDM向M1型極化；(4)GL可在24 h內(nèi)顯著上調(diào)BMDM中iNOS的基因表達(dá),下調(diào)Argl的表達(dá),并誘導(dǎo)iNOS的蛋白表達(dá)和NO的合成,表明GL能夠促進(jìn)BMDM中NO的合成,提商細(xì)胞的抗感染能力；(5)GL可增強BMDM吞噬FITC-Dextran和E.coli K88的能力,并顯著降低ST和E.coli K88在細(xì)胞內(nèi)的存活,表明GL能夠增強BMDM的吞噬、殺菌能力；(6)GL在60 min內(nèi)不能激活BMDM中STAT1信號分子,可激活NF-κB、ERK、JNK和p38 MAPK信號分子,其中NF-κB和JNK介導(dǎo)了BMDM中M1型特征分子NO和IL-6的產(chǎn)生,而ERK介導(dǎo)了IL-10的分泌,表明NF-κB和JNK信號通路參與了GL誘導(dǎo)的M1型巨噬細(xì)胞極化,而ERK通路通過調(diào)節(jié)抗炎細(xì)胞因子IL-10的分泌對上述過程進(jìn)行調(diào)控；(7)GL可顯著下調(diào)IL-4誘導(dǎo)的M2型巨噬細(xì)胞特征分子Arg1、FIZZ1、MR和Ym1的基因表達(dá),提示GL能夠抑制IL-4誘導(dǎo)的M2型巨噬細(xì)胞的產(chǎn)生；(8)GL可顯著上調(diào)IL-4誘導(dǎo)的M2型巨噬細(xì)胞表達(dá)M1型特征分子iNOS、TNF-α、IL-12p40和IL-6,提示GL可促進(jìn)IL-4誘導(dǎo)的M2型巨噬細(xì)胞向M1型轉(zhuǎn)化；(9)GL可顯著下調(diào)IL-4誘導(dǎo)的M2型極化相關(guān)信號分子PPAR-γ和KLF4的基因表達(dá),提示GL可能通過降低PPAR-γ和KLF4的表達(dá),抑制IL-4誘導(dǎo)的M2型巨噬細(xì)胞極化。(四)GL對雞HD11巨噬細(xì)胞系免疫功能的影響本研究以100μg/mL GL處理雞HD11巨噬細(xì)胞系,研究GL對其免疫功能的激活情況。結(jié)果如下：(1)GL對雞HD11巨噬細(xì)胞系的安全濃度范圍為0-400 μg/mL; (2) GL可顯著增強HD11細(xì)胞吞噬FITC-Dextran和ST的能力,顯著降低胞內(nèi)ST數(shù)量；(3)GL在12h內(nèi)可顯著上調(diào)HD11細(xì)胞表面分子CD40、CD80、CD83和CD197及細(xì)胞因子IFN-γ、IL-6和IL-10的基因表達(dá)；(4)GL可顯著提高細(xì)胞中iNOS和NOX-1的基因表達(dá)以及NO和H202的產(chǎn)生水平。上述結(jié)果表明,GL能夠激活雞HD11巨噬細(xì)胞系,提商其吞噬、殺菌和抗原遞呈能力。綜上所述,50-200μg/mL GL不具備直接殺死和抑制ST生長的能力,其主要通過激活小鼠DC和巨噬細(xì)胞,促進(jìn)DC功能的成熟和巨噬細(xì)胞的M1型極化,改善腸道菌群結(jié)構(gòu)和提高免疫功能等途徑增強小鼠抗ST感染的能力。同時本研究還發(fā)現(xiàn)GL能夠激活雞HD11巨噬細(xì)胞系,提高其吞噬和殺滅胞內(nèi)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楂撴潵婧愭爲(wèi)紿佺姸緇嗚優(yōu)(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.緇撴灉鍙戠幇錛，

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