褐藻胶及其衍生物的免疫抗炎及抗阿尔兹海默症的机制研究

发布时间：2017-12-28 19:21

本文关键词：褐藻胶及其衍生物的免疫抗炎及抗阿尔兹海默症的机制研究　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：褐藻胶是由互为C5差向异构体的聚甘露糖醛酸(PM)和聚古罗糖醛酸(PG)相互嵌合而成的线性大分子,是一类广泛存在于各种褐藻中的酸性多糖。据大量文献报道,褐藻胶及其衍生物具有多种生物活性。本课题以褐藻胶、硒化甘露糖醛酸(Se-PM)、酶解甘露糖醛酸寡糖(MOS)为原料,分别研究它们的促吞噬作用、抗炎、抗阿尔兹海默症(AD)等方面的生物活性。有以下四个工作:(1)本课题研究褐藻胶促进小鼠巨噬细胞RAW264.7吞噬作用的机制。研究发现,褐藻胶能够显著提高RAW264.7细胞对金纳米颗粒、荧光微球、免疫球蛋白G(IgG)调理的金黄色葡萄球菌的吞噬作用。究其机制,形态学分析表明褐藻胶能够明显提高RAW264.7细胞面积。此外,深入研究发现褐藻胶能够显著上调RAW264.7细胞中Toll样受体4(TLR4)基因和蛋白的表达,当用TLR4的特异性抑制剂TAK-242处理细胞及利用shRNA干扰技术将RAW264.7细胞的TLR4基因下调后,褐藻胶促进RAW264.7细胞吞噬金纳米颗粒的作用被显著性抑制。研究还发现,褐藻胶能够激活RAW264.7细胞核转录因子κB(NF-κB)信号通路和p38丝裂原活化蛋白激酶(MAPK)信号通路,当用p38 MAPK和NF-κB的抑制剂处理细胞后,褐藻胶促进巨噬细胞吞噬IgG调理的金黄色葡萄球菌的作用有明显的降低。(2)本课题研究Se-PM的抗炎活性及分子机制。研究发现,Se-PM能够有效抑制LPS诱导的小鼠巨噬细胞RAW264.7细胞炎症介质,如一氧化氮(NO)、前列腺素E2(PGE2)、活性氧簇(ROS)的生成,诱导型一氧化氮合酶(iNOS)、环氧合酶-2(COX-2)的表达,细胞因子,如肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、IL-6、IL-12的分泌,且均呈浓度依赖性。深入研究发现,Se-PM可以显著抑制LPS诱导的RAW264.7细胞NF-κB信号通路和MAPK信号通路的过度激活。并且,Se-PM还能够显著抑制LPS诱导的原代小鼠巨噬细胞炎症介质,如NO、TNF-α、IL-1β和IL-6的生成。另外,本课题构建LPS诱导的败血性休克小鼠模型和卡拉胶诱导的气囊炎症小鼠模型,在动物水平上研究发现,Se-PM能够抑制上述两种炎症模型的小鼠腹水、血清、气囊浸出液中TNF-α和IL-6的分泌。(3)本课题研究MOS通过自噬途径降低AD模型细胞的Aβ生成。研究发现,MOS可以降低N2a-sw-APP695细胞中β-淀粉样蛋白(Aβ)的分泌、淀粉样前体蛋白(APP)和β分泌酶(BACE1)的表达,抑制3×Transgene(3×Tg,APPswe/PS1M146V/TauP301L)小鼠原代神经元细胞中APP和BACE1的表达。并且,MOS还可以降低哺乳动物雷帕霉素靶蛋白(mTOR)的激活,提高Beclin-1、微管相关蛋白轻链3(LC3)-II、组织蛋白酶(Cat)-D的表达,抑制p62的表达,进而提高N2a-sw-APP695细胞的自噬水平。(4)本课题研究MOS通过自噬途径降低Tau蛋白磷酸化。研究发现,MOS可以降低HEK293/Tau细胞和3×Tg小鼠原代神经元细胞中Tau蛋白在丝氨酸202、262、396、404位点的磷酸化水平,以及总Tau的表达水平,还可以抑制糖原合成酶激酶3β(GSK3β)的活性。深入研究还发现,MOS可以降低3×Tg小鼠原代神经元细胞mTOR的激活,提高Beclin-1、LC3-II、Cat-D的表达,降低p62的表达,进而提高细胞自噬,促进自噬体和溶酶体的融合。本论文发现褐藻胶及其衍生物在促吞噬作用、抗炎、抗AD方面具有良好的生物活性,具有开发成各类保健食品或药物的潜力。
[Abstract]:Alginate is by mutual C5 epimer of Polymannuronic acid (PM) and poly guluronate (PG) linear macromolecules mutually embedded together, is a kind of acidic polysaccharide alginate in widely exists in various. According to a large number of reports, the alginate and its derivatives have a variety of bioactivity. In this study, alginate, seleno manuronic acid (Se-PM) and mannan uronic acid oligosaccharides (MOS) were used as raw materials to study their phagocytosis, anti inflammation and anti Alzheimer's disease (AD), respectively. There are four tasks as follows: (1) the mechanism of the phagocytosis of murine macrophage RAW264.7 by algin is studied in this study. It is found that alginate can significantly enhance the phagocytosis of RAW264.7 cells on Staphylococcus aureus, which is modulated by gold nanoparticles, fluorescent microspheres and immunoglobulin G (IgG). The morphological analysis showed that brown alga could significantly increase the area of RAW264.7 cells. In addition, we found that alginate can significantly increase RAW264.7 cell Toll like receptor 4 (TLR4) gene and protein expression, when using the TLR4 and the cells treated with TAK-242 specific inhibitor shRNA interference TLR4 gene of RAW264.7 cells decreased after alginate promotes RAW264.7 cell phagocytosis of gold nanoparticles is significant suppression. The study also found that alginate can activate RAW264.7 nuclear transcription factor kappa B (NF- K B) signaling pathway and p38 mitogen activated protein kinase (MAPK) signaling pathway, when using the p38 MAPK and NF- kappa B inhibitor treatment cells, alginate IgG promote macrophage phagocytosis of opsonized Staphylococcus aureus have obvious effect reduce. (2) this topic studies the anti-inflammatory activity and molecular mechanism of Se-PM. The study found that Se-PM can effectively inhibit the mouse macrophage RAW264.7 cells LPS induced by inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), reactive oxygen species (ROS) generation of inducible nitric oxide synthase (iNOS) and cyclooxygenase -2 (COX-2) on the surface of cells, such as tumor necrosis factor. Factor alpha (TNF- alpha), interleukin 1 beta (IL-1 beta), the secretion of IL-6 and IL-12, and were in a concentration dependent manner. It was found that Se-PM could significantly inhibit the overactivation of NF- kappa B signaling pathway and MAPK signaling pathway in RAW264.7 cells induced by LPS. Moreover, Se-PM can also significantly inhibit the LPS induced macrophage inflammatory mediators, such as the formation of NO, TNF- alpha, IL-1 beta and IL-6. In addition, the construction of airbag inflammation in mice model of septic shock mice model and carrageenan induced by LPS induction, the study found in the animal level, secretion of TNF- and IL-6 Se-PM can inhibit the two inflammatory model mice ascites, serum, balloon in leaching solution. (3) the study of MOS reduces the A beta production of AD model cells by autophagy pathway. It is found that MOS can reduce the expression of beta amyloid protein (A beta), the expression of amyloid precursor protein (APP) and beta secretase (BACE1) in N2a-sw-APP695 cells, and inhibit the expression of APP and BACE1 in primary neurons of 3 * Transgene (3 x Tg, APPswe/PS1M146V/TauP301L) mice. Moreover, MOS can also reduce the activation of mammalian target of rapamycin (mTOR), increase the expression of Beclin-1, microtubule associated protein light chain 3 (LC3) -II and cathepsin (Cat) -D, inhibit the expression of p62, and further improve the autophagy level of N2a-sw-APP695 cells. (4) this topic studies MOS to reduce the phosphorylation of Tau protein by autophagy pathway. It is found that MOS can reduce the phosphorylation level of Tau protein at serine 202, 262, 396, 404 sites, and the expression level of total Tau in HEK293/Tau cells and 3 * Tg mouse primary neurons, and also inhibit the activity of glycogen synthase kinase 3 GSK3 (GSK3 beta). Further studies also showed that MOS could reduce the activation of mTOR in primary neurons of 3 * Tg mice, increase the expression of Beclin-1, LC3-II and Cat-D, reduce the expression of p62, further enhance autophagy, and promote the fusion of autophagosome and lysosome. It is found that alginate and its derivatives have good bioactivity in promoting phagocytosis, anti-inflammatory and anti AD, and have potential to develop various kinds of health foods or drugs.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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