肠道菌群介导海带多糖干预高脂饮食诱导小鼠胰岛素抵抗的调控机制

发布时间：2018-07-14 10:54

【摘要】：海带(Laminaria japonica)是一种常见的海洋蔬菜,多糖是其主要的生物活性物质。课题组前期研究发现,海带多糖可以改善由高脂饮食引起的小鼠胰岛素抵抗(Insulin resistance)。已有研究表明,肠道菌群(gut microbiota)产生的LPS是胰岛素抵抗病理形成的主要机制之一。基于此,本论文以一种化学结构和生物活性明确的海带多糖均一组分LJP61A为实验材料,重点研究海带多糖通过肠道菌群介导抑制高脂暴露诱发胰岛素抵抗的调控机制。论文获得的实验结果主要有:(1)LJP61A可以显著改善小鼠由高脂饮食引起的胰岛素抵抗:HFD高脂饮食会引起小鼠胰岛素敏感性降低、葡萄糖耐量下降,并产生胰岛素抵抗。胰岛素耐量实验与口服葡萄糖耐量实验的结果表明LJP61A可以显著改善小鼠的胰岛素敏感性及葡萄糖耐受性。此外,LJP61A能够显著降低小鼠空腹葡萄糖含量、胰岛素含量以及HOMA-IR值。通过western-blot分析,我们还发现海带多糖LJP61A能够显著降低肝脏组织与脂肪组织中p-AKT、p-IRS1的蛋白表达水平。这些结果均表明LJP61A可以显著改善高脂饮食暴露诱发的胰岛素抵抗。(2)LJP61A可以显著抑制高脂饮食诱导胰岛素抵抗小鼠的炎症反应:HFD会导致炎症因子的过量分泌,使得机体产生持续的低度炎症,而这种炎症反应是导致机体产生胰岛素抵抗的关键因素之一。本实验采用高脂饮食HFD诱导C57BL小鼠胰岛素抵抗的实验模型,研究了LJP61A对小鼠炎症反应的干预作用。通过RT-qPCR分析,我们发现LJP61A显著改善了肝脏组织与脂肪组织中TNF-α、IL-6、IL-10、IL-1β、PAI-1mRNA表达水平,以及血清中TNF-α、IL-6、IL-1β的含量,表明LJP61A可以下调NFκB和MAPK通路下游关键效应分子的表达。通过western-blot分析,我们发现LJP61A能够显著降低肝脏组织与脂肪组织中p-NFκBp65、p-P38、p-JNK、p-IκB、p-ERK1/2的蛋白表达水平,说明LJP61A抑制了调控因子NF-κB信号通路,并通过抑制JNK、ERK、P38的活化而抑制MAPK信号通路。这些结果均表明LJP61A可以显著抑制高脂饮食诱导胰岛素抵抗小鼠的炎症反应。(3)LJP61A能显著下调高脂饮食诱导的小鼠肠道通透性及内毒素血症:HFD高脂饮食会导致小鼠肠道通透性的增加,进而引起内毒素血症以及慢性炎症。本次实验结果表明,海带多糖LJP61A可以显著增大肠道闭合蛋白(Occludin)、闭锁连接蛋白(ZO-1)的表达量,以及Occludin、ZO-1 mRNA的表达水平。另外,LJP61A显著减少了HFD小鼠血清中LPS的含量,并且降低了肝脏组织与脂肪组织中TLR4蛋白的表达量。这些结果表明LJP61A可以显著下调高脂饮食诱导的小鼠肠道通透性及内毒素血症。(4)LJP61A对高脂饮食改变小鼠肠道菌群构成具有干预作用:HFD饮食会导致肠道菌群的失调以及益生菌数量的下降,进而引起小鼠肠道紧密性发生变化。本次试验发现高脂饮食显著提高了Firmicutes-to-Bacteroidetes比例,且LJP61A干预浓度为200mg/kg/day时厚壁菌门细菌的含量最低,拟杆菌门含量最高。另外,LJP61A的干预使Akkermansia、Bifidobacterium、Lactobacillus等肠道益生菌的相对丰度显著提高。这些结果表明LJP61A对高脂饮食改变小鼠肠道菌群构成具有干预作用,可使其菌群结构更加接近于正常对照组小鼠。以上结果表明,LJP61A通过调节HFD诱导小鼠的肠道菌群结构,改善小鼠肠道通透性以及内毒素血症,调节炎症反应,从而达到干预胰岛素抵抗的目的。
[Abstract]:Laminaria japonica is a common marine vegetable and polysaccharide is its main bioactive substance. Previous studies have found that kelp polysaccharide can improve insulin resistance (Insulin resistance) caused by high fat diet (Insulin resistance). Studies have shown that the LPS produced by the intestinal flora (gut microbiota) is the pathology of insulin resistance. In this paper, based on this, this paper uses a chemical structure and biological activity clear LJP61A as the experimental material, focusing on the regulation mechanism of inhibiting high fat exposure through the intestinal flora mediated by the intestinal flora. The results of the paper are as follows: (1) LJP61A can be significant Improve insulin resistance induced by high fat diet in mice: HFD high fat diet can cause insulin sensitivity to decrease, glucose tolerance and insulin resistance. The results of insulin tolerance test and oral glucose tolerance test show that LJP61A can significantly improve insulin sensitivity and glucose tolerance in rats. LJP61A can significantly reduce the fasting glucose content, insulin content and HOMA-IR value in mice. Through Western-blot analysis, we also found that the Laminaria Polysaccharide LJP61A significantly reduced the protein expression levels of p-AKT and p-IRS1 in the liver and adipose tissues. These results suggest that LJP61A can significantly improve the exposure to high fat diet exposure. Insulin resistance. (2) LJP61A can significantly inhibit the inflammatory response induced by high fat diet induced insulin resistance in mice: HFD leads to excessive secretion of inflammatory factors and causes the body to produce persistent low degree of inflammation, which is one of the key factors leading to the body's insulin resistance. This experiment uses a high fat diet HFD to induce C57BL The experimental model of insulin resistance in mice was used to study the effect of LJP61A on the inflammatory response in mice. Through RT-qPCR analysis, we found that LJP61A significantly improved the level of TNF- alpha, IL-6, IL-10, IL-1 beta, PAI-1mRNA expression in the liver tissue and adipose tissue, as well as the content of TNF- alpha, IL-6, IL-1 beta in the serum. Through Western-blot analysis, we found that LJP61A can significantly reduce the protein expression level of p-NF kappa Bp65, p-P38, p-JNK, p-I kappa B, and p-ERK1/2 in the liver and adipose tissue. It shows that LJP61A inhibits the NF- kappa B signal pathway, and inhibits the activation by inhibiting the activation. These results all indicate that LJP61A can significantly inhibit the inflammatory response in insulin resistant mice induced by high fat diet. (3) LJP61A can significantly reduce the intestinal permeability and endotoxemia in mice induced by high fat diet. HFD high fat diet may lead to the increase of intestinal permeability in mice, and then cause endotoxemia and chronic inflammation. The results showed that LJP61A could significantly increase the intestinal closed protein (Occludin), the expression of interlocking connexin (ZO-1), and the expression level of Occludin and ZO-1 mRNA. In addition, LJP61A significantly reduced the content of LPS in the serum of HFD mice, and reduced the expression of TLR4 protein in the liver and adipose tissues. The results showed that LJP61A could significantly reduce the intestinal permeability and endotoxemia in mice induced by high fat diet. (4) LJP61A has an intervention effect on the formation of intestinal flora in mice with high fat diet. The HFD diet will lead to the imbalance of intestinal flora and the decrease of the number of probiotics, and then lead to the change of intestinal tightness in mice. In the present high fat diet, the proportion of Firmicutes-to-Bacteroidetes was significantly increased, and the content of bacilli was the lowest when the concentration of LJP61A was 200mg/kg/day, and the highest content of the bacilli was found. In addition, the intervention of LJP61A significantly increased the relative abundance of intestinal probiotics, such as Akkermansia, Bifidobacterium, Lactobacillus and so on. These results showed LJP61A. The results showed that LJP61A could improve the intestinal permeability and endotoxemia and regulate the inflammatory response by regulating the intestinal microflora structure of mice by regulating HFD, and thus interfering with insulin resistance. The purpose.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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