基于肠道菌群调控下副干酪乳杆菌G15和Q14对Ⅱ型糖尿病的机制研究

发布时间：2018-03-01 06:12

  本文关键词： Ⅱ型糖尿病 益生菌 肠道菌群 肠道粘膜屏障 短链脂肪酸　出处：《哈尔滨工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着肠道菌群在糖尿病发病中的作用逐渐被揭示,益生菌在防治糖尿病中的作用备受学者关注。前期课题组从分离自西北牧区传统发酵食品中的乳酸菌中,筛选出副干酪乳杆菌(Lactobacillus paracasei subsp.paracasei)G15和副干酪乳杆菌(Lactobacillus casei)Q14,初步证实副干酪乳杆菌G15和Q14对Ⅱ型糖尿病改善明显,并发现肠道菌群在其中起着关键的调控作用。据此,本研究将进一步揭示副干酪乳杆菌G15和Q14如何通过调控肠道菌群来发挥抗糖尿病的作用。采用高脂饮食结合链脲霉素建立Ⅱ型糖尿病(T2D)大鼠模型。实验共分为五组,分别为正常组(N)、糖尿病组(D)、二甲双胍治疗组(M)、G15治疗组(G)和Q14治疗组(Q)。整个实验历时13周。采用ELISA法测定血清学相关指标;气相色谱法测定短链脂肪酸;RT-PCR法测定肠道菌群、短链脂肪酸受体、紧密连接蛋白以及炎症因子;免疫组织化学和HE染色分析肠道通透性和屏障功能。副干酪乳杆菌G15和Q14对T2D大鼠体重、糖耐量、胰岛素抵抗以及血脂水平具有明显的改善作用。T2D模型组大鼠的体重明显下降,G15和Q14治疗组显著改善了糖尿病大鼠的体重减轻,二甲双胍也显示较明显的改善作用。T2D大鼠的葡萄糖耐受能力显著下降,各治疗组葡萄糖耐量有了不同程度的改善。此外,G15和Q14还显著改善了T2D引起的胰岛素以及胰高血糖素的增加,改善了糖尿病大鼠胰岛β细胞的受损症状。G15和Q14作用也缓解了Ⅱ型糖尿病大鼠模型血脂水平的增加。副干酪乳杆菌G15和Q14通过影响肠道菌群-短链脂肪酸-G蛋白偶联受体通路来发挥抗糖尿病的作用。结果显示:G15和Q14改善了大鼠的肠道菌群紊乱,增加了乙酸产生菌(乳酸菌、双歧杆菌)和丁酸产生菌(柔嫩梭菌、普利沃菌)的含量,并增加乙酸和丁酸的含量。G15和Q14进一步上调短链脂肪酸受体GPR43的表达而对GPR41受体无明显作用,并且促进GPR43下游胃肠激素GLP-1、PYY分泌。副干酪乳杆菌G15和Q14通过改善肠道粘膜结构、紧密连接蛋白体系、粘膜通透性以及炎性指标的炎性机制来发挥抗糖尿病作用。G15和Q14明显降低了革兰氏阴性菌(G-)、拟杆菌和肠杆菌的含量,并且伴随着T2D大鼠血浆内毒素(LPS)水平的降低。结肠粘膜病理学检查发现,糖尿病大鼠结肠上皮细胞连接破损、细胞部分被破坏,且杯状细胞数量减少;G15和Q14可不同程度的修复肠粘膜屏障的受损。G15和Q14显著改善了紧密连接蛋白(ZO-1、Occludin、Muc2)的表达。G15和Q14进一步降低了糖尿病大鼠IL-1β、IL-6和IL-8的表达。综上,副干酪乳杆菌G15和Q14能够改善Ⅱ型糖尿病大鼠的糖耐量以及胰岛素抵抗,其作用机制有如下两方面:(1)以肠道菌群为核心,副干酪乳杆菌G15和Q14通过肠道菌群-SCFA-G蛋白偶联受体通路发挥抗糖尿病的作用;(2)副干酪乳杆菌G15和Q14通过改善肠道粘膜结构、紧密连接蛋白体系、粘膜通透性以及炎性指标的炎性机制来发挥抗糖尿病作用。本研究进一步解析G15和Q14通过肠道菌群改善T2D大鼠的深入机制,为益生菌用于糖尿病的防治奠定了理论基础;并从饮食角度防控Ⅱ型糖尿病提供了新思路。
[Abstract]:With the role in the pathogenesis of diabetes intestinal flora has been revealed, the role of probiotics in the prevention and treatment of diabetes in the concern of scholars. The research group from lactic acid bacteria isolated from traditional fermented food in the pastoral area of the northwest, screened L.paracasei (Lactobacillus paracasei subsp.paracasei) G15 and L.paracasei (Lactobacillus casei Q14) preliminary confirmed, L.paracasei G15 and Q14 on type II diabetes improved significantly, and found that the intestinal flora plays a key role. Therefore, this study will further reveal the pair of Lactobacillus casei strains G15 and Q14 how to play the anti diabetic effects by regulating intestinal flora by high-fat diet combined with. Streptozotocin to establish type II diabetes mellitus (T2D) rat model. Rats were divided into five groups, including normal group (N), diabetic group (D), metformin group (M), G15 treatment group (G) and Q14 treatment The treatment group (Q). The experiment lasted 13 weeks. Serum related indexes were measured by ELISA method; Determination of short chain fatty acid gas chromatography; RT-PCR method for determination of intestinal microflora, short chain fatty acid receptor, tight junction proteins and inflammatory factors; analysis of intestinal permeability and barrier function of immunohistochemistry and HE staining. Lactobacillus casei G15 and Q14 on T2D rats, glucose tolerance, insulin resistance and blood lipid levels has obvious improvement effect of.T2D model rats body weight decreased significantly, G15 and Q14 in treatment group significantly improved the body weight of diabetic rats reduced glucose tolerance, metformin also showed obvious improvement of.T2D rats in the treatment group significantly decreased glucose tolerance have different degrees of improvement. In addition, G15 and Q14 also significantly improved T2D induced by insulin and glucagon increased, improved rat pancreatic diabetes The island of beta cell damage symptoms of.G15 and Q14 to alleviate the increased level of serum lipid in rats model of type II diabetes. L.paracasei G15 and Q14 through the intestinal flora - short chain fatty acid -G protein coupled receptor pathway to play the anti diabetic effects. The results showed that G15 and Q14 improved intestinal disorders flora of rats, increase of acetic acid producing bacteria (Lactobacillus, Bifidobacterium) and butyrate producing bacteria (Clostridium leptum, Puliwo bacteria) content, and increase the expression of acetic acid and butyric acid content of.G15 and Q14 further increases in short chain fatty acid receptor GPR43 but had no obvious effect on the GPR41 receptor, and promote GPR43 downstream gastrointestinal hormone GLP-1, secretion of PYY. G15 and Q14 dry buttermilk bacillus by improving the structure of intestinal mucosa, tight junction protein system, and mucosal permeability in inflammatory markers of inflammatory mechanisms play anti diabetic effect of.G15 and Q14 decreased significantly. Gram negative bacteria (G-), the content of coli and Enterobacter, and accompanied by the plasma endotoxin in T2D rats (LPS). The decrease of the level of colonic mucosal pathological examination found that colonic epithelial cells in diabetic rats with damaged cells were damaged, and the decrease in the number of goblet cells; G15 and Q14 can be damaged and.G15 Q14 repair of intestinal mucosa barrier in different degree was improved obviously with the tight junction protein (ZO-1, Occludin, Muc2) on the expression of.G15 and Q14 further decreased in diabetic rats IL-1 beta, the expression of IL-6 and IL-8. In summary, dry weight and insulin resistance in G15 and L.CASEI Q14 can improve the glucose in type 2 diabetic rats resistance, the mechanism has the following two aspects: (1) to the intestinal flora as the core, L.paracasei G15 and Q14 play the anti diabetic effects of intestinal flora by -SCFA-G protein coupled receptor pathway; (2) the L.paracasei and Q14 by G15 Improve the structure of intestinal mucosa, tight junction protein system, play the anti diabetic effect of mucosal permeability and inflammatory index of inflammatory mechanisms. This study further mechanism further analytical G15 and Q14 through improving intestinal flora in T2D rats, as probiotics for the prevention and treatment of diabetes and laid a theoretical foundation; provides new ideas and Prevention from diet point of type II diabetes.

【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
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本文编号：1550737