乳酸杆菌对LPS诱导肠道炎症的免疫调节作用及机制研究
发布时间：2018-10-13 14:30
【摘要】：乳酸杆菌是应用最为广泛的益生菌,在肠道稳态调控中发挥重要作用。仔猪肠道应激多发,乳酸菌等益生菌对肠道的稳恒调节起着重要的作用。因此,阐明乳酸杆菌对肠道免疫调节功能,解析其免疫调节的关键信号分子及信号网络,将为益生菌调控肠道稳恒提供提供科学依据,对仔猪肠道健康具有重要的意义。本试验以公认的标准益生菌——鼠李糖乳酸杆菌(Lactobacillus rhamnousus GG,LGG)为代表,设计了两个试验。试验一:构建断奶仔猪肠道炎症模型,连续灌胃1x1010CFU/mL乳酸杆菌两周,腹腔注射来源于大肠杆菌的脂多糖(LPS)来诱导炎症,分析鼠李糖乳酸杆菌预处理对LPS诱导肠道炎症的免疫调节作用及机制。实验二:以LGG为标准菌株,构建小鼠炎症模型,连续灌胃108CFU/mL鼠李糖乳酸杆菌两周后,腹腔注射LPS诱发炎症。采用Western blot技术、免疫组化技术检测肠道组织中炎症的相关信号通路关键蛋白的表达水平,采用高通量测序分析肠道的微生物群系,同时用气相色谱检测乳酸杆菌对代谢途径的调节作用。实验一的研究结果表明::1)与单纯LPS刺激仔猪相比,LGG预处理仔猪能显著降低LPS诱导的细胞炎症因子IL-6,TNF-α和显著升高抗炎因子IL-10的表达量;细胞信号通路分子ERK1/2,p38MAPK和NF-κBp65的磷酸化水平显著降低,但I-κBα表达量显著升高(P0.05)。2)与单纯LPS刺激仔猪相比,在属水平上,LGG能够减少梭菌、链球菌和乳杆菌,在门水平上能减少厚壁菌门和增加变形菌门。3)与对照相比,LPS能降低具有抗炎作用的熊果苷。实验二的结果表明:1)与单纯LPS刺激小鼠相比,LGG连续灌胃2周的小鼠接受LPS刺激后,NF-κB基因敲除小鼠肠道炎症细胞因子IL-6和TNF-α的表达量显著升高;有趣的是p38基因敲除小鼠则出现相反的结果。2)与单纯LPS刺激小鼠相比,野生型小鼠的细胞信号通路分子p38MAPK和NF-κBp65的磷酸化水平升高,与之前的研究结果相反,有趣的时NF-κB基因敲除小鼠的ERK1/2,p38MAPK和NF-κBp65的磷酸化水平显著升高(P0.05)。p38基因敲除小鼠的ERK1/2,p38MAPK和NF-κBp65的磷酸化水平没有显著差异。6)与单纯LPS刺激小鼠相比,在属水平上,LGG能够减少贺氏菌属,在门水平上能减少变形菌门和增加变形菌门厚壁菌门。由此可见,鼠李糖乳酸杆菌LGG能够对LPS诱导的肠道炎症发挥免疫调节作用,通过抑制细胞MAPK和NF-κB信号通路激活,降低炎症因子,从而介导肠道免疫调节作用。
[Abstract]:Lactobacillus is the most widely used probiotics and plays an important role in the regulation of intestinal homeostasis. The intestinal stress of piglets is frequent, and probiotics such as lactic acid bacteria play an important role in the stable regulation of intestinal tract. Therefore, to elucidate the function of Lactobacillus on intestinal immune regulation and to analyze its key signal molecules and signal networks will provide a scientific basis for probiotic regulation of intestinal stability and play an important role in the intestinal health of piglets. In this experiment, two experiments were designed, represented by the standard probiotics-Lactobacillus rhamnoides (Lactobacillus rhamnousus GG,LGG). Experiment 1: the model of intestinal inflammation in weaned piglets was established, and the inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli for two weeks by intragastric administration of Lactobacillus 1x1010CFU/mL. To analyze the immunomodulatory effect and mechanism of lactobacillus rhamnoides pretreatment on intestinal inflammation induced by LPS. Experiment 2: using LGG as the standard strain, the inflammatory model of mice was established. After two weeks of continuous intragastric administration of Lactobacillus rhamnoides 108CFU/mL, LPS was injected intraperitoneally to induce inflammation. Western blot technique and immunohistochemical technique were used to detect the expression of key proteins related to inflammatory signaling pathway in intestinal tissue. High throughput sequencing was used to analyze the microflora of intestinal tract. At the same time, the regulation of Lactobacillus on metabolic pathway was detected by gas chromatography. The results of experiment 1 showed that: 1) compared with LPS alone, LGG pretreated piglets could significantly reduce the expression of LPS induced cytokine IL-6,TNF- 伪 and increase the expression of anti-inflammatory factor IL-10; The phosphorylation levels of ERK1/2,p38MAPK and NF- 魏 Bp65 decreased significantly, but the expression of I- 魏 B 伪 increased significantly (P0.05). Compared with LPS alone, LGG decreased the levels of clostridium, streptococcus and lactobacillus. Compared with the control, LPS could reduce the arbutin with anti-inflammatory effect. The results of experiment 2 showed that: 1) the expression of NF- 魏 B gene knockout inflammatory cytokines IL-6 and TNF- 伪 in NF- 魏 B knockout mice was significantly higher than that in mice stimulated by LPS for 2 weeks. Interestingly, p38 knockout mice showed the opposite results. 2) the phosphorylation levels of p38MAPK and NF- 魏 Bp65 in wild-type mice were higher than those in mice stimulated by LPS alone, contrary to previous studies. Interestingly, the phosphorylation levels of ERK1/2,p38MAPK and NF- 魏 Bp65 in NF- 魏 B knockout mice were significantly increased (P0.05). There was no significant difference in phosphorylation levels between ERK1/2,p38MAPK and NF- 魏 Bp65 in p38 knockout mice. At the gate level, it can reduce the phylum of Proteus and increase the phylum of the phylum. It can be concluded that Lactobacillus rhamnoides LGG can play an immunomodulatory role on the intestinal inflammation induced by LPS. By inhibiting the activation of MAPK and NF- 魏 B signaling pathway, and reducing the inflammatory factors, Lactobacillus rhamnoides can mediate the intestinal immune regulation.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816
本文编号：2268958
[Abstract]:Lactobacillus is the most widely used probiotics and plays an important role in the regulation of intestinal homeostasis. The intestinal stress of piglets is frequent, and probiotics such as lactic acid bacteria play an important role in the stable regulation of intestinal tract. Therefore, to elucidate the function of Lactobacillus on intestinal immune regulation and to analyze its key signal molecules and signal networks will provide a scientific basis for probiotic regulation of intestinal stability and play an important role in the intestinal health of piglets. In this experiment, two experiments were designed, represented by the standard probiotics-Lactobacillus rhamnoides (Lactobacillus rhamnousus GG,LGG). Experiment 1: the model of intestinal inflammation in weaned piglets was established, and the inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli for two weeks by intragastric administration of Lactobacillus 1x1010CFU/mL. To analyze the immunomodulatory effect and mechanism of lactobacillus rhamnoides pretreatment on intestinal inflammation induced by LPS. Experiment 2: using LGG as the standard strain, the inflammatory model of mice was established. After two weeks of continuous intragastric administration of Lactobacillus rhamnoides 108CFU/mL, LPS was injected intraperitoneally to induce inflammation. Western blot technique and immunohistochemical technique were used to detect the expression of key proteins related to inflammatory signaling pathway in intestinal tissue. High throughput sequencing was used to analyze the microflora of intestinal tract. At the same time, the regulation of Lactobacillus on metabolic pathway was detected by gas chromatography. The results of experiment 1 showed that: 1) compared with LPS alone, LGG pretreated piglets could significantly reduce the expression of LPS induced cytokine IL-6,TNF- 伪 and increase the expression of anti-inflammatory factor IL-10; The phosphorylation levels of ERK1/2,p38MAPK and NF- 魏 Bp65 decreased significantly, but the expression of I- 魏 B 伪 increased significantly (P0.05). Compared with LPS alone, LGG decreased the levels of clostridium, streptococcus and lactobacillus. Compared with the control, LPS could reduce the arbutin with anti-inflammatory effect. The results of experiment 2 showed that: 1) the expression of NF- 魏 B gene knockout inflammatory cytokines IL-6 and TNF- 伪 in NF- 魏 B knockout mice was significantly higher than that in mice stimulated by LPS for 2 weeks. Interestingly, p38 knockout mice showed the opposite results. 2) the phosphorylation levels of p38MAPK and NF- 魏 Bp65 in wild-type mice were higher than those in mice stimulated by LPS alone, contrary to previous studies. Interestingly, the phosphorylation levels of ERK1/2,p38MAPK and NF- 魏 Bp65 in NF- 魏 B knockout mice were significantly increased (P0.05). There was no significant difference in phosphorylation levels between ERK1/2,p38MAPK and NF- 魏 Bp65 in p38 knockout mice. At the gate level, it can reduce the phylum of Proteus and increase the phylum of the phylum. It can be concluded that Lactobacillus rhamnoides LGG can play an immunomodulatory role on the intestinal inflammation induced by LPS. By inhibiting the activation of MAPK and NF- 魏 B signaling pathway, and reducing the inflammatory factors, Lactobacillus rhamnoides can mediate the intestinal immune regulation.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816
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