Probiotics-derived soluble proteins regulate intestinal inflammation

益生菌衍生的可溶性蛋白质调节肠道炎症

• 批准号: 8586657
• 负责人: FANG YAN
• 金额: $ 0.15万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8586657
• 关键词: Address; Amidohydrolases; Animal Model; Apoptosis; Apoptotic; Bacteria; Bacterial Proteins; Biological; Biological Assay; Cell Survival; Cell physiology; Chimeric Proteins; Chromosomes; Clinical Trials; Colitis; Colon; Cysteine; Deletion Mutagenesis; Disease; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Goals; Health; Histidine; Homeostasis; Human; Hydrogels; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Inhibition of Apoptosis; Interleukin-10; Interleukin-9; Interleukins; Intestinal Diseases; Intestines; Investigation; Knowledge; Lactobacillus; Lactobacillus casei rhamnosus; Life; Ligands; Matrix Metalloproteinases; Mentored Research Scientist Development Award; Metalloproteases; Modeling; Molecular; Mus; Mutate; Pathway interactions; Pectins; Peptide Hydrolases; Phosphotransferases; Pouchitis; Prevention; Probiotics; Production; Proteins; Receptor Activation; Relapse; Research; Research Proposals; Signal Pathway; Signal Transduction; Sodium Dextran Sulfate; Structure; Study models; System; Systems Integration; Testing; Therapeutic; Therapeutic Agents; Ulcerative Colitis; United States National Institutes of Health; Zein; abstracting; bacterial genetics; base; clinical application; cytokine; disorder prevention; gastrointestinal; human KSR protein; in vivo; insight; intercellular communication; kinase inhibitor; microorganism; mouse model; mutant; novel; novel therapeutics; prevent; receptor expression; response; transcriptional coactivator p75

Project Summary/Abstract Lactobacillus rhamnosus GG (LGG) is one of the best-studied probiotic bacteria in clinical trials for treating and/or preventing several intestinal disorders, includig inflammatory bowel disease (IBD). However, the clinical application of LGG and other probiotics is limited by the paucity of information regarding their mechanisms of action. We have successfully purified and cloned two novel LGG-derived soluble proteins (p40 and p75) that prevent cytokine-induced apoptosis through activating Akt in intestinal epithelial cells. We focus on p40, a secreted protein which exerts more potent effects than p75, and have found that p40 activates epidermal growth factor (EGF) receptor, an known upstream signaling pathway regulating Akt and cell survival. Since increased production of inflammatory cytokines and epithelial cell apoptosis are two major pathogenic fcators for IBD, the goal of this research proposal is to define mechanisms by which p40 regulates intestinal epithelial cell function and determine the effects of p40 on intestinal inflammation. We will test the hypothesis that p40 prevents and/or treats intestinal inflammation through activating anti-apoptotic signals to inhibit cytokine-induced intestinal epithelial cell apoptosis. Three Specific Aims are proposed to address this hypothesis: Aim 1. To define p40-regulated signaling pathways for Akt activation and inhibition of cytokine- induced apoptosis in intestinal epithelial cells. We will focus on determining the requirement of EGF receptor activation by p40 for Akt activation and inibition of apoptosis using intestinal epithelial cells lacking EGF receptor expression. To further invstigate the mechanism of EGF receptor activation by p40, we will identify p40-stimulated EGF receptor ligand release using ELISA assays. Aim 2. To determine the structure-functional requirements of p40 for LGG-regulated signaling pathways and survival of intestinal epithelial cells. We will precisely define the functional domain using deletion mutagenesis. Then we will generate p40 functional domain and mutant p40 with the functional domain deletion fusion proteins and determine their in vitro and in vivo effects on signaling and intestinal inflammation. The requirement of p40 for LGG's regulatory effects will be determined by inactivating p40 from the LGG chromosome using a single-crossover insertional integration system and comparing effects of wild-type to mutated LGG on cell signaling and survival. Aim 3. To define the in vivo effects of p40 on intestinal inflammation in animal models of colitis. We will determine the optimal conditions for delivering p40 to the colon using the specific colon delivery strategy, the pectin/zein hydrogel system. The effects of p40 on prevention and/or treatment of inflammation and intestinal epithelial apoptosis will be detected in two mouse models of colitis, interleukin-10 and kinase suppressor of Ras double deficiency- elicited colitis and dextran sodium sulfate-induced colitis. The requirement of EGF receptor for p40's effects on inflammation will be analyzed using a EGF receptor kinase inhibitor and EGF receptor defective mice. Our long-term goal is to use p40 as a novel therapeutic agent for human intestinal inflammatory disorders.

项目摘要/摘要 鼠尾草乳杆菌GG（LGG）是治疗临床试验中最久经考验的益生菌之一 和/或预防几种肠道疾病，包括炎症性肠病（IBD）。但是，临床 LGG和其他益生菌的应用受到有关其机制的信息的限制 行动。我们已经成功纯化并克隆了两个新型LGG衍生的可溶性蛋白（p40和p75） 通过激活肠上皮细胞中的Akt来防止细胞因子诱导的凋亡。我们专注于P40，一个 分泌的蛋白质比P75发挥更有效作用，并且发现P40激活表皮 生长因子（EGF）受体，这是一种调节AKT和细胞存活的已知上游信号通路。 由于炎性细胞因子和上皮细胞凋亡的产生增加是两个主要的 IBD的致病FCators，该研究建议的目的是定义P40调节的机制 肠上皮细胞功能并确定p40对肠炎的影响。我们将测试 p40可以通过激活抗凋亡信号来阻止和/或治疗肠道炎症的假设 抑制细胞因子诱导的肠上皮细胞凋亡。提出了三个具体目标来解决这个问题 假设：目的1。定义P40调节的信号通路，以进行Akt激活和抑制细胞因子 - 诱发肠上皮细胞的凋亡。我们将专注于确定EGF受体的需求 使用缺乏EGF的肠上皮细胞激活p40，以激活Akt激活和凋亡 受体表达。为了进一步研究p40的EGF受体激活的机制，我们将确定 P40刺激的EGF受体配体使用ELISA分析释放。目标2。确定结构功能 P40对LGG调节的信号通路和肠上皮细胞存活的要求。我们将 精确地使用缺失诱变定义功能域。然后，我们将生成P40功能 具有功能域缺失融合蛋白的结构域和突变体p40，并确定其体外和 体内对信号传导和肠炎的影响。 P40对LGG的调节效应的要求将 通过使用单跨插入式积分从LGG染色体中灭活p40来确定 系统和比较野生型与突变的LGG对细胞信号传导和存活的影响。目标3。定义 p40对结肠炎动物模型的体内影响。我们将确定最佳 使用特定的结肠递送策略，果胶/锌水凝胶将P40传递到结肠的条件 系统。 p40对预防和/或治疗炎症和肠上皮细胞凋亡的影响 将在两种小鼠结肠炎模型，白介素10和Ras双缺乏症的激酶抑制剂中检测到 引起结肠炎和葡萄糖硫酸钠诱导的结肠炎。 EGF受体对P40对P40的影响的要求 将使用EGF受体激酶抑制剂和EGF受体有缺陷的小鼠分析炎症。我们的 长期目标是将P40用作人类肠道炎症性疾病的新型治疗剂。