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: 7582872
• 负责人: FANG YAN
• 金额: $ 36.84万
• 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/7582872
• 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; 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; bacterial genetics; base; clinical application; cytokine; disorder prevention; gastrointestinal; human KSR protein; in vivo; insight; kinase inhibitor; microorganism; mouse model; mutant; novel; novel therapeutics; prevent; public health relevance; receptor expression; response; transcriptional coactivator p75

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE This proposal will provide fundamental knowledge of understanding the mechanisms by which p40, a secreted protein from a probiotic bacterium, Lactobacillus GG, regulates intestinal epithelial cell function and will dedermine the in vivo effects of p40 on preventing and/or treating intestinal inflammation using animal models of colitis. Therefore, the findings from this proposal will support a scientific basis for a potential therapeutic application of p40 for preventing and/or treating human gastrointestinal inflammatory disorders.

描述（由申请人提供）：Rhamnosus gg（LGG）是治疗和/或预防多种肠道疾病的临床试验中最久经考验的益生菌之一，包括炎症性肠病（IBD）。但是，LGG和其他益生菌的临床应用受到有关其作用机理的信息的限制。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），​​它们通过在肠上皮细胞中激活AKT来防止细胞因子诱导的凋亡。我们专注于P40，这是一种分泌的蛋白质，比P75发挥更有效的作用，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。由于炎症细胞因子和上皮细胞凋亡的产生增加是IBD的两个主要致病fcators，因此该研究建议的目的是确定p40调节肠道上皮细胞功能的机制，并确定p40对肠道炎症的影响。我们将通过激活抗凋亡信号来抑制细胞因子诱导的肠上皮细胞细胞凋亡来预防和/或治疗肠道炎症的假设。提出了三个具体目的来解决这一假设：目的1。定义P40调节的信号传导途径，以激活Akt激活和抑制肠上皮细胞中细胞因子诱导的凋亡。我们将重点介绍使用缺乏EGF受体表达的肠上皮细胞来确定p40对p40激活EGF受体激活的要求。为了进一步通过p40侵入EGF受体激活的机理，我们将使用ELISA分析识别P40刺激的EGF受体配体释放。目标2。确定p40对于LGG调节的信号通路和肠上皮细胞存活的结构功能要求。我们将使用缺失诱变来精确定义功能域。然后，我们将使用功能域缺失融合蛋白生成P40功能域和突变体P40，并确定其体外和体内对信号传导和肠炎的影响。 P40对LGG调节效应的需求将通过使用单跨插入式整合系统从LGG染色体失活的P40来确定，并比较野生型对突变的LGG对细胞信号传导和存活率的影响。目标3。定义p40对结肠炎动物模型中肠道炎症的体内影响。我们将使用特定的结肠输送策略，即果胶/Zein水凝胶系统来确定将P40传递到结肠的最佳条件。 P40对炎症和肠上皮细胞凋亡的预防和/或治疗的影响将在两种小鼠结肠炎，白细胞介素-10和Ras双功能引起的结肠炎和硫酸钠硫酸钠诱导的结肠炎的小鼠模型中检测到。 EGF受体对P40对炎症的影响的需求将使用EGF受体激酶抑制剂和EGF受体缺陷的小鼠分析。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。公共卫生相关性该提案将提供理解理解的基本知识，p40是益生菌细菌的分泌蛋白，GG乳酸乳杆菌的分泌蛋白，调节肠上皮细胞功能，并将使用动物模型的动物模型来预防和/或治疗肠道炎症对预防和/或治疗肠道炎症的体内影响。因此，该提案的发现将支持p40潜在治疗应用于预防和/或治疗人类胃肠道炎症性疾病的科学基础。