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: 7759167
• 负责人: FANG YAN
• 金额: $ 36.47万
• 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/7759167
• 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(申请人提供)：鼠李糖乳杆菌GG(LGG)是临床试验中研究最多的益生菌之一，用于治疗和/或预防多种肠道疾病，包括炎症性肠病(IBD)。然而，由于缺乏对其作用机制的了解，LGG和其他益生菌的临床应用受到限制。我们已经成功地纯化和克隆了两个新的LGG衍生的可溶性蛋白(p40和p75)，它们通过激活Akt来阻止细胞因子诱导的肠上皮细胞凋亡。我们专注于p40，这是一种比p75更有效的分泌性蛋白，并发现p40激活了表皮生长因子(EGF)受体，这是一个已知的上游信号通路，调节Akt和细胞生存。由于炎性细胞因子的产生增加和上皮细胞凋亡是IBD的两个主要致病因素，本研究的目的是明确p40调节肠上皮细胞功能的机制，并确定p40在肠炎症中的作用。我们将验证p40通过激活抗凋亡信号来抑制细胞因子诱导的肠上皮细胞凋亡来预防和/或治疗肠炎症的假设。针对这一假说提出了三个具体的目标：目的1.明确p40调节的Akt激活和抑制细胞因子诱导的肠上皮细胞凋亡的信号通路。我们将着重于利用缺乏EGF受体表达的肠上皮细胞来确定p40对Akt激活和凋亡的抑制所需的EGF受体。为了进一步探讨p40激活EGF受体的机制，我们将用ELISA法鉴定p40刺激的EGF受体配体的释放。目的2.确定p40在LGG调控的信号通路和肠上皮细胞存活中的结构和功能需求。我们将使用删除突变来精确定义功能结构域。然后，我们将构建p40功能结构域和带有功能结构域缺失融合蛋白的突变体p40，并确定它们在体外和体内对信号转导和肠道炎症的影响。P40对LGG调节作用的需求将通过使用单交叉插入整合系统从LGG染色体上灭活p40，并比较野生型和突变型LGG对细胞信号和存活的影响来确定。目的3.明确p40对结肠炎动物模型肠道炎症的体内作用。我们将使用特定的结肠传递策略，即果胶/玉米醇溶蛋白水凝胶系统，确定将p40传递到结肠的最佳条件。P40在预防和/或治疗炎症和肠上皮细胞凋亡方面的作用将在两种结肠炎小鼠模型上进行检测，即白介素10和激酶抑制因子RAS双缺乏诱导的结肠炎和葡聚糖硫酸钠诱导的结肠炎。利用表皮生长因子受体激酶抑制剂和表皮生长因子受体缺陷小鼠，分析表皮生长因子受体对p40‘S在炎症中的作用的需求。我们的长期目标是使用p40作为一种治疗人类肠道炎症性疾病的新药物。公共卫生相关性这项建议将为理解p40的机制提供基础知识，p40是益生菌乳杆菌GG的一种分泌蛋白，通过调节肠道上皮细胞功能，并将确定p40在使用结肠炎动物模型预防和/或治疗肠炎方面的体内作用。因此，这项提案的发现将为p40预防和/或治疗人类胃肠炎性疾病的潜在治疗应用提供科学基础。