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）：鼠李糖乳杆菌GG（LGG）是临床试验中研究最好的益生菌之一，用于治疗和/或预防几种肠道疾病，包括炎症性肠病（IBD）。然而，LGG和其他益生菌的临床应用受到有关其作用机制的信息缺乏的限制。我们已经成功地纯化和克隆了两种新的LGG衍生的可溶性蛋白（p40和p75），通过激活肠上皮细胞中的Akt来防止精氨酸诱导的凋亡。我们专注于p40，一种比p75发挥更有效作用的分泌蛋白，并发现p40激活表皮生长因子（EGF）受体，这是一种已知的调节Akt和细胞存活的上游信号通路。由于炎症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，因此本研究的目标是确定p40调节肠上皮细胞功能的机制，并确定p40对肠道炎症的影响。我们将检验p40通过激活抗凋亡信号来抑制奎宁诱导的肠上皮细胞凋亡从而预防和/或治疗肠道炎症的假设。提出了三个具体目标来解决这一假设：目标1。目的：探讨p40调控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。我们将集中于确定表皮生长因子受体激活p40 Akt激活和inibition的细胞凋亡缺乏表皮生长因子受体表达的肠上皮细胞的要求。为了进一步研究p40激活EGF受体的机制，我们将使用ELISA测定鉴定p40刺激的EGF受体配体释放。目标二。确定p40对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精确地定义功能结构域。然后，我们将产生p40功能域和功能域缺失的突变型p40融合蛋白，并确定它们在体外和体内对信号传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灭活LGG染色体上的p40并比较野生型与突变LGG对细胞信号传导和存活的影响来确定。目标3。确定p40在结肠炎动物模型中对肠道炎症的体内作用。我们将确定使用特定的结肠递送策略，果胶/玉米醇溶蛋白水凝胶系统将p40递送至结肠的最佳条件。将在两种结肠炎小鼠模型中检测p40对预防和/或治疗炎症和肠上皮细胞凋亡的作用，所述结肠炎小鼠模型为Ras双重缺陷引起的结肠炎的白介素-10和激酶抑制剂和葡聚糖硫酸钠引起的结肠炎。将使用EGF受体激酶抑制剂和EGF受体缺陷小鼠分析p40对炎症作用的EGF受体需求。我们的长期目标是使用p40作为一种新的治疗剂，用于人类肠道炎症性疾病。公共卫生相关性本提案将提供理解p40（来自益生菌乳杆菌GG的分泌蛋白）调节肠上皮细胞功能的机制的基础知识，并将使用结肠炎动物模型确定p40对预防和/或治疗肠道炎症的体内作用。因此，本提案的发现将为p40预防和/或治疗人类胃肠道炎症性疾病的潜在治疗应用提供科学依据。