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: 8247770
• 负责人: FANG YAN
• 金额: $ 32.72万
• 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/8247770
• 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.

项目概要/摘要 鼠李糖乳杆菌 GG (LGG) 是临床试验中研究最充分的益生菌之一，用于治疗 和/或预防多种肠道疾病，包括炎症性肠病（IBD）。然而，临床 LGG 和其他益生菌的应用因缺乏有关其作用机制的信息而受到限制。 行动。我们已经成功纯化并克隆了两种新型 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细胞凋亡。我们专注于 p40， 分泌蛋白比 p75 发挥更有效的作用，并发现 p40 激活表皮 生长因子 (EGF) 受体，一种调节 Akt 和细胞存活的已知上游信号通路。 由于炎症细胞因子产生的增加和上皮细胞凋亡是两个主要的 IBD 的致病因子，本研究计划的目标是确定 p40 的调节机制 肠上皮细胞功能并确定 p40 对肠道炎症的影响。我们将测试 假设 p40 通过激活抗凋亡信号来预防和/或治疗肠道炎症 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：目标 1. 定义用于 Akt 激活和细胞因子抑制的 p40 调节信号通路 诱导肠上皮细胞凋亡。我们将重点确定EGF受体的需求 使用缺乏 EGF 的肠上皮细胞通过 p40 激活 Akt 并抑制细胞凋亡 受体表达。为了进一步研究 p40 激活 EGF 受体的机制，我们将鉴定 使用 ELISA 检测 p40 刺激的 EGF 受体配体释放。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需求。我们将 使用缺失诱变精确定义功能域。然后我们将生成p40功能 域和突变体 p40 与功能域缺失融合蛋白并测定其体外和体内 对信号传导和肠道炎症的体内影响。 p40对于LGG监管效果的要求将 通过使用单交换插入整合使 LGG 染色体上的 p40 失活来确定 系统并比较野生型和突变型 LGG 对细胞信号传导和存活的影响。目标 3. 定义 p40 对结肠炎动物模型肠道炎症的体内影响。我们将确定最优的 使用特定的结肠递送策略（果胶/玉米醇溶蛋白水凝胶）将 p40 递送至结肠的条件 系统。 p40对炎症和肠上皮细胞凋亡的预防和/或治疗作用 将在两种结肠炎小鼠模型中检测到白细胞介素10和Ras激酶抑制因子双重缺陷—— 诱发结肠炎和右旋糖酐硫酸钠诱发的结肠炎。 p40 作用需要 EGF 受体 将使用 EGF 受体激酶抑制剂和 EGF 受体缺陷小鼠来分析炎症。我们的 长期目标是利用p40作为人类肠道炎症性疾病的新型治疗剂。