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: 8055059
• 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/8055059
• 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; 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

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，LGG 和其他益生菌的临床应用因其作用机制的信息匮乏而受到限制。我们成功纯化并克隆了两种新型 LGG 衍生的可溶性蛋白（p40 和 p75），它们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。我们关注 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 IBD 的两个主要致病因素，因此本研究计划的目标是确定 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抑制细胞因子诱导的肠上皮细胞凋亡来预防和/或治疗肠道炎症的假设。提出了三个具体目标来解决这一假设： 目标 1. 定义 p40 调节的信号通路，以激活 Akt 并抑制肠上皮细胞中细胞因子诱导的细胞凋亡。我们将重点利用缺乏 EGF 受体表达的肠上皮细胞来确定 p40 激活 EGF 受体对 Akt 激活和抑制细胞凋亡的要求。为了进一步研究 p40 激活 EGF 受体的机制，我们将使用 ELISA 测定来鉴定 p40 刺激的 EGF 受体配体释放。目标 2. 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定义功能域。然后我们将生成p40功能域和具有功能域缺失融合蛋白的突变体p40，并确定它们对信号传导和肠道炎症的体外和体内影响。 LGG 调节作用对 p4​​0 的需求将通过使用单交换插入整合系统使 LGG 染色体上的 p40 失活并比较野生型和突变型 LGG 对细胞信号传导和存活的影响来确定。目标 3. 确定 p40 对结肠炎动物模型肠道炎症的体内影响。我们将使用特定的结肠递送策略（果胶/玉米醇溶蛋白水凝胶系统）确定将 p40 递送至结肠的最佳条件。将在两种小鼠结肠炎模型、白细胞介素10和Ras双缺陷激酶抑制剂诱发的结肠炎和葡聚糖硫酸钠诱发的结肠炎中检测p40对预防和/或治疗炎症和肠上皮细胞凋亡的作用。将使用 EGF 受体激酶抑制剂和 EGF 受体缺陷小鼠来分析 p40 对炎症的影响需要 EGF 受体。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。公共健康相关性 该提案将为了解 p40（益生菌乳杆菌 GG 的一种分泌蛋白）调节肠上皮细胞功能的机制提供基础知识，并将确定 p40 在预防和/或治疗肠道炎症方面的体内作用使用结肠炎动物模型。因此，该提案的研究结果将为 p40 用于预防和/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。