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: 8440768
• 负责人: FANG YAN
• 金额: $ 31.58万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440768
• 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.

项目总结/摘要 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）。但临床 LGG和其他益生菌的应用受到关于其作用机制的信息缺乏的限制。 行动上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来防止苦参碱诱导的细胞凋亡。我们专注于p40，a 分泌的蛋白质，其发挥比p75更有效的作用，并且已经发现p40激活表皮 生长因子（EGF）受体，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两个主要的 IBD的致病因子，这项研究的目的是确定p40调节IBD的机制。 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通过激活抗细胞凋亡信号来预防和/或治疗肠道炎症， 抑制苦参碱诱导肠上皮细胞凋亡。为解决这一问题，提出了三个具体目标 假设：目标1。为了确定p40调节的Akt激活和细胞因子抑制的信号通路， 诱导肠上皮细胞凋亡。我们将重点关注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测定的p40刺激的EGF受体配体释放。目标2.为了确定结构-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 使用缺失突变精确定义功能结构域。然后我们将生成p40函数 结构域和突变型p40与功能结构域缺失的融合蛋白，并确定其在体外和在 对信号传导和肠道炎症的体内影响。p40对LGG调节作用的要求将 通过使用单交叉插入整合使LGG染色体的p40失活来确定 系统中，并比较野生型与突变型LGG对细胞信号传导和存活的影响。目标3.来定义 p40对结肠炎动物模型中肠道炎症的体内作用。我们将确定最佳的 使用特定的结肠递送策略，果胶/玉米醇溶蛋白水凝胶， 系统p40在炎症防治和肠上皮细胞凋亡中的作用 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- 诱发结肠炎和葡聚糖硫酸钠诱发结肠炎。EGF受体对p40作用的需求 使用EGF受体激酶抑制剂和EGF受体缺陷小鼠分析炎症。我们 长期目标是使用p40作为人肠道炎性疾病的新型治疗剂。