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.

项目摘要/摘要 鼠李糖乳杆菌(lactobacillusrhamnosus gg，lgg)是临床试验中研究最多的益生菌之一。 和/或预防几种肠道疾病，包括炎症性肠病(IBD)。然而，临床上 由于缺乏对免疫球蛋白和其他益生菌作用机制的了解，限制了其应用。 行动。我们已经成功地纯化并克隆了两个新的LGG衍生的可溶性蛋白(p40和p75)。 通过激活肠上皮细胞内Akt来预防细胞因子诱导的细胞凋亡。我们关注的是p40，a 一种比p75更有效的分泌蛋白，并发现p40能激活表皮。 生长因子(EGF)受体，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的凋亡是两个主要的 IBD的致病因子，本研究计划的目的是定义p40调节机制。 肠上皮细胞功能，并测定p40在肠道炎症中的作用。我们将测试 假设p40通过激活抗凋亡信号来预防和/或治疗肠炎 抑制细胞因子诱导的肠上皮细胞凋亡。为了解决这一问题，我们提出了三个具体目标 假设：目的1.确定p40调节的Akt激活和抑制细胞因子的信号通路。 诱导肠上皮细胞凋亡。我们将重点确定EGF受体的需求 P40对缺乏EGF的肠上皮细胞Akt激活和细胞凋亡的抑制作用 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 酶联免疫吸附试验检测P40刺激的EGF受体配体释放。目的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要求。我们会 利用缺失突变精确定义功能结构域。然后我们将生成p40泛函 结构域和突变体p40与功能结构域缺失融合蛋白的融合，并测定它们的体外和体内 活体对信号和肠道炎症的影响。P40对LGG的调节效果的要求将 通过使用单交叉插入整合从LGG染色体中灭活p40来确定 系统和比较野生型和突变型lgg对细胞信号和存活的影响。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递送到结肠的条件 系统。P40在防治炎症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，白介素10和RAS双缺乏的激酶抑制物- 诱发结肠炎和葡聚糖硫酸钠诱导的结肠炎。表皮生长因子受体对p40‘S作用的要求 炎症将使用EGF受体激酶抑制剂和EGF受体缺陷小鼠进行分析。我们的 长期目标是使用p40作为一种新的治疗人类肠炎性疾病的药物。