Use of Probiotic Conditioned Media to Protect Against Necrotizing Enterocolitis

使用益生菌条件培养基预防坏死性小肠结肠炎

• 批准号: 7532220
• 负责人: Erika C Claud
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7532220
• 关键词: Affect; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Bacteria; Binding; Blood flow; Childhood; Clinical; Clinical Trials; Complex; Conditioned Culture Media; Confocal Microscopy; Cytoprotection; Data; Diagnosis; Diagnostic; Disease; Drug Formulations; Enteral Feeding; Enterocolitis; Enzyme-Linked Immunosorbent Assay; Equilibrium; Etiology; Evaluation; Functional disorder; Goals; Heat shock proteins; Histopathologic Grade; Histopathology; Host Defense Mechanism; Immunocompromised Host; Incidence; Infection; Inflammation; Inflammatory; Inflammatory Response; Injury; Intestines; Investigation; Ischemia; Lactobacillus plantarum; Life; Liquid substance; Malondialdehyde; Measurement; Measures; Mediating; Microbe; Modeling; Morbidity - disease rate; NF-kappa B; NFKB Signaling Pathway; NIH Program Announcements; Necrosis; Necrotizing Enterocolitis; Neonatal; Nitric Oxide; Nuclear; Outcome; Oxidants; Pathway interactions; Permeability; Peroxidase; Physiological; Predisposition; Premature Infant; Prematurity of fetus; Probiotics; Production; Property; Proteasome Inhibition; Protein Isoforms; Proteins; Protocols documentation; Public Health; Radiologic Finding; Rattus; Regulation; Risk; Risk Factors; Sepsis; Severities; Solutions; Specific qualifier value; Specificity; Staining method; Stains; Stress; Testing; Tight Junctions; Tissues; Ubiquitination; Western Blotting; Xanthine Oxidase; concept; cytokine; fluorescein isothiocyanate dextran; improved; in vivo; intestinal epithelium; microbial; mortality; multicatalytic endopeptidase complex; protein expression; response

DESCRIPTION (provided by applicant): Necrotizing enterocolitis (NEC), an inflammatory bowel necrosis that primarily afflicts preterm infants after the initiation of enteral feeds, is likely the result of synergy of several risk factors including altered intestinal barrier function leading to an exaggerated inflammatory response to gut microbial flora as well as altered intestinal blood flow regulation leading to ischemia and tissue injury from oxidant-mediated damage. In response to a NIH program announcement (PA-06-316) for investigations into mechanisms of probiotic formulation for treatment of pediatric illnesses, we propose to test the hypothesis that conditioned media (CM) obtained by filtering the broth used to grow the probiotic bacteria Lactobacillus plantarum can decrease the incidence and severity of NEC by improving intestinal host defense mechanisms, including (A) enhancement of intestinal barrier function, (B) induction of cytoprotective heat shock proteins and protection against ischemia-mediated oxidant injury, and (C) modulation of intestinal inflammatory responses by inhibition of NF-kB activation/proteasome function. To control for specificity of effect, conditioned media from other gut bacteria which do not display the same type of bioactivity (e.g. E.coli) will be used as controls. As stated in the program announcement, the intent of this proposal is to utilize a complex formulation of natural bioactive components synthesized and secreted by the probiotics in a solution that is bacteria-free. We will test our hypothesis through the following specific aims: (1) Establish that probiotic CM (instead of live probiotic bacteria) decreases the incidence and severity of NEC by using a rat NEC model. (2) Investigate the mechanism of CM protection. Specifically, we will evaluate heat shock protein production (Hsp) by Western blot and ELISA, and also evaluate differences in oxidant-mediated injury by measuring oxidant markers such as xanthine oxidase, malondialdehyde, and nitric oxide production. Barrier function will be measured by in vivo permeability studies using FITC-dextran. Expression of specific tight junction proteins will be measured by confocal microscopy and western blot. L.plantarum CM-induced NF-kB/proteasome inhibition will be measured by a sequential evaluation of the NF-kB signaling pathway including NF-kB binding activity and nuclear localization, IkB degradation and ubiquitination, proteasome activity, and cytokine release. We propose that L. plantarum-CM, which is bacteria-free and thus safer to use, will have the capacity to alter the balance of intestinal cytoprotective and pro-inflammatory influences and thus protect against intestinal injury in NEC. Since there is no treatment for this devastating disease, limiting the initiation or propagation of intestinal injury and understanding the mechanisms involved could significantly improve outcome. PUBLIC HEALTH RELEVANCE: There is no specific treatment for necrotizing enterocolitis (NEC), and while clinical trials suggest that probiotics are protective, no mechanism has been delineated. Furthermore, bioactive factors in bacteria-free probiotic CM may provide a safer alternative to conventional probiotic use. Understanding how probiotic CM may limit the initiation or propagation of intestinal injury in this devastating disease could significantly improve outcome.

描述（申请人提供）：坏死性小肠结肠炎（NEC）是一种炎症性肠坏死，主要影响开始肠内喂养后的早产儿，可能是多种危险因素协同作用的结果，包括肠道屏障功能改变导致对肠道微生物菌群的炎症反应过度，以及肠道血流调节改变导致缺血和组织损伤。 氧化剂介导的损伤。为了响应 NIH 计划公告 (PA-06-316)，调查益生菌制剂治疗儿科疾病的机制，我们建议测试以下假设：通过过滤用于生长益生菌植物乳杆菌的肉汤获得的条件培养基 (CM) 可以通过改善肠道宿主防御机制来降低 NEC 的发病率和严重程度，包括 (A) 增强 肠道屏障功能，(B) 诱导细胞保护性热休克蛋白并防止缺血介导的氧化损伤，以及 (C) 通过抑制 NF-kB 激活/蛋白酶体功能来调节肠道炎症反应。为了控制效果的特异性，来自不表现出​​相同类型生物活性的其他肠道细菌（例如大肠杆菌）的条件培养基将用作对照。正如计划公告中所述，该提案的目的是利用益生菌在无菌溶液中合成和分泌的天然生物活性成分的复杂配方。我们将通过以下具体目标来检验我们的假设：（1）通过使用大鼠 NEC 模型，确定益生菌 CM（而不是活益生菌）可降低 NEC 的发生率和严重程度。 (2)研究CM保护机制。具体来说，我们将通过蛋白质印迹和 ELISA 评估热休克蛋白 (Hsp) 的产生，并通过测量氧化剂标记物（如黄嘌呤氧化酶、丙二醛和一氧化氮的产生）来评估氧化剂介导的损伤的差异。屏障功能将通过使用 FITC-葡聚糖的体内渗透性研究来测量。特定紧密连接蛋白的表达将通过共聚焦显微镜和蛋白质印迹来测量。植物乳杆菌 CM 诱导的 NF-kB/蛋白酶体抑制将通过对 NF-kB 信号通路的连续评估进行测量，包括 NF-kB 结合活性和核定位、IkB 降解和泛素化、蛋白酶体活性和细胞因子释放。我们认为，植物乳杆菌-CM 是无菌的，因此使用起来更安全，将有能力改变肠道细胞保护和促炎影响的平衡，从而防止 NEC 中的肠道损伤。由于这种毁灭性的疾病无法治疗，限制肠道损伤的发生或传播并了解相关机制可以显着改善结果。 公众健康相关性：坏死性小肠结肠炎 (NEC) 尚无特效治疗方法，虽然临床试验表明益生菌具有保护作用，但尚未阐明其机制。此外，无菌益生菌 CM 中的生物活性因子可能为传统益生菌使用提供更安全的替代品。了解益生菌 CM 如何限制这种破坏性疾病中肠道损伤的发生或传播，可以显着改善预后。