Use of Probiotic Conditioned Media to Protect Against Necrotizing Enterocolitis

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，在开始肠道喂养后主要困扰早产儿，可能是几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过度炎症反应，以及肠道血流调节改变导致缺血和氧化剂介导的损伤造成的组织损伤。针对美国国立卫生研究院(NIH)关于研究益生菌制剂治疗儿科疾病的机制的计划公告(PA-06-316)，我们建议测试一种假设，即通过过滤用于培养植物乳杆菌的益生菌发酵液获得的条件培养液(CM)可以通过改善肠道宿主防御机制来降低NEC的发生率和严重性，包括(A)增强肠道屏障功能，(B)诱导细胞保护性热休克蛋白和防止缺血介导的氧化损伤，以及(C)通过抑制NF-kB激活/蛋白酶体功能调节肠道炎症反应。为了控制效果的特异性，来自其他不具有相同生物活性的肠道细菌(如大肠杆菌)的条件培养液将被用作对照。正如计划公告中所述，这项提议的目的是利用由益生菌在无细菌的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验证我们的假设：(1)通过建立大鼠NEC模型，建立益生菌CM(代替活的益生菌)降低NEC的发病率和严重性。(2)探讨CM的保护机制。具体地说，我们将通过Western印迹和ELISA法评估热休克蛋白产生(HSP)，并通过检测氧化剂标记物如黄嘌呤氧化酶、丙二醛和一氧化氮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。通过共聚焦显微镜和蛋白质印迹检测特定紧密连接蛋白的表达。通过对核因子-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等核因子-kB信号通路的序贯评估，来衡量植物乳杆菌CM诱导的核因子-kB/蛋白酶体抑制。我们认为，植物乳杆菌-CM是无菌的，因此使用更安全，将有能力改变肠道细胞保护和促炎影响的平衡，从而保护NEC的肠道损伤。由于目前还没有治疗这种毁灭性疾病的方法，限制肠道损伤的开始或传播并了解其中的机制可能会显著改善预后。 与公共卫生相关：目前还没有针对坏死性小肠结肠炎(NEC)的特效治疗方法，虽然临床试验表明益生菌具有保护作用，但还没有明确的机制。此外，无菌益生菌CM中的生物活性因子可能为传统益生菌的使用提供一种更安全的替代方案。了解益生菌CM如何限制这种毁灭性疾病中肠道损伤的启动或传播，可以显著改善预后。

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.