Use of Probiotic Conditioned Media to Protect Against Necrotizing Enterocolitis

描述（由申请人提供）：坏死性小肠结肠炎（NEC）是一种炎症性肠坏死，主要发生在开始肠内喂养后的早产儿，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肠道微生物菌群的炎症反应夸大，以及肠道血流调节改变导致氧化介导的损伤引起缺血和组织损伤。为了响应NIH项目公告（PA-06-316），研究益生菌制剂治疗儿科疾病的机制，我们提出验证这样一个假设，即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)细胞保护性热休克蛋白的诱导和对缺血介导的氧化损伤的保护，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，将使用来自其他肠道细菌的条件培养基，这些细菌不表现出相同类型的生物活性（例如大肠杆菌）。正如项目公告中所述，本提案的目的是利用由益生菌在无细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的来验证我们的假设：(1)通过大鼠NEC模型，建立益生菌CM（而不是活的益生菌）降低NEC的发病率和严重程度。(2)探讨CM保护机制。具体来说，我们将通过Western blot和ELISA来评估热休克蛋白的产生（Hsp），并通过测量氧化标记物（如黄嘌呤氧化酶、丙二醛和一氧化氮的产生）来评估氧化介导损伤的差异。屏障功能将通过使用fitc -葡聚糖进行体内渗透性研究来测量。特异性紧密连接蛋白的表达将通过共聚焦显微镜和western blot检测。植物草cm诱导的NF-kB/蛋白酶体抑制将通过NF-kB信号通路的顺序评估来测量，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是无细菌的，因此使用更安全，可能有能力改变肠道细胞保护和促炎影响的平衡，从而防止NEC的肠道损伤。由于这种毁灭性疾病没有治疗方法，限制肠道损伤的开始或传播并了解所涉及的机制可以显著改善结果。

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.