Probiotics: Potential Therapeutic Roles in Diarrhea

描述(由申请人提供)：腹泻疾病仍然是全球儿童死亡的主要原因(约120万/年)。在发展中国家，即使在西方国家，IBD引起的腹泻以及食源性病原体或医院获得性感染引起的腹泻，除了死亡率和发病率很高外，仍然是一个重大的医疗挑战。因此，需要更好和新的治疗方式。在这方面，在我们之前的资助周期中的研究为潜在的基于嗜酸乳杆菌(LA)的益生菌改善腹泻的方法提供了开创性的体外和体内证据。我们证明，LA介导的止泻作用的机制是刺激肠道对盐的吸收，包括上调NHE3和DRA(关键的肠顶膜Na+/H+和Cl-/HCO3-交换器)的活性。最重要的是，LA的无细菌培养上清(CS)在刺激氯化钠吸收方面同样有效，定义了一种利用细菌衍生的生物活性分子而不是活细菌来治疗腹泻疾病的新治疗方式。我们的研究进一步表明，LA r LA-CS诱导的Cl-/HCO3-交换活性的增强与顶膜上DRA水平的增加及其转录机制有关。然而，调控LA或LA-CS诱导的DRA运输的变化以及在正常生理和疾病状态下在转录水平上调节DRA表达的详细细胞和分子事件尚不清楚。因此，基于我们已发表的报道和本文提供的强有力的初步数据，我们假设嗜酸乳杆菌无菌培养上清液通过多种机制刺激肠上皮细胞Cl-/HCO3-交换活性，涉及改变DRA的细胞内转运和DRA表达。我们还假设LA或LA-CS可以对抗IBD模型中DRA的抑制。我们将利用体外和体内模型以及最先进的成像和先进的分子生物学技术，通过一套系统的方法来测试这些假设。在特定目标1中提出的研究将探讨LA-CS诱导的DRA在Caco 2细胞单层中的转运的机制，并阐明脂筏、细胞骨架和PDZ蛋白的作用；特定目标2将集中于阐明LA-CS诱导的DRA在Caco 2细胞中转录的机制；以及特定目标3的研究将在包括IBD模型在内的体内小鼠模型中检查LA的短期和长期治疗对DRA功能和膜靶向机制的影响。这些研究不仅将加深我们对益生菌LA促进吸收/止泻作用的机制的了解，而且还将突出LA-CS中分泌的生物活性分子在对抗IBD相关性腹泻方面的潜力。

DESCRIPTION (provided by applicant): Diarrheal diseases remain a leading cause of deaths in children (~1.2 million/year) worldwide. In addition to its significant mortality and morbidity i developing countries, even in western countries, diarrhea associated with IBD and infection by food-borne pathogens or hospital acquired infections remains a major healthcare challenge. Therefore, better and novel treatment modalities are needed. In this regard, studies during our previous funding cycle provided pioneering in vitro and in vivo evidence for a potential probiotic Lactobacillus acidophilus (LA) based approach for the amelioration of diarrhea. We demonstrated that the mechanism of LA-mediated antidiarrheal effects involved stimulation of intestinal NaCl absorption, involving upregulation of the activities of NHE3 and DRA (the key intestinal apical membrane Na+/H+ and Cl-/HCO3- exchangers, respectively). Most importantly, the bacteria-free culture supernatant (CS) of LA was equally effective in stimulating NaCl absorption, defining a novel therapeutic modality to utilize bacteria-derived bioactive molecules, rather than live bacteria, for treating diarrheal disorders. Our studies further indicated that LA r LA- CS-induced enhancement of Cl-/HCO3- exchange activity involved increased levels of DRA on the apical membrane as well as its increased expression via transcriptional mechanisms. However, detailed cellular and molecular events governing LA- or LA-CS-induced alterations in DRA trafficking and regulation of DRA expression at the transcriptional level under normal physiology and under disease states are not known. Therefore, based on our published reports and strong preliminary data presented here we hypothesized that bacteria-free culture supernatant of L. acidophilus stimulates intestinal epithelial Cl-/HCO3- exchange activity via multiple mechanisms involving alterations of both intracellular trafficking of DRA as well as DRA expression. We also hypothesize that LA or LA-CS can combat the repression of DRA in models of IBD. We will test these hypotheses by systematic set of approaches utilizing both in vitro and in vivo models and state-of-the-art imaging and advanced molecular biology techniques. Studies proposed in Specific Aim 1 will investigate the mechanisms of LA-CS induced modulation of DRA trafficking in Caco2 cell monolayers and elucidate the role of lipid rafts, cytoskeletal and PDZ proteins; Specific Aim 2 will focus on elucidating mechanisms of LA-CS-induced stimulation of DRA transcription in Caco2 cells; and studies in Specific Aim 3 will examine the effects of short-term as well as long-term LA treatment on DRA function and membrane targeting mechanisms in in-vivo mouse models including IBD models. These studies will not only enhance our understanding of the mechanisms underlying proabsorptive/antidiarrheal role of probiotic LA but will also highlight the potential of the secreed bioactive molecules in LA-CS in combating the IBD associated diarrhea.