Probiotics: Potential Therapeutic Roles in Diarrhea

描述（由申请人提供）：腹泻病仍然是全球儿童死亡的主要原因（约120万/年）。除了在发展中国家，甚至在西方国家，与IBD和食源性病原体感染或医院获得性感染相关的腹泻的死亡率和发病率很高之外，它仍然是一个主要的卫生保健挑战。因此，需要更好和新颖的治疗方式。在这方面，在我们之前的资助周期中的研究为潜在的益生菌嗜酸乳杆菌（LA）改善腹泻的方法提供了开创性的体外和体内证据。我们证明了la介导的止泻作用的机制涉及刺激肠道NaCl吸收，包括NHE3和DRA活性的上调（NHE3和DRA分别是关键的肠尖膜Na+/H+和Cl-/HCO3-交换物）。最重要的是，LA的无细菌培养上清（CS）在刺激NaCl吸收方面同样有效，这定义了一种利用细菌衍生的生物活性分子而不是活细菌治疗腹泻疾病的新治疗方式。我们的研究进一步表明，LA或LA- cs诱导的Cl-/HCO3-交换活性的增强与根尖膜上DRA水平的增加以及其转录机制的表达增加有关。然而，在正常生理和疾病状态下，控制LA-或LA- cs诱导的DRA运输改变和转录水平上DRA表达调节的详细细胞和分子事件尚不清楚。因此，根据我们发表的报告和本文提供的强有力的初步数据，我们假设嗜酸乳杆菌的无细菌培养上清通过多种机制刺激肠上皮细胞Cl-/HCO3-交换活性，包括改变DRA的细胞内运输和DRA的表达。我们还假设LA或LA- cs可以对抗IBD模型中DRA的抑制。我们将通过系统的方法，利用体外和体内模型、最先进的成像和先进的分子生物学技术来检验这些假设。在Specific Aim 1中提出的研究将探讨LA-CS诱导的Caco2细胞单层中DRA运输的调节机制，并阐明脂筏、细胞骨架和PDZ蛋白的作用；特异性目标2将重点阐明la - cs诱导的Caco2细胞中DRA转录的刺激机制；Specific Aim 3的研究将研究短期和长期LA治疗对包括IBD模型在内的体内小鼠模型中DRA功能和膜靶向机制的影响。这些研究不仅将增强我们对益生菌LA的促吸收/止泻作用机制的理解，而且将强调LA- cs分泌的生物活性分子在对抗IBD相关腹泻中的潜力。

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.