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）。除了其在发展中国家的显著死亡率和发病率之外，甚至在西方国家，与IBD相关的腹泻和食源性病原体感染或医院获得性感染仍然是主要的医疗保健挑战。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基于益生菌嗜酸乳杆菌（LA）的改善腹泻的潜在方法提供了开创性的体外和体内证据。我们证明了LA介导的抗溃疡作用的机制涉及刺激肠NaCl吸收，涉及上调NHE 3和NHE 4（分别是关键的肠顶膜Na+/H+和Cl-/HCO 3-交换剂）的活性。最重要的是，LA的无菌培养物上清液（CS）在刺激NaCl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治疗牙周病的新治疗方式。我们的研究进一步表明，LA或LA-CS诱导的Cl-/HCO 3-交换活性的增强涉及顶端膜上的增加的水平以及其通过转录机制增加的表达。然而，在正常生理和疾病状态下，在转录水平上，控制LA或LA-CS诱导的β-内酰胺运输和β-内酰胺表达调节的改变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强有力的初步数据，我们假设，无菌培养上清液的L。嗜酸乳杆菌通过多种机制刺激肠上皮Cl-/HCO 3-交换活性，所述机制包括改变胞内转运的α-内酰胺酶以及α-内酰胺酶表达。我们还假设LA或LA-CS可以在IBD模型中对抗对Escherichia coli的抑制。我们将测试这些假设的系统设置的方法，利用在体外和体内模型和国家的最先进的成像和先进的分子生物学技术。具体目标1中提出的研究将研究LA-CS诱导调节Caco 2细胞单层中的β-内酰胺转运的机制，并阐明脂筏、细胞骨架和PDZ蛋白的作用;具体目标2将侧重于阐明LA-CS诱导刺激Caco 2细胞中β-内酰胺转录的机制;和特定目标3中的研究将检查短期以及长期LA治疗对包括IBD模型在内的体内小鼠模型中的膜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/抗腹泻作用的机制的理解，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.