Identification of Lactobacillus Secreted Factors in Regulation of Pgp

描述(申请人提供)：由ABCB1基因编码的P-糖蛋白(P-gp/mdr1)在肠上皮细胞顶端大量表达。PGP通过介导外源性物质和细菌毒素从粘膜外流到肠腔，参与肠上皮的保护作用。Pgp功能和表达失调与炎症性肠病(IBD)和结肠炎等肠道疾病的发病机制有关。在这一点上，人类MDR1基因的多态与溃疡性结肠炎和克罗恩病患者肠道P-糖蛋白表达减少有关。此外，mdr1缺陷小鼠会自发发展成类似于人类UC的严重结肠炎。此外，在DSS诱导的实验性结肠炎小鼠模型中，Pgp的功能和表达也出现了下降。因此，阐明增强P-糖蛋白功能的调控机制对肠上皮的保护至关重要。解开这些机制可能有益于肠道炎症的治疗。在这方面，我们最近发现，益生菌，嗜酸乳杆菌(24小时)培养上清(CS)增加了人肠上皮细胞中Pgp的功能和表达。此外，在小鼠身上进行的平行研究表明，对活的嗜酸乳杆菌的反应，回肠和结肠中Pgp的表达显著增加。嗜酸乳杆菌还可抑制DSS结肠炎小鼠结肠中Pgp基因和蛋白的表达，抑制炎症反应，进一步提示嗜酸乳杆菌上调Pgp表达可能是其抑制炎症反应的部分原因。嗜酸乳杆菌CS通过PI3K、ERK1/2 MAPK通路和Pgp启动子活性的刺激在基因转录水平调控Pgp功能。然而，嗜酸乳杆菌培养上清液中存在的促进Pgp功能、表达和启动子活性的分泌型生物活性因子(S)的身份尚不清楚。因此，我们在AIMS 1a-c中提出的研究将集中在分泌型生物活性因子(S)的理化性质的基础上，并通过蛋白质组和脂体学分析进行进一步的表征。AIMS 1D和E将检测已鉴定的蛋白(S)或脂类(S)对Pgp活性和表达的作用效果，以及刺激Pgp基因表达的分子机制。因此，本研究旨在鉴定嗜酸乳杆菌分泌的生物活性因子(S)，并确定所鉴定的蛋白(S)或脂类(S)调节Pgp功能和表达的分子机制，将极大地加深我们对肠道Pgp功能机制的理解，并可能为新的、更有效的缓解肠道炎症的治疗方法提供基础。由于使用LIVE 细菌在治疗肠道疾病中可能是一个潜在的风险，使用分泌的生物活性因子(S)而不是活的细菌将为它们的治疗使用带来一种创新的方法。 公共卫生相关性：肠腔壁健康的紊乱导致与各种肠道疾病相关的肠道炎症的发展，如炎症性肠病(IBD)和结肠炎。P-糖蛋白(P-gp)通过介导药物/异物和细菌毒素从肠壁进入肠腔，在保护肠道屏障中发挥重要作用。在IBD患者和实验性结肠炎小鼠模型中，Pgp的功能和表达都有所下降。我们最近发现，嗜酸乳杆菌的益生菌培养上清可以增加肠道上皮细胞的Pgp功能和表达，这可能有助于益生菌在炎症中的有益作用。本申请中提出的研究将确定调节PGP的新的分泌型生物活性因子(S)，并可能作为潜在的治疗药物用于预防和治疗IBD。

DESCRIPTION (provided by applicant): P-glycoprotein (P-gp/MDR1), encoded by the ABCB1 gene is abundantly expressed on the apical surface of intestinal epithelial cells. Pgp is involved in the protection of intestinal epithelia by mediating the efflux of xenobiotics and bacterial toxis from the mucosa into the lumen. Dysregulation of Pgp function and expression have been implicated in the pathogenesis of intestinal disorders, such as inflammatory bowel diseases (IBD) and colitis. In this regard, polymorphisms in the human MDR1 gene have been associated with reduced intestinal P-glycoprotein expression in patients with ulcerative colitis and Crohn's disease. Also, MDR1- deficient mice spontaneously develop severe colitis resembling human UC. Further, a decrease in function and expression of Pgp has been shown in experimental mouse model of DSS-induced colitis. Therefore, it is critical to delineate the regulatory mechanisms that increase P-glycoprotein function in order to protect the intestinal epithelium. Unraveling such mechanisms may be beneficial in the treatment of intestinal inflammation. In this regard, we have recently shown that probiotic, Lactobacillus acidophilus (24 h) culture supernatant (CS) increased Pgp function and expression in human intestinal epithelial Caco2 cells. Also, in parallel studies in mice demonstrated a significant increase in Pgp expression in the ileum and colon in response to live L. acidophilus bacteria. L. acidophilus also blocked the reduced expression of Pgp mRNA and protein expression as well as inflammation in the colon of DSS colitis mice further suggesting that suppression of inflammation could be in part due to the up-regulation of Pgp by L. acidophilus. The increase in Pgp function in Caco2 cells by L. acidophilus CS occurred via PI3K, Erk1/2 MAPK pathways and stimulation of Pgp promoter activity indicating modulation at the level of gene transcription. However, the identity of the secreted bioactive factor (s) present in the culture supernatant of L. acidophilus that increase Pgp function, expression and promoter activity are not known. Therefore, our studies proposed in Aims 1 a-c will focus on identifying the secreted bioactive factor (s) on the basis of their physicochemical properties and further characterization by proteomic and lipidomic analysis. Aims 1d & e will test the functional efficacy of the identified protein (s) or lipid (s) on Pgp actvity and expression and the molecular mechanisms involved in the stimulation of Pgp gene expression. Therefore, our studies aimed at identifying the bioactive factor (s) secreted by L. acidophilus and determining the molecular mechanisms underlying the regulation of Pgp function and expression by the identified protein (s) or lipid (s) will greatly enhance our understanding of the mechanisms of intestinal Pgp function and may provide the basis for new and more efficacious treatment modalities for alleviating intestinal inflammation. Since using live bacteria could be a potential risk in the treatment of gut disorders, using secreted bioactive factor (s) instead of live bacteria would lead to an innovative approach for their therapeutic usage. PUBLIC HEALTH RELEVANCE: Disturbances in the intestinal luminal wall health leads to the development of intestinal inflammation associated with various intestinal diseases such as inflammatory bowel diseases (IBD) and colitis. P-glycoprotein (Pgp) plays a critical role in the protection of the intestinal barrier by mediating the removal of drugs/xenobiotics and bacterial toxins from the intestinal wall into the gut cavity. A decrease in function and expression of Pgp has been shown in patients with IBD and in experimental mouse model of colitis. We have recently shown that the probiotic, L. acidophilus culture supernatant increases Pgp function and expression in intestinal epithelial cells, that could contribute to the beneficial effects of probitics in inflammation. The studies proposed in this application will identify the novel secreted bioactive factor (s) that modulate Pgp and could be used as potential therapeutic agents in the prevention and treatment of IBD.