Identification of Lactobacillus Secreted Factors in Regulation of Pgp

描述（申请人提供）：p -糖蛋白（P-gp/MDR1），由ABCB1基因编码，在肠上皮细胞的顶端表面大量表达。Pgp通过介导外源性药物和细菌毒素从粘膜进入肠腔，参与肠上皮的保护。Pgp功能和表达的失调与肠道疾病的发病机制有关，如炎症性肠病（IBD）和结肠炎。在这方面，人类MDR1基因的多态性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为类似人类UC的严重结肠炎。此外，在dss诱导的结肠炎实验小鼠模型中，Pgp的功能和表达也有所下降。因此，阐明提高p -糖蛋白功能的调控机制以保护肠上皮是至关重要的。揭示这种机制可能对肠道炎症的治疗有益。在这方面，我们最近发现益生菌，嗜酸乳杆菌（24 h）培养上清（CS）增加了Pgp在人肠上皮Caco2细胞中的功能和表达。此外，在小鼠的平行研究中显示，在活的嗜酸乳杆菌的作用下，回肠和结肠中Pgp的表达显著增加。嗜酸乳杆菌还能抑制DSS结肠炎小鼠结肠中Pgp mRNA和蛋白表达的减少以及炎症的发生，进一步表明炎症的抑制可能部分是由于嗜酸乳杆菌上调Pgp。L. acidophilus CS通过PI3K、Erk1/2 MAPK通路和Pgp启动子活性的刺激，在基因转录水平上调节Pgp在Caco2细胞中的功能。然而，嗜酸乳杆菌培养上清液中分泌的能增加Pgp功能、表达和启动子活性的生物活性因子的身份尚不清楚。因此，我们在Aims 1 a-c中提出的研究将侧重于根据其物理化学性质鉴定分泌的生物活性因子，并通过蛋白质组学和脂质组学分析进一步表征。目的1d和e将测试所鉴定的蛋白或脂质对Pgp活性和表达的功能功效，以及刺激Pgp基因表达的分子机制。因此，我们的研究旨在鉴定嗜酸乳杆菌分泌的生物活性因子，并确定所鉴定的蛋白或脂质调节Pgp功能和表达的分子机制，将大大提高我们对肠道Pgp功能机制的认识，并可能为新的更有效的治疗方式提供基础，以减轻肠道炎症。自从使用live

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.