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The airway epithelium in human plays a central role as the first line of defense against environmental contaminants. Most respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. Several muco-active agents have been used to treat hypersecretion symptoms in patients. Current muco-active reagents control mucus secretion by modulating either airway inflammation, cholinergic parasympathetic nerve activities or by reducing the viscosity by cleaving crosslinking in mucin and digesting DNAs in mucus. However, none of the current medication regulates mucus secretion by directly targeting airway goblet cells. The major hurdle for screening potential muco-active agents that directly affect the goblet cells, is the unavailability of in vivo model systems suitable for high-throughput screening. In this study, we developed a high-throughput in vivo model system for identifying muco-active reagents using Xenopus laevis embryos. We tested mucus secretion under various conditions and developed a screening strategy to identify potential muco-regulators. Using this novel screening technique, we identified narasin as a potential muco-regulator. Narasin treatment of developing Xenopus embryos significantly reduced mucus secretion. Furthermore, the human lung epithelial cell line, Calu-3, responded similarly to narasin treatment, validating our technique for discovering muco-active reagents.
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29470529
???displayArticle.pmcLink???PMC5823443 ???displayArticle.link???PLoS One
Fig 1. The composition of the muco-ciliary epithelium of Xenopus laevis embryo.A. Immunofluorescence image of mucus secreting goblet cells and multiciliated cells in Xenopus embryonic epidermis. The membrane was stained using membrane GFP (green), Cilia were detected using α-tubulin (red) and actin by anti-actin antibody (green).
Fig 2. High throughput screening strategy for muco-active reagents from the compounds library using Xenopus embryos.A. Schematic showing the drug screening procedure. Sibling Xenopus embryos were incubated with individual compounds in the library. Then, the media was collected after washing off the compound from the media. The mucus in the media was detected using WGA-HRP and mucus level was analyzed by measuring the optical density (OD) of the HRP substrates. B. Schematic showing the mucus detection procedure using WGA-HRP in ELLA. WGA-HRP can detect the glycosylated region of mucus in the media. C. Dose-dependent linear correlation of OD values with the amount of media used for mucus detection in ELLA.
Fig 3. High throughput screening yielded putative muco-active reagents from the compounds library.A. The result of natural compound library screening. Data were analyzed by calculating the Z-score which indicates how far an individual score is away from the mean of a data set. Four putative muco-active reagents which have a Z-score over 4 or below -4 were selected for further analysis. B. The screening results of a random chemical library from NCBI. The random chemical library did not yield any muco-active reagents.
Fig 4. Putative muco-active reagents affecting mucus secretion level.The mucus level in the media upon the treatment of increasing doses of individual chemicals was normalized to those of control embryos and plotted on the graph (left panel, n = 5). The secreted mucus levels at 50uM of each natural compound were plotted (right panel, n = 39). P-values were calculated from student’s t-test. A. Phorbol 12,13-dibutyrate is a known activator of PKC and promotes mucus secretion. B. Bicuculline is a GABA antagonist and inhibits mucus secretion. In the presence of bicuculline, mucus secretion was strongly inhibited (rectangle line); however, mucus secretion recovered after removing bicuculline (circle line). C. Podophyllotoxin is a known microtubule polymerization inhibitor with anti-viral and anti-tumor activities. Podophyllotoxin strongly inhibited mucus secretion from the Xenopus embryonic epithelium. D. An ionophoric coccidiostat, narasin inhibits mucus secretion.
Fig 5. Bicuculline reversibly inhibits mucus secretion.Mucus secretion was analyzed in the presence of 50uM of bicuculline (A) or after changing the media without bicuculline at every 30 min (B). Mucus secretion was restored within 1 hour after removing bicuculline. P-values were calculated from studentâs t-test (n = 4).
Fig 6. Ionophoric antibiotics narasin inhibits mucus secretion.A. The mucus granules were stained with WGA-Alexa 488 to visualize retained mucus granules. Narasin treated goblet cells (last panel) displayed severe retention of mucus granules. B. Ionophoric antibiotics, Monensin and Nigericin, inhibit mucus secretion similar to narasin. C. Narasin treatment increased intracellular levels of glycosylated protein.
Fig 7. Narasin inhibits mucus secretion in Calu-3 cells.A. Narasin-treated Calu-3 cells were stained with WGA (green) and Muc5ac (Red). Narasin-treated Calu-3 cells accumulated Muc5ac-positive puncta and WGA-stained glycoproteins in the cytosol. B. The number and average size of WGA stained signal was plotted graphically. C. The number of Muc5ac stained puncta was measured and represented graphically. D. Although the PERK level was slightly increased, ER stress was not strongly induced by narasin in Calu-3 cells. Thapsigargin induced strong ER stress response but mucus secretion was not affected. E. Narasin did not severely induce ER stress responses in Xenopus embryos. P-values were calculated from studentâs t-test using the triplicate Q-PCR data set. A.U.; Arbitrary unit.
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