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postgraduate thesis: Polyphenol-mediated protective effects against bacterial translocation across intestinal epithelial cells and their mechanisms
Title | Polyphenol-mediated protective effects against bacterial translocation across intestinal epithelial cells and their mechanisms |
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Authors | |
Issue Date | 2015 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Ling, K. [凌加豪]. (2015). Polyphenol-mediated protective effects against bacterial translocation across intestinal epithelial cells and their mechanisms. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5699899. |
Abstract | An intact intestinal barrier maintains gut health by keeping pathogens at bay. Normally, the intestinal barrier is very effective —the luminal side is heavily infested with more than 1012microbes per milliliter of fecal materials, yet the basolateral side (portal blood and mesenteric lymph nodes) is virtually sterile. However, insults from the external environment, such as food toxins, drugs and pathogens, can undermine intestinal integrity. A compromised intestinal barrier (leaky gut) is prone to bacterial translocation, predisposing one to various gastrointestinal disorders. Polyphenols (PPs) are widely present in plant-based foods and beverages, with daily human consumption estimated at up to several hundred milligrams. Therefore, intestinal epithelial cells (IECs) are constantly and directly exposed to a high dose of PPs. However, the effect of PPs on the intestinal barrier remains largely unexplored. The present study aimed to identify dietary PPs that can improve gut health by reducing bacterial translocation across IEC monolayers. Principal action mechanism(s) were characterized.
Cell-based in vitroassays were performed using polarized porcine IECs, IPEC-J2, cultured on permeable membrane supports. Translocation of Escherichia coliacross the cell monolayers was first determined by agar plate counting. Further mechanistic analyses —applying electrophysiology assay, unidirectional tracer flux assay, antibacterial assay, quantitative reverse transcriptase polymerase chain reaction, protein immunoblotting, enzyme-linked immunosorbent assay, and Luminex multiplex assay —were then undertaken to characterize PP-mediated protective effects in normal and deoxynivalenol (DON)-damaged cell monolayers.
Selected dietary PPs, including chlorogenic acid, epigallocatechin gallate (EGCG), quercetin, resveratrol (RES) and rutin, were screened. It was revealed that EGCG and RES, present in green tea and red wine respectively, significantly reduced E. colitranslocation. In further mechanistic investigation, the two PPs were shown to function differently: RES enhanced the physical barrier (strengthening tight junctions), while EGCG enhanced the immunological barrier (inducing antimicrobial peptides secretion). In detail, RES promoted the assembly of claudin-4 to the tight junction complex in normal cells, and protected against DON-induced barrier dysfunction by preventing dissociation of claudin-4 from actin cytoskeleton, through modulation of IL-6 and IL-8 secretion in a mitogen activated protein kinase (MAPK)-dependent response. Meanwhile, EGCG reduced bacterial translocation, in normal and DON-damaged cells, by stimulating the release of cytoplasmic β-defensin-1 via a MAPK-independent pathway, as well as upregulating gene expression and production ofβ-defensin-2 via a p38 MAPK signaling pathway.
The present study reveals, for the first time, that PPs can reduce bacterial translocation across IECs. Our findings have highlighted a great potential of dietary intervention with PPs to uphold intestinal integrity. |
Degree | Doctor of Philosophy |
Subject | Polyphenols Epithelial cells Intestines - Microbiology |
Dept/Program | Biological Sciences |
Persistent Identifier | http://hdl.handle.net/10722/236746 |
HKU Library Item ID | b5699899 |
DC Field | Value | Language |
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dc.contributor.author | Ling, Ka-ho | - |
dc.contributor.author | 凌加豪 | - |
dc.date.accessioned | 2016-12-02T23:27:08Z | - |
dc.date.available | 2016-12-02T23:27:08Z | - |
dc.date.issued | 2015 | - |
dc.identifier.citation | Ling, K. [凌加豪]. (2015). Polyphenol-mediated protective effects against bacterial translocation across intestinal epithelial cells and their mechanisms. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5699899. | - |
dc.identifier.uri | http://hdl.handle.net/10722/236746 | - |
dc.description.abstract | An intact intestinal barrier maintains gut health by keeping pathogens at bay. Normally, the intestinal barrier is very effective —the luminal side is heavily infested with more than 1012microbes per milliliter of fecal materials, yet the basolateral side (portal blood and mesenteric lymph nodes) is virtually sterile. However, insults from the external environment, such as food toxins, drugs and pathogens, can undermine intestinal integrity. A compromised intestinal barrier (leaky gut) is prone to bacterial translocation, predisposing one to various gastrointestinal disorders. Polyphenols (PPs) are widely present in plant-based foods and beverages, with daily human consumption estimated at up to several hundred milligrams. Therefore, intestinal epithelial cells (IECs) are constantly and directly exposed to a high dose of PPs. However, the effect of PPs on the intestinal barrier remains largely unexplored. The present study aimed to identify dietary PPs that can improve gut health by reducing bacterial translocation across IEC monolayers. Principal action mechanism(s) were characterized. Cell-based in vitroassays were performed using polarized porcine IECs, IPEC-J2, cultured on permeable membrane supports. Translocation of Escherichia coliacross the cell monolayers was first determined by agar plate counting. Further mechanistic analyses —applying electrophysiology assay, unidirectional tracer flux assay, antibacterial assay, quantitative reverse transcriptase polymerase chain reaction, protein immunoblotting, enzyme-linked immunosorbent assay, and Luminex multiplex assay —were then undertaken to characterize PP-mediated protective effects in normal and deoxynivalenol (DON)-damaged cell monolayers. Selected dietary PPs, including chlorogenic acid, epigallocatechin gallate (EGCG), quercetin, resveratrol (RES) and rutin, were screened. It was revealed that EGCG and RES, present in green tea and red wine respectively, significantly reduced E. colitranslocation. In further mechanistic investigation, the two PPs were shown to function differently: RES enhanced the physical barrier (strengthening tight junctions), while EGCG enhanced the immunological barrier (inducing antimicrobial peptides secretion). In detail, RES promoted the assembly of claudin-4 to the tight junction complex in normal cells, and protected against DON-induced barrier dysfunction by preventing dissociation of claudin-4 from actin cytoskeleton, through modulation of IL-6 and IL-8 secretion in a mitogen activated protein kinase (MAPK)-dependent response. Meanwhile, EGCG reduced bacterial translocation, in normal and DON-damaged cells, by stimulating the release of cytoplasmic β-defensin-1 via a MAPK-independent pathway, as well as upregulating gene expression and production ofβ-defensin-2 via a p38 MAPK signaling pathway. The present study reveals, for the first time, that PPs can reduce bacterial translocation across IECs. Our findings have highlighted a great potential of dietary intervention with PPs to uphold intestinal integrity. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.subject.lcsh | Polyphenols | - |
dc.subject.lcsh | Epithelial cells | - |
dc.subject.lcsh | Intestines - Microbiology | - |
dc.title | Polyphenol-mediated protective effects against bacterial translocation across intestinal epithelial cells and their mechanisms | - |
dc.type | PG_Thesis | - |
dc.identifier.hkul | b5699899 | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Biological Sciences | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_b5699899 | - |
dc.identifier.mmsid | 991018964919703414 | - |