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Improvement of Human Oral Epithelial Barrier Function and Tight Junctions by Micronutrients Elizabeth Rybakovsky, Mary Carmen Valenzano, Rachael Deis, Katherine M. DiGuilio, Sunil Thomas, and James M. Mullin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04203 • Publication Date (Web): 25 Nov 2017 Downloaded from http://pubs.acs.org on November 27, 2017

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Journal of Agricultural and Food Chemistry

Improvement of Human Oral Epithelial Barrier Function and Tight Junctions by Micronutrients

Elizabeth Rybakovsky, Mary Carmen Valenzano, Rachael Deis, Katherine M. DiGuilio, Sunil Thomas and James M. Mullin* Lankenau Institute for Medical Research, 100 E. Lancaster Ave., Wynnewood, PA 19096 USA [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

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Running Title: Micronutrient Enhancement of Oral Epithelial Tight Junctions

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This research did not receive any specific grant from funding agencies in the public, commercial or notfor-profit sectors. The authors declare no competing financial interest.

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*Corresponding author: James M. Mullin, Lankenau Institute for Medical Research, 100 E. Lancaster Ave., Wynnewood, PA 19096, phone: 484-476-2703, fax: 484-476-2205, [email protected]

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1 ACS Paragon Plus Environment


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Abstract

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The oral epithelium represents a major interface between an organism and its external

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environment. Improving this barrier at the molecular level can provide an organism added

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protection from microbial-based diseases. Barrier function of the Gie-3B11 human gingival

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epithelial cell culture model is enhanced by the micronutrients zinc, quercetin, retinoic acid and

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acetyl-11-keto-β-boswellic acid, as observed by a concentration-dependent increase in

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transepithelial electrical resistance and a decrease in transepithelial 14C-D-mannitol

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permeability. With this improvement of tight junction (TJ) barrier function (reduced leak)

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comes a pattern of micronutrient-induced changes in TJ claudin abundance that is specific to

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each individual micronutrient, along with changes in claudin subcellular localization. These

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micronutrients were effective not only when administered simultaneously to both cell surfaces

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but also to the apical surface alone, the surface to which the micronutrients would be

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presented in routine clinical use. Biomedical implications of micronutrient enhancement of the

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oral epithelial barrier are discussed.

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Keywords: gingival; tight junction; claudin; zinc; quercetin; retinoic acid; AKBA


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Introduction

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Epithelial mucosal layers perform many varied functions in the body, but their primary function is

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their simplest and most ubiquitous — a physical barrier between a luminal compartment in contact with

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the apical epithelial surface, and interstitial fluid/ vasculature – an antiluminal compartment - facing the

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epithelium’s basal-lateral surface. This barrier is formed fundamentally by two elements: the cells per

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se and the circumferential, continuous tight junction (TJ) (zonula occludens) complex that surrounds

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every epithelial cell like a gasket. For epithelia that engage intensely in absorptive functions, such as in

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the intestinal tract or the renal tubules, the barrier function has an obvious role in preventing “back

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leak” of salts/nutrients/water that were just reabsorbed by the epithelial cells, a short circuiting of a

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highly energy-dependent process. However, for those epithelia and for others that are not principally

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engaged in absorptive or secretory functions, such as the esophagus or the urinary bladder, the barrier

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function has another key role.

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Luminal compartments typically are in open communication with the external environment, and as

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such can contain an enormous array of potential pathogens as well as harmful antigenic substances. A

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vital (secondary) role of the epithelial barrier is to keep the antigens and pathogens that populate

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luminal compartments sequestered there so they do not cross the barrier and engage the immune

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system in an unregulated manner. Such unregulated presentation can result in an inflammatory

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response, sometimes chronically, as exemplified by inflammatory bowel diseases. This secondary role of

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the barrier thus keeps microbial pathogens out of the systemic circulation, which in clinical settings

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could prevent sepsis. This could likewise also prevent these pathogens from triggering an immune

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response thereby preventing or reducing inflammation. It is noteworthy that the vast majority of

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human diseases of microbial origin begin with a pathogen contacting the luminal surface of an epithelial

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barrier and then exerting a variety of evolutionary mechanisms to get across the barrier and infect the

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organism systemically. In addition to this eventuality of pathogens inducing leak, there is also the 3 ACS Paragon Plus Environment


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situation of pathogens able to take advantage of leakiness, i.e. of already existing compromises in

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epithelial barrier function. Given that both neoplasia and inflammation induce leakiness in TJ seals and

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in barriers generally,1 this would be a quite common occurrence clinically.

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Many things can go into the mouth of an organism, and this makes the oral epithelium a particular

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hot spot with regard to the above-mentioned infectious disease-related barrier function. With the

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possible exception of the epidermis and the upper airways, no other epithelia see the variety and

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quantity of pathogens with which the oral epithelia are presented. Accordingly, understanding the

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means by which the oral epithelium regulates its TJ seals and responds to injury of all kinds is a

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compelling undertaking from the viewpoint of infectious disease.

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The gingival epithelia of the oral cavity are not one epithelial cell type but many (as reviewed

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excellently in Groeger and Meyle).2 There are keratinized, stratified squamous epithelia (gingiva and

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papillae), as well as stratified squamous non-keratinized epithelia (oral sucular epithelium). The

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junctional epithelium is squamous, but non-keratinized and non-stratifying.

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The epithelial cell culture Gie-3B11, used in the studies reported here, originated from a biopsy of

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normal gingival tissue of the upper jaw.3 Immortalization of cells of the primary cultures was done by

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means of transfecting genes coding for the human papillomavirus (HPV) oncoproteins E6 and E7. As

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reported by Groger et al.,3 Gie-3B11 cells can form a functional polarized epithelium with a measurable

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transepithelial electrical resistance (Rt). It is worth noting that unlike the more widely used renal

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(MDCK) and intestinal (CACO-2) epithelial cell culture models, the Rt of Gie-3B11 reaches a maximal

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value only a few days after seeding a confluent density of cells, then declines to 20% of peak Rt only a

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few days later. The cytokeratin expression pattern of Gie-3B11 is similar to that of primary human

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gingival epithelial cells.2


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Encompassing the work of many laboratories worldwide, there is now full realization that a select

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group of micronutrients and nutraceuticals have the capability to induce structural changes in epithelial

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TJ complexes that result in reduced leakiness across many different epithelial barriers.4-13 These

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substances range from dietary constituents such as the procyanidins of grape seeds to metabolic

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endproducts of the gastrointestinal microbiome such as butyrate.

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findings is the observation that retinoic acid can significantly improve the barrier function of the Gie-

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3B11 cell layer, while also enhancing expression of the TJ proteins claudin-4 and occludin.16 Our current

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studies represent a further look into micronutrient-mediated modification of this oral epithelial barrier

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model, with a specific focus on the TJ complex. The clinical relevance of such studies will be in future

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micronutrient-based oral prophylaxis that would improve oral epithelial barrier function basally and/or

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reduce the induced leakiness that comes from various pathophysiologies.

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Materials and Methods

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Cell culture

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14, 15

Included among this growing list of

The Gie-3B11 cell culture, an immortalized human gingival keratinocyte cell line, was purchased

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from Applied Biological Materials (Richmond, BC, Canada), and was used between passages 2 and 16.

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When cells reached confluence, they were passaged by trypsinization (0.25% trypsin and 2.21 mM EDTA

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[Mediatech, Inc [a Corning subsidiary, Manassas, VA, USA]) on a weekly basis. Cells were routinely

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seeded at 2 x 105 cells/Falcon 75cm2 culture flask with 25 ml of Prigrow IV medium (Applied Biological

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Materials) supplemented with 2mM L-Glutamine (Mediatech, Inc.) and 5% fetal bovine serum (Seradigm

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(VWR, Inc., Radnor, PA, USA). Cultures were incubated at 37°C in 95% air/5% CO2 atmosphere.

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Media Supplementation with Nutraceuticals

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A zinc stock solution was made from zinc sulfate (Thermo Fisher Scientific, Waltham, MA, USA) in distilled, deionized water. Theaflavin (Camellia Sinensis) (Sigma-Aldrich Corp., St. Louis, MO, USA),



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retinoic acid (Sigma-Aldrich), and acetyl-11-keto-β-boswellic acid (AKBA) (Enzo Life Sciences, Inc.,

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Farmingdale, NY, USA) stock solutions were made in absolute ethanol. The retinoic acid stock solution

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required heating to approximately 37°C with vortexing for complete solubilization. A berberine (Sigma-

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Aldrich) stock solution was made in distilled, deionized water. For quercetin, a stock solution was not

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made. Instead quercetin (Sigma-Aldrich) was dissolved directly in culture medium to its working

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concentration and heated to no higher than 40°C for complete solubilization at a 400 µM concentration.

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Serial dilutions in culture medium were then made from the 400 µM concentration. Proper solvent

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controls were performed in all experiments.

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Transepithelial Electrophysiology and Permeability

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Cells were seeded into sterile Millipore Millicell polycarbonate (PCF) units (30 mm diameter with 0.4

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µm pore size) (EMD Millipore, Burlington, MA, USA) on day 0 at a seeding density of 5 x 105 cells/4.2 cm2

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insert. Three or four sterile Millicell PCF inserts were placed into a 100 mm petri dish. On day 1, all cell

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layers were refed (2 ml apical/15 ml basal-lateral) with control medium containing 50 U/ml penicillin

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and 50 µgms/ml streptomycin. All nutraceutical treatments were performed 3 days post-seeding of cell

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layers, and exposure time was 48 hours (with the exception of berberine, which was a 17-hour exposure

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time based upon earlier observations in CACO-2 gastrointestinal epithelia.7 Electrophysiological

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measurements and radiotracer flux studies with 14C-D-mannitol followed.

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On the day of transepithelial permeability experiments, the cell layers were refed with fresh control

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medium and allowed to incubate for 1.5 to 2 hours prior to electrophysiological readings. Rt was

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measured using 1 sec, 40 µamp direct current pulses, and calculated using Ohm’s law. As soon as

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electrical measurements were completed, the basal-lateral medium was aspirated and replaced with 15

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ml of medium containing 0.1 mM, 0.1 µCi/ml 14C-D- mannitol (PerkinElmer, Inc., Boston, MA, USA) and

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incubated at 37°C for 90 min. Triplicate basal-lateral medium samples were taken for liquid scintillation 6 ACS Paragon Plus Environment

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counting (LSC) for specific activity determination. Duplicate samples were taken from the apical side for

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LSC to determine mannitol flux rates. The flux rate (Jm) (in cpm/min/cm2) was calculated for the 14C-D-

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mannitol diffusing across the cell layer. Using the measured specific activity (cpm/µmole), flux was then

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expressed as pmoles/min/cm2.

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Immunoblot Analyses of Tight Junctional Proteins

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Cells were harvested from Millicell PCF rings by first washing 5 times in cold phosphate buffered

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saline, then adding 600 ul of lysis buffer with protease and phosphatase inhibitors to each PCF, scraping

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the cell layer, collecting the suspension, flash-freezing, then storing at -80oC. Whole cell lysates were

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prepared by sonication and ultracentrifugation. Samples of these lysates were analyzed by PAGE

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analysis using a 4%-20% gradient Tris-glycine gel (Invitrogen, a division of Thermo Fisher Scientific) at

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125V for 1 hour and 40 minutes. Precision Plus Kaleidoscope Protein Standards (Bio-Rad, Inc., Hercules,

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CA, USA) were also included on each gel. Proteins were transferred at 30V for 2 hours from the gel to a

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PVDF membrane. The membranes were then washed 3 times with PBS-T (0.3% Tween 20) for 10

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minutes each and blocked with 5% milk/PBS-T overnight at 4oC. Membranes were incubated with the

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specific primary antibody (anti-claudin-1, -2, -4, -5, from Thermo Fisher Scientific), at 0.5 ug/ml in 5%

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milk/PBS-T for 2 hours at room temperature (RT). The membranes were again washed 3 times, 10

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minutes each with PBS-T, then incubated with secondary antibody (rabbit anti-mouse or goat anti-rabbit

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IgG labeled with horseradish peroxidase, from Southern Biotech, Birmingham, AL, USA) for 1 hour at RT.

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The membranes were washed 4 times, 10 minutes each with PBS-T, then treated for 1 minute with

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Western Lightning Plus-ECL chemiluminesence reagents (PerkinElmer). The membranes were then

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exposed to Hyblot CL autoradiography film (Denville Scientific, Holliston, MA, USA), which was

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developed in a Kodak M35A X-OMAT processor. Band densities were quantified on the BioRad 7 ACS Paragon Plus Environment


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ChemiDoc Imaging System. Band densities of normalized nutraceutical-treated cell samples were

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compared to normalized averages of corresponding control cell sample densities. All anti-claudin

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antibodies used in these studies were obtained from Thermo Fisher Scientific.

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Confocal Immunofluoresence

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Gie-3B11 cells cultured on glass cover slips were washed 3 times with PBS (15 seconds per wash)

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and fixed in ice-cold acetone for 5 minutes. The fixed cells were washed twice in PBS, 5 minutes per

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wash, and blocked with 5% normal goat serum in PBS/0.1% Tween 20 for 30 minutes. After blocking,

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tissues were treated 45 minutes at room temperature with primary antibody (mouse anti-human

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claudin 2; 5 ug/ml) diluted in blocking buffer. Following three 5-minute washes with PBS/0.1% Tween

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20, cells were incubated 45 minutes at room temperature with Cy3-conjugated secondary antibody

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diluted in blocking buffer (1:500). After washing 3 times as before, cell layers were stained with DAPI

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and mounted using Fluoromount (Southern Biotech) mounting medium and then viewed under confocal

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microscopy (Nikon Eclipse TI). Images were taken from different fields on each slide.

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Results

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Only a few days after seeding at approximately 1/3 confluent cell density (confluent density = 3.3 x

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105 cells/cm2), cell layers of Gie-3B11 epithelia will form a functional epithelial barrier with TJs, and a

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measureable Rt typically 50-60 ohms x cm2. There is, however, no observable transepithelial potential

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difference (voltage) because of the lack of any significant short-circuit current associated with these cell

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layers, due presumably to the absence of significant transepithelial ion transport (in contrast to

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absorptive gastrointestinal epithelial cell culture models such as CACO-2). Again, unlike CACO-2 (whose

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Rt value will increase steadily as a function of days post-seeding until a plateau level is reached

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approximately 7 to 14 days post-seeding), Gie-3B11 cell layers’ Rt values maximize 5 to7 days post-

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seeding; and instead of plateauing, they then decrease, as observed by other investigators.17 This


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transience in maximal Rt values is the reason why all permeability studies reported here were performed

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5 to 7 days post-seeding.

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We next examined the effects of various micronutrients that have been reported to enhance

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epithelial barrier function in other differentiated epithelial cell culture models. We employed treatment

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times and concentration ranges that were reported to produce maximal effect in these various other

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epithelial models, and found that zinc, quercetin, retinoic acid and the triterpene, AKBA, all produced

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enhanced epithelial barrier function (seen as either an increase in Rt or a decrease in Jm, or both) after

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simultaneous micronutrient exposure to both epithelial cell surfaces (Table 1). Theaflavins and

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berberine (berberine having had significant beneficial effects in the CACO-2 epithelial barrier model)7

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were without an observable effect here on Gie-3B11. 150 µM zinc increased Rt values by an average of

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38%, but at that concentration simultaneously induced transepithelial leakiness to the nonelectrolyte

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probe, 14C-D-mannitol. 50 µM zinc did not significantly alter Rt but did significantly reduce transepithelial

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leak of 14C-D-mannitol. 100, 200 and 400 µM quercetin dramatically reduced transepithelial leak to 14C-

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D-mannitol and produced simultaneous elevation of Rt at the higher concentrations (an average 42%

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increase in Rt at 400 µM). 15 µM retinoic acid significantly elevated Rt and decreased 14C-D-mannitol

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leak, a finding consistent with observations reported by Groeger et al.16 For the case of the triterpene,

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AKBA, a complete concentration curve is shown here. As indicated in Figure 1, 10 µM AKBA (on both cell

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surfaces) yielded the maximal increase in Rt, with higher concentrations inducing an Rt decrease.

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However 10 µM AKBA induced leak to 14C-D-mannitol, whereas at 2 µM AKBA, a maximal decrease in Jm

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— plus an increase in Rt — was observed. In summary, these four micronutrients are capable of inducing

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an altered and enhanced barrier function in this oral epithelial model, which can result in decreased

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passive leak across the barrier to both electrolytes and nonelectrolytes.



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To determine if these induced improvements in barrier function were due in part to alterations in

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Gie-3B11 TJ complexes, we also examined these compounds’ effects on a series of claudin proteins that

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comprise actual elements of the barrier complex to passive paracellular leak. Specifically, induced

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changes in the abundance of claudins-1, -2, -4 and -5 were examined in total cell lysates of the above-

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treated cell layers (Figure 2). The harvested cell layers were obtained from the same physiological

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experiments reported in Table 1. Results are reported here for 50 µM zinc, 400 µM quercetin, 15 µM

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retinoic acid and 2 µM AKBA. These concentrations were chosen based upon the greatest increase in Rt

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that associated with either a decrease in Jm or at least no associated Jm increase. These were therefore

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concentrations that were causing improved barrier function to both electrolytes and nonelectrolytes. As

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shown in Figure 2, zinc caused only a minor but significant decrease in claudin-1 along with a more

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potent decrease in claudin-5. Claudins-2 and -4 were unchanged by zinc treatment. Quercetin produced

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no changes in claudins-1, -4 or -5, but induced a highly significant 60% increase in claudin-2. AKBA

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produced no change in claudins-1, -2 or -5, while inducing a small (20%) but highly significant increase in

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claudin-4. Retinoic acid produced the most substantial changes in the claudins that we analyzed, with

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significant decreases in claudins-1 and -2 and significant increases in claudins-4 and -5. Therefore the

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observed improvement in barrier function by these various micronutrients reported in Table 1 was

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occurring with a unique pattern of induced changes in abundance of TJ proteins.

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As shown in Figure 3, exposure of Gie-3B11 cell layers to micronutrients could not only change the

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abundance of specific TJ proteins but could change their subcellular localization as well. Quercetin, for

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example, not only upregulated claudin-2 after 48 hours of continuous exposure (as was shown in the

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western immunoblot results of Figure 2), but caused a pronounced change in the subcellular localization

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of claudin-2 as well. Quercetin caused not only increased claudin-2 expression, but also a dramatic

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translocation of claudin-2 to the border region of the cells. Figure 3 shows a change in claudin-2


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localization from a punctate, cytoplasmic distribution in control cells to a perijunctional distribution that

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occurs simultaneously with the upregulation.

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An orally-administered micronutrient (delivered as an oral liquid, spray or lozenge) could result in

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very high micronutrient concentrations being delivered specifically to the apical surface of oral epithelia

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in situ (relative to the concentrations achieved in the bloodstream following small-intestinal absorption

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of the micronutrient). We questioned, however, whether apical-only administration of micronutrients

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could produce effects as favorable as those reported in Table 1 (for simultaneous apical and basal-lateral

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exposure) since membrane transporters for these substances may not exist on the apical cell surface.

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However, as shown in Figure 4, apical-only administration of retinoic acid (50 µM) achieved a significant

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increase in Rt and a dramatic decrease in transepithelial mannitol leak. Apical-only administration of zinc

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(500 µM) and quercetin (200 µM) also achieved statistically significant (25%-35%) decreases in

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transepithelial mannitol leak. Apical-only (100 µM) AKBA did not decrease mannitol leak (data not

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shown), but did produce an over 50% increase in Rt. Apical-only administration of certain

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micronutrients is, therefore, capable of inducing oral epithelial barrier enhancement to small

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electrolytes and nonelectrolytes, just as exposure to both cell surfaces induced. However, the effects on

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barrier function from apical-only administration require higher concentrations of the micronutrient than

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those concentrations observed to be maximally effective from both cell surfaces. A complete

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concentration curve is shown for the case of apically-administered AKBA (Figure 5), highlighting that

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higher concentrations of AKBA were required at the apical surface to produce similar effects on barrier

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function compared to those resulting from AKBA exposure to both cell surfaces (Figure 1). Moreover,

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the (higher) concentrations of micronutrients that produced compromised (decreased) barrier function

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when presented simultaneously to both cell surfaces (e.g., 50 µM retinoic acid inducing 162% of normal

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mannitol leak [Table 1]), could have beneficial action (improved barrier function) when delivered to the

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apical surface only (Figure 4). 11 ACS Paragon Plus Environment


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Discussion

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The purpose of this study was to determine the potential of specific micronutrients for improving

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the barrier function of the oral mucosa. The research identified zinc, quercetin, retinoic acid and the

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boswellic acid derivative, AKBA, as possessing efficacy in this regard, as well as possessing the underlying

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capability of being able to induce compositional changes in oral epithelial TJs. These effects indicate the

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possible utility of these micronutrients for improving the oral mucosal epithelial barrier as part of a

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regular clinical prophyllaxis.

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Our data (Table 1) show that zinc, retinoic acid, quercetin and AKBA can all enhance Gie-3B11

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barrier function, an effect that has already been reported for retinoic acid.16 For quercetin, this barrier

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enhancement took the form of not only an increased Rt but also a decreased Jm at a 400 µM quercetin

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concentration (on both cell surfaces) in a 48-hour exposure. These two independent measurements of

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barrier function, Rt and Jm, are highly informative, not only because they are measured independently

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but because they also reflect paracellular permeability to two very different molecular species and are

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likely governed by different claudins or claudin-claudin interactions in the TJ barrier. Rt is reflective of

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permeability to Na+ and Cl- ions, whereas Jm is reflective of permeability to small nonelectrolytes. For

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retinoic acid, a 15 µM concentration was optimal in terms of both elevating Rt and decreasing Jm. For

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AKBA, a range of concentrations (0.5 µM to10 µM) produced these dual indications of enhanced barrier

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function (Figure 1). For zinc, a 50 µM concentration significantly reduced transepithelial mannitol leak,

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whereas 150 µM zinc significantly increased transepithelial electrical resistance. Theaflavins and

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berberine (a compound that produced dramatic improvement of gastrointestinal barrier function)7 were

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without significant effects here at the concentrations shown to be efficacious on other epithelial cell


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layers. This difference is not surprising given the tissue specificity previously reported for TJ effects by

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micronutrients.7, 8

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Given the fact that each of these micronutrients act via quite distinct signaling pathways, it is not

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surprising that each produced distinct effects on claudin proteins. AKBA is known to induce and activate

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nrf2 as well as induce the tyrosine phosphatase, SHP-1.18, 19 Retinoic acid has been reported to have the

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opposite effects on SHP-1.20 Quercetin has been reported to upregulate nrf2 but there are no reports

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that its downstream signaling involves SHP-1.21 Zinc has been reported to inhibit SHP-1,22 although it has

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– like AKBA – been reported to activate the nrf2 pathway.23 Considering only these two signaling

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elements (and undoubtedly many others are involved as well), it would not be surprising that these four

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micronutrients have diverse effects on claudin proteins. What is fascinating and promising is that these

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diverse effects on claudin barrier proteins all result in improved barrier function.

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We also examined the results of apical-only exposure to these micronutrients (Figure 4). This is an

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important modality, because in a clinical situation, topical exposure of any agent to an epithelial

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mucosal surface is by definition an apical-only exposure. Oral administration of micronutrients as a

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liquid, lozenge or spray would be an apical-only exposure to the oral epithelia. This is a principal

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consideration in any discussion of potential in vivo therapies that could emanate from investigations like

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those presented here. Consider that oral administration of a micronutrient to the buccal, gingival, or

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lingual epithelium in the form of a liquid, lozenge or oral spray, would perhaps be most efficient in terms

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of drug delivery to the epithelial cells, if the compound could be taken up from the apical surface of the

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epithelium. This would circumvent the need for duodenal/jejunal absorption of the compound (with its

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own complexities), then dilution of the compound in the systemic circulation, followed by blood-borne

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delivery of the compound to the back (basal-lateral) epithelial surface. In apical-only exposures to the

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Gie-3B11 epithelia, we used concentrations higher than those presented to the basal-lateral surface,



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since epithelia in general tolerate significantly higher concentrations of bioactive substances on their

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apical surface, above levels that would be toxic on the basal-lateral surface (unpublished observations).

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Retinoic acid, quercetin and AKBA were again all significantly effective from the apical surface. Apical-

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only AKBA scored very dramatic effects on Rt, whereas apical-only quercetin and retinoic acid very

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dramatically reduced transepithelial mannitol leak. In addition, apical-only administration of a very high

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zinc concentration (500 µM) also significantly reduced mannitol leak.

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Only where there is an already existing compromise in oral epithelial barrier function (mechanical

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injury, inflammation-induced damage, etc.) would an orally administered agent have immediate access

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to both epithelial cell surfaces, and even then, only at the site of the compromise. A compound so

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administered will also undergo dilution in interstitial fluid. Perhaps in part owing to their significant

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hydrophobicity (ability to diffuse into lipid membranes), it seems that AKBA, retinoic acid and quercetin

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can all be effective from the apical cell surface. The effectiveness of 500 µM zinc from the apical surface

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is due either to the presence of apical zinc transporters or some degree of apically-administered zinc

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diffusing through the TJ complex to the site of basal-lateral zinc transporters, thereby allowing zinc entry

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into the cell.

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Although our data clearly show that retinoic acid, quercetin, zinc and AKBA can enhance Gie-3B11

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barrier function in the epithelial barrier’s basal state (Table 1), in future work we need to determine if

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these three compounds are equally efficacious in preventing or reducing the barrier leakiness induced

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by proinflammatory agents such as cytokines and hydrogen peroxide. Different types of transepithelial

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leak can be brought about by each cytokine, individually, and by cytokine combinations.24 Prior work

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from other research groups suggests the micronutrients under study here may be effective in reducing

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cytokine-mediated leak. Quercetin has been shown to have beneficial effects on barrier function in both

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renal and gastrointestinal epithelia.8, 25 It has been theorized that quercetin may achieve these effects


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not only by inhibition of oxidative stress but also by positive actions on mitochondrial bioenergetics.12

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Quercetin has demonstrated the ability to counteract the barrier disruptive action of the

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proinflammatory cytokine TNF-α, and to have dramatic effects on the TJ protein claudin-4 in intestinal

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epithelia.26, 27 Quercetin has even demonstrated protective action against the barrier disruptive effects

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of hydrogen peroxide, an effect that may involve quercetin’s action on the phosphorylation state of

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Erk1/2.28 An effect of quercetin on the phosphorylation state of PKC-δ has also been invoked.29

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Retinoic acid may possibly be inducing a generalized differentiation program in the Gie-3B11 cells,

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with suppression of Notch signaling being one element.30 Conversely, the free radical scavenging actions

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of retinoic acid may also be in play here, relieving oxidative stress-induced impairment of barrier

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function.31 It is worth noting that retinoic acid at similar concentrations has also been reported to

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compromise — not enhance — barrier function in other epithelial cell types,32, 33 although as reported

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by Groeger et al.,16 retinoic acid is generally favorable for barrier function.34, 35

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AKBA was shown earlier to support barrier function in human gastrointestinal epithelial cell layers

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by decreasing the deleterious effects of hydrogen peroxide, TNF-α and interferon-ϒ.10 This protective

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action was accompanied by a decrease in the level of NF-κB phosphorylation produced by the

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proinflammatory stimuli. In related studies, AKBA has been shown to inhibit leukotriene biosynthesis, as

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well as the synthesis and secretion of TNF-α, IL-1, IL-2, IL-6 and interferon-ϒ.36

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These functional effects of the micronutrients on Gie-3B11 barrier function correlated with unique

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patterns of induced changes in abundance of several TJ proteins. At concentrations that produced

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optimal improvement of barrier function, retinoic acid produced significant decreases in claudins-1 and -

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2, and significant increases in claudins-4 and -5 (Figure 2). Quercetin, on the other hand, produced a

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significant increase only in claudin-2, while zinc produced small but significant decreases in claudins-1

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and -5. AKBA produced a significant change (increase) in claudin-4. Claudins -1, -2, -4 and -5 are, 15 ACS Paragon Plus Environment


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however, only a partial representation of the integral TJ proteins that may constitute the paracellular

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barrier in these cell layers. AKBA, for example, also produced significant (30%) increases in claudins-3

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and -7 in Gie-3B11 cell layers in our hands (data not shown). It also needs to be emphasized that these

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immunoblot-based results are derived from total cell lysates, and this composite homogenate of

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different cell compartments may obscure the magnitude of these various compounds’ effects. As shown

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in Figure 3, quercetin is seen to have a much more dramatic upregulatory action on claudin-2 in an

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immunofluorescent confocal image, where it can be observed that quercetin also produces an induced

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translocation of claudin-2 from primarily cytosolic localization to perijunctional localization. Lastly, we

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are leaving unaddressed the issue of micronutrient-induced changes in the phosphorylation state of

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these TJ proteins, which can also be in play as TJ permeability changes are occurring .37

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In this paper we have emphasized the ability of certain micronutrients/nutraceuticals to enhance

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one or more aspects of TJ barrier function. The significance of such reduced leak for epithelial absorptive

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processes and for protecting a tissue from chronic inflammation — and an organism from sepsis — has

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been discussed. However, micronutrients’ more basic ability to simply alter the TJ complex structurally

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needs emphasizing as well. As shown in Figure 2, each compound tested produced almost signature-like

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changes in the TJ complex, with some claudins being upregulated, others downregulated, and others

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unchanged in total abundance. Although Figure 2 represents only a partial view of the compositional

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changes induced in the TJ complex, it serves to show that the individual micronutrients have truly

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distinct, unique effects on the TJ complex. This could have far reaching implications for prophylaxis in

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certain infectious diseases. There are subclasses of pathogens that can themselves, or via their secreted

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products, bind to specific TJ components as part of their means of infecting the epithelial cell or the

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organism at large.1, 38-42 Clostridium perfringens enterotoxin has a distinct affinity for claudins -3, -4 and -

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19, as part of its overall induction of transepithelial gastrointestinal leakiness.43 On the viral level,

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hepatitis C has a demonstrated affinity for claudin-1 as part of its mechanism of invading the epithelial 16 ACS Paragon Plus Environment

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cell.44 We would hypothesize that because of their large size, these pathogens or pathogen components

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can have their binding to the epithelial cell layer affected not simply by the abundance of their specific

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claudin-binding “receptor,” but also the overall claudin “neighborhood” around their binding partner.

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Modification of these “neighborhoods” by micronutrient/nutraceutical-induced compositional changes

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of TJ complexes may have effects on the ability of such pathogens to adhere to and invade across

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epithelial barriers.40 This implies that certain micronutrients/nutraceuticals, or combinations thereof,

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may be prophylactically effective in helping to reduce the frequency and/or severity of certain

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infections. It is less certain, but still possible, that micronutrient/nutraceutical-based modification of TJs

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may also interfere with the ability of other pathogens such as HIV that do not bind directly to TJ

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complexes but still induce TJ leakiness following infection of the epithelial cell.45 Reduction of viral

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penetration across a micronutrient-treated barrier tissue may result in a lower viral load, and

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subsequently, decreased morbidity. The ability of certain micronutrients to induce compositional

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changes in TJ complexes and thereby decrease pathogen binding and translocation across epithelial

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barriers may make them a unique, novel and untapped anti-microbial resource.

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The very fact that different micronutrients give rise to different patterns of claudin changes and yet

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give rise to tighter (better) barrier function is the singular beauty of micronutrients with regard to

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barrier protection. TJ barrier function cannot be reduced to simple (and often incorrect) axioms that if

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e.g. claudin-2 rises or claudin-5 falls, the end result must be poorer barrier function. The very nature of

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the homo- and heterodimer action of claudins would strongly suggest that it cannot be so simple.46

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Compounding this combinational functionality of TJ claudins, the very numerousness of TJ constituents

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(e.g. the very high number of constituent claudin components and other proteins as well) likely allows

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for a myriad of unique, distinct enhanced (tighter) states in the junctional complex. With over twenty

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known claudin proteins, how many homodimer and heterodimer combinations can exist within a

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junctional complex? The accomplishment of the feat of multitudinous improved TJ states induced by a 17 ACS Paragon Plus Environment


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wide variety of very chemically distinct micronutrients, speaks to a very long history of co-evolution of

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organisms and their epithelial barriers with such dietary and environmental micronutrients. This is a

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history that biomedical research and therapeutics would be wise to investigate.

386 387 388 389 390 391 392 393 394 395 396

Abbreviations: AKBA: acetyl-11-keto-β-boswellic acid Jm: transepithelial mannitol flux LSC: liquid scintillation counting PAGE: polyacrylamide gel electrophoresis PBS: phosphate buffered saline Rt: transepithelial electrical resistance TJ: tight junction

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Grants, sponsors and funding sources: None

398 399

Acknowledgements

400 401 402

The authors are very grateful to Ms. Kate Ciavarelli for her invaluable assistance in preparing the manuscript and its Figures for publication. We also wish to thank Ms. Nicole Buleza of Drexel University for her help in editing the final version of the manuscript.

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19. Kunnumakkara, A. B.; Nair, A. S.; Sung, B.; Pandey, M. K.; Aggarwal, B. B. Boswellic acid blocks signal transducers and activators of transcription 3 signaling, proliferation, and survival of multiple myeloma via the protein tyrosine phosphatase SHP-1. Molecular cancer research 2009, 7 (1), 118-128. 20. Chen, Y.; Wang, W.; Liu, F.; Tang, L.; Tang, R.; Li, W. 9-cis-retinoic acid improves sensitivity to platelet-derived growth factor-BB via RXRÎ± and SHP-1 in diabetic retinopathy. Biochemical and biophysical research communications 2015, 465 (4), 810-816. 21. Li, X.; Wang, H.; Gao, Y.; Li, L.; Tang, C.; Wen, G.; Zhou, Y.; Zhou, M.; Mao, L.; Fan, Y. Protective effects of quercetin on mitochondrial biogenesis in experimental traumatic brain injury via the Nrf2 signaling pathway. PLoS One 2016, 11 (10), e0164237. 22. Haase, H.; Maret, W. Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin/insulin-like growth factor-1 signaling. Experimental cell research 2003, 291 (2), 289298. 23. Fan, Y.; Zhang, X.; Yang, L.; Wang, J.; Hu, Y.; Bian, A.; Liu, J.; Ma, J. Zinc inhibits high glucoseinduced NLRP3 inflammasome activation in human peritoneal mesothelial cells. Molecular Medicine Reports 2017, 16 (4), 5195-5202. 24. DiGuilio, K. M.; Mercogliano, C. M.; Born, J.; Ferraro, B.; To, J.; Mixson, B.; Smith, A.; Valenzano, M. C.; Mullin, J. M. Sieving characteristics of cytokine- and peroxide-induced epithelial barrier leak: Inhibition by berberine. World J Gastrointest Pathophysiol 2016, 7 (2), 223-34. 25. Valenzano, M. C.; DiGuilio, K. M.; Mullin, J. M. Comparative Effects of the Micronutrients Zinc, Quercetin, Berberine, Indole and Butyrate on CACO-2 Barrier Function and Tight Junction Composition. PlosOne 2015, July 30. 26. Amasheh, M.; Schlichter, S.; Amasheh, S.; Mankertz, J.; Zeitz, M.; Fromm, M.; Schulzke, J. D. Quercetin enhances epithelial barrier function and increases claudin-4 expression in Caco-2 cells. J Nutr 2008, 138 (6), 1067-73. 27. Amasheh, M.; Luettig, J.; Amasheh, S.; Zeitz, M.; Fromm, M.; Schulzke, J. D. Effects of quercetin studied in colonic HT-29/B6 cells and rat intestine in vitro. Ann N Y Acad Sci 2012, 1258, 100-7. 28. Chuenkitiyanon, S.; Pengsuparp, T.; Jianmongkol, S. Protective effect of quercetin on hydrogen peroxide-induced tight junction disruption. Int J Toxicol 2010, 29 (4), 418-24. 29. Suzuki, T.; Hara, H. Quercetin enhances intestinal barrier function through the assembly of zonula [corrected] occludens-2, occludin, and claudin-1 and the expression of claudin-4 in Caco-2 cells. J Nutr 2009, 139 (5), 965-74. 30. Zanetti, A.; Affatato, R.; Centritto, F.; Fratelli, M.; Kurosaki, M.; Barzago, M. M.; Bolis, M.; Terao, M.; Garattini, E.; Paroni, G. All-trans-retinoic Acid Modulates the Plasticity and Inhibits the Motility of Breast Cancer Cells: ROLE OF NOTCH1 AND TRANSFORMING GROWTH FACTOR (TGFbeta). J Biol Chem 2015, 290 (29), 17690-709. 31. Molina-Jijon, E.; Rodriguez-Munoz, R.; Namorado Mdel, C.; Bautista-Garcia, P.; Medina-Campos, O. N.; Pedraza-Chaverri, J.; Reyes, J. L. All-trans retinoic acid prevents oxidative stress-induced loss of renal tight junction proteins in type-1 diabetic model. J Nutr Biochem 2015, 26 (5), 441-54. 32. Ortiz-Melo, M. T.; Sanchez-Guzman, E.; Gonzalez-Robles, A.; Valdes, J.; Gomez-Flores, E.; CastroMunozledo, F. Expression of claudins -2 and -4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier. Biol Open 2013, 2 (2), 132-43. 33. Hatakeyama, S.; Ishida, K.; Takeda, Y. Changes in cell characteristics due to retinoic acid; specifically, a decrease in the expression of claudin-1 and increase in claudin-4 within tight junctions in stratified oral keratinocytes. J Periodontal Res 2010, 45 (2), 207-15. 34. Rong, J.; Liu, S. Effect of all-trans retinoic acid on the barrier function in human retinal pigment epithelial cells. Biochem Biophys Res Commun 2011, 407 (3), 605-9.


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Figure captions Figure 1. The concentration-dependent effects of AKBA on Gie-3B11 barrier function. Cell layers were treated with AKBA on both cell surfaces for 48 hours, beginning 4 days post-seeding, before measurements were performed. Results shown represent the mean + standard error for n = 7 cell layers (for Rt) or n = 4 cell layers (for mannitol flux). * P < 0.05 **P < 0.01, ***P < 0.001, one-way ANOVA (Tukey). Figure 2. Micronutrient-specific effects on claudin abundance in Gie-3B11 cell layers. Cell layers used in the barrier function studies of Table 1 were harvested from their filter supports and total cell lysates analyzed for specific tight junctional proteins in Western immunoblots as described in Materials and Methods. Cell layers were treated with 50 uM zinc, 400 uM quercetin, 15 uM retinoic acid or 2 uM AKBA as described. Panel A: The actual immunoblot bands observed for claudins -1, -2, -4 and -5 are shown for 4 control cell layers (lanes 1-4) and 4 micronutrient-treated cell layers (lanes 5-8). Panel B: Band density was quantitated by optical density. Results shown represent the mean of 4 cell layers + standard error. ** P < 0.01, *** P < 0.001 for two-sided Student’s t-test. Figure 3. Immunofluorescent localization of claudin-2 in Gie-3B11 cell layers before and after exposure to quercetin. Cell layers were exposed to 400 uM quercetin for 48 hours followed by fixation and immunostaining as described in Materials and Methods. Preparations were double-labeled with DAPI to indicate nuclei location and an anti-claudin-2 primary antibody. Claudin-2 can be seen to not only upregulate in the presence of quercetin, but change its intracellular localization from a punctate, cytosolic distribution to a more perijunctional distribution. Panels A & B: control cell layer at 200X and 400X respectively. Panel C: quercetin-treated cell layer (200X). Figure 4. Apical-only micronutrient exposures that improve Gie-3B11 barrier function. Left Panel: transepithelial electrical resistance; Right Panel: transepithelial mannitol diffusion rate. Data shown represent the percent of normalized control ± standard error for n = 8 cell layers. * indicates P < 0.05, **indicates P

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