A Dual Zinc plus Arginine formulation attenuates the pathogenic properties of Porphyromonas gingivalis and protects gingival keratinocyte barrier function in an in vitro model

Amel Ben Lagha, Ying Yang, Harsh M Trivedi, James G Masters, Daniel Grenier, Amel Ben Lagha, Ying Yang, Harsh M Trivedi, James G Masters, Daniel Grenier

Abstract

Background and objectives: Porphyromonas gingivalis, a late colonizer of the periodontal biofilm, has been strongly associated with the chronic form of periodontitis. The aim of this study was to investigate the effects of a Dual Zinc plus Arginine formulation (aqueous solution and dentifrice) on the pathogenic properties of P. gingivalis and the barrier function of an in vitro gingival epithelium model.

Results: The Dual Zinc plus Arginine aqueous solution and dentifrice inhibited the hemolytic and proteolytic activities of P. gingivalis. The Dual Zinc plus Arginine aqueous solution and dentifrice enhanced the barrier function of an in vitro gingival epithelium model as determined by a time-dependent increase in transepithelial electrical resistance and decrease in paracellular permeability. This was associated with an increased immunolabeling of two important tight junction proteins: zonula occludens-1 and occludin. The deleterious effects of P. gingivalis on keratinocyte barrier function as well as the ability of the bacterium to translocate through a gingival epithelium model were attenuated in the presence of either Dual Zinc plus Arginine aqueous solution or dentifrice.

Conclusions: The Dual Zinc plus Arginine formulation may offer benefits for patients affected by periodontal disease through its ability to attenuate the pathogenic properties of P. gingivalis and promote keratinocyte barrier function.

Keywords: Arginine; Porphyromonas gingivalis; dentifrice; epithelial barrier; gingipain; periodontal disease; periodontitis; toothpaste; zinc.

Conflict of interest statement

The authors report no conflicts of interest related to this study. Y. Yang, H.M. Trivedi, and J.G. Masters work as scientists for Colgate-Palmolive Co.

© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

Figures

Figure 1.
Figure 1.
Effects of the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, and the regular fluoride dentifrice on collagen degradation by P. gingivalis. Results are expressed as the means ± SD of triplicate assays two independent experiments. *, significant decrease (p < 0.05) compared to untreated control cells.
Figure 2.
Figure 2.
Time- and dose-dependent effects of the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, and the regular fluoride dentifrice on gingival keratinocyte tight junction integrity, as determined by monitoring TER. A 100% value was assigned to the TER values at time 0. Results are expressed as the means ± SD of triplicate assays. *, significant increase (p < 0.05) compared to untreated control cells. φ, significant increase (p < 0.05) compared to the regular fluoride dentifrice.
Figure 3.
Figure 3.
Time- and dose-dependent effects of the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, and the regular fluoride dentifrice on the paracellular permeability of gingival keratinocytes to FITC-dextran 4 (FD-4). Results are expressed as the means ± SD of triplicate assays. *, significant decrease (p < 0.05) compared to untreated control cells.
Figure 4.
Figure 4.
Immunofluorescence staining of the tight junction proteins occludin and zonula occludens-1 of gingival keratinocytes treated for 48 h with the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, and the regular fluoride dentifrice. Arrows indicate discontinuities in protein labeling. This analysis was performed three times; a representative experiment and a representative field of this experiment is presented.
Figure 5.
Figure 5.
Time- and dose-dependent protective effects of the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, and the regular fluoride dentifrice against P. gingivalis-mediated damage of gingival keratinocyte tight junction integrity as determined by monitoring TER values. A 100% value was assigned to the TER values at time 0. Results are expressed as the means ± SD of triplicate assays. *, significant increase (p < 0.001) compared to P. gingivalis-infected cells not treated with compounds. Φ, significant decrease (p < 0.05) compared to non-stimulated control cells. φ, significant increase (p < 0.05) compared to the regular fluoride dentifrice.
Figure 6.
Figure 6.
Protective effects of the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, and the regular fluoride dentifrice on the paracellular permeability of gingival keratinocytes to FITC-dextran 4 (FD-4) compromised by P. gingivalis. Results are expressed as the means ± SD of triplicate assays. *, significant decrease (p < 0.05) compared to P. gingivalis-stimulated cells.
Figure 7.
Figure 7.
Immunofluorescence staining of the tight junction proteins occludin and zonula occludens-1 in gingival keratinocytes following a 48-h treatment with P. gingivalis (MOI = 104) in the absence and presence of the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, or the regular fluoride dentifrice. Arrows indicate discontinuities in protein labeling. This analysis was performed three times; a representative experiment and a representative field of this experiment is presented.
Figure 8.
Figure 8.
Effects of the Dual Zinc plus Arginine aqueous solution, the Dual Zinc plus Arginine dentifrice, and the regular fluoride dentifrice on the invasion of a gingival keratinocyte barrier by P. gingivalis. Results are expressed as the means ± SD of triplicate assays. *, significant decrease (p < 0.001) compared to P. gingivalis-infected cells not treated with the compounds.

References

    1. Haffajee AD, Socransky SS.. Microbial etiological agents of destructive periodontal diseases. Periodontol 2000. 1994;5:78–12.
    1. Darveau RP. Periodontitis: a polymicrobial disruption of host homeostasis. Nat Rev Microbiol. 2010;8:481–490.
    1. Bostanci N, Belibasakis GN. Porphyromonas gingivalis: an invasive and evasive opportunistic oral pathogen. FEMS Microbiol Lett. 2012;333:1–9.
    1. How KY, Song KP, Chan KG. Porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. Front Microbiol. 2016;7:53.
    1. Hajishengallis G, Lamont RJ. Beyond the red complex and into more complexity: the polymicrobial synergy and dysbiosis (PSD) model of periodontal disease etiology. Mol Oral Microbiol. 2012;27:409–419.
    1. Guo Y, Nguyen KA, Potempa J. Dichotomy of gingipains action as virulence factors: from cleaving substrates with the precision of a surgeon’s knife to a meat chopper-like brutal degradation of proteins. Periodontol 2000. 2010;54:15–44.
    1. Mysak J, Podzimek S, Sommerova P, et al. Porphyromonas gingivalis: major periodontopathic pathogen overview. J Immunol Res. 2014:476068.
    1. Dale BA. Periodontal epithelium: a newly recognized role in health and disease. Periodontol 2000. 2002;30:70–78.
    1. Groeger SE, Meyle J. Epithelial barrier and oral bacterial infection. Periodontol 2000. 2015;69:46–67.
    1. Forster C. Tight junctions and the modulation of barrier function in disease. Histochem Cell Biol. 2008;130:55–70.
    1. Groeger S, Doman E, Chakraborty T, et al. Effects of Porphyromonas gingivalis infection on human gingival epithelial barrier function in vitro. Eur J Oral Sci. 2010;118:582–589.
    1. Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation. Nat Rev Immunol. 2015;15:30–44.
    1. Han YW, Wang X. Mobile microbiome: oral bacteria in extra-oral infections and inflammation. J Dent Res. 2013;92:485–491.
    1. Delgado E, Garcia-Godoy F, Montero-Aguilar M, et al. A clinical investigation of a Dual Zinc plus Arginine dentifrice in reducing established dental plaque and gingivitis over a six-month period of product use. J Clin Dent. 2018;29:A33–A40.
    1. Prasad KV, Therathil SG, Agnihotri A, et al. The effects of two new Dual Zinc plus Arginine dentifrices in reducing oral bacteria in multiple locations in the mouth: 12-hour whole mouth antibacterial protection for whole mouth health. J Clin Dent. 2018;29:A25–A32.
    1. Groeger S, Michel J, Meyle J. Establishment and characterization of immortalized human gingival keratinocyte cell lines. J Periodontal Res. 2008;43:604–614.
    1. Gumbiner B, Simons K. A functional assay for proteins involved in establishing an epithelial occludin barrier: identification of a uvomorulin-like polypeptide. J Cell Biol. 1986;102:457–468.
    1. Ben Lagha A, Groeger S, Meyle J, et al. Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis. Pathog Dis. 2018;76:FTY030.
    1. Marquis A, Genovese S, Epifano F, et al. The plant coumarins auraptene and lacinartin as potential multifunctional therapeutic agents for treating periodontal disease. BMC Complement Altern Med. 2012;12:80.
    1. Bodet C, Chandad F, Grenier D. Hemoglobin and LPS act in synergy to amplify the inflammatory response. J Dent Res. 2007;86:878–882.
    1. Houle MA, Grenier D, Plamondon P, et al. The collagenase activity of Porphyromonas gingivalis is due to Arg-gingipain. FEMS Microbiol Lett. 2003;221:181–185.
    1. Imamura T. The role of gingipains in the pathogenesis of periodontal disease. J Periodontol. 2003;74:111–118.
    1. Nishikata M, Yoshimura F, Nodasaka Y. Possibility of Bacteroides gingivalis hemagglutinin possessing protease activity revealed by inhibition studies. Microbiol Immunol. 1989;33:75–80.
    1. Krauser JA, Potempa J, Travis J, et al. Inhibition of arginine gingipains (RgpB and HRgpA) with benzamidine inhibitors: zinc increases inhibitory potency. Biol Chem. 2002;383:1193–1198.
    1. Manus LM, Daep CA, Begum-Gafur R, et al. Enhanced in vitro zinc bioavailability through rational design of a Dual Zinc plus Arginine dentifrice. J Clin Dent. 2018;29:A10–A19.
    1. Meyle J, Gultig K, Rascher G, et al. Transepithelial electrical resistance and tight junctions of human gingival keratinocytes. J Periodontal Res. 1999;34:214–222.
    1. Ben Lagha A, Grenier D. Black tea theaflavins attenuate Porphyromonas gingivalis virulence properties, modulate gingival keratinocyte tight junction integrity and exert anti-inflammatory activity. J Periodontal Res. 2017;52:458–470.
    1. Ben Lagha A, LeBel G, Grenier D. Dual action of highbush blueberry proanthocyanidins on Aggregatibacter actinomycetemcomitans and the host inflammatory response. BMC Complement Altern Med. 2018;18(1):10.
    1. Rybakovsky E, Valenzano MC, Deis R, et al. Improvement of humanoralepithelialbarrier function and of tight junctions by micronutrients. J Agric Food Chem. 2017;65:10950–10958.
    1. Dale BA, Fredericks LP. Antimicrobial peptides in the oral environment: expression and function in health and disease. Curr Issues Mol Biol. 2011;7:119–134.
    1. Lombardo Bedran TB, Feghali K, Zhao L, et al. Green tea extract and its major constituent, epigallocatechin-3-gallate, induce epithelial beta-defensin secretion and prevent beta-defensin degradation by Porphyromonas gingivalis. J Periodontal Res. 2013;49:615–623.
    1. Andrian E, Grenier D, Rouabhia M. Porphyromonas gingivalis-epithelial cell interactions in periodontitis. J Dent Res. 2006;85:392–403.
    1. Sakanaka A, Takeuchi H, Kuboniwa M, et al. Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells. Microb Pathog. 2016;94:42–47.
    1. Katz J, Sambandam V, Wu JH, et al. Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes. Infect Immun. 2000;68:1441–1449.
    1. Sarkar P, Saha T, Sheikh IA, et al. Zinc ameliorates intestinal dysfunctions in shigellosis by reinstating claudin−2 and −4 on the membranes. Am J Physiol Gastrointest Liver Physiol. 2019;316:G229–G246.
    1. Lamont RJ, Chan A, Belton CM, et al. Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun. 1995;63:3878–3885.
    1. Nakagawa I, Inaba H, Yamamura T, et al. Invasion of epithelial cells and proteolysis of cellular focal adhesion components by distinct types of Porphyromonas gingivalis fimbriae. Infect Immun. 2006;74:3773–3782.

Source: PubMed

Sponsoren und Mitarbeiter

Krankheiten

Drogeninterventionen

3
Abonnieren