PRACA ORYGINALNA / ORIGINAL ARTICLE

GALUSAN EPIGALLOKATECHINY ZAPOBIEGA USZKODZENIU TKANKI ŁĄCZNEJ OZĘBNEJ I GRUCZOŁÓW ŚLINOWYCH W SZCZURZYM MODELU UOGÓLNIONEGO STANU ZAPALNEGO

Alina M. Yelins’ka, Olena O. Shvaykovs’ka, Vitalii O. Kostenko

Ukrainian Medical Stomatological Academy, Poltava, Ukraine

ABSTRACT

Introduction: The connective tissue remodeling is essential for periodontal and salivary glands (SG) pathology. Recently there has been demonstrated the number of pharmacological effects of green tea (Camellia sinensis) such as antioxidant, anti-inflammatory, anti-aging, antibacterial, antiviral and DNA-protective effects, associated with the presence of epigallocatechin-3-gallate (EGCG) as an inducer of the Keap1 / Nrf2 / antioxidant response element signaling pathway. However, the EGCG effects on the components of soft connective tissues of periodontium and SG are still unclear.

The aim: To investigate the effect of EGCG on markers of disruption of periodontal and submandibular SG connective tissues in rats during the conditions of experimental systemic inflammation (SI).

Materials and methods: The studies were conducted on 30 white rats of the Wistar line, divided into 3 groups: the 1st included intact animals, the 2nd was made up of animals after induced SI (by intraperitoneal administration of lipopolysaccharide Salmonella typhi), and the 3rd included animals, which were injected EGCG (production of Sigma-Aldrich, Inc., USA) intraperitoneally in a dose of 21.1 mg / kg 3 times a week, starting on the 30th day of SI induction. The level of collagenolysis was assessed by the content of free hydroxyproline (FHP). The process of depolymerization of proteoglycans and sialoglycoproteins was evaluated by determining their monomers, glycosaminoglycans (GAGs) and N-acetylneuraminic acid (NANA) respectively. The molar roots exposure index (MREI) was calculated.

Results: Administering EGCG reduced the content of FHP by 33.3 % (p<0.01), the content of GAGs by 39.4% (p<0.02), and content of NANA by 34.3% (p<0.001) in the soft periodontal tissues compared with the relevant findings in the second group of the animals. In this condition the concentration of these compounds in the calcified components of periodontium (alveolar bone) lowered as well: FHP – by 41.9% (p<0.001), GAGs – by 41.0% (p<0.001), NANA – by 53.3% (p<0.001), MREI reduced to 27.1+1.6, i.e. by 27.7% (p<0.01) compared with the relevant findings in the second group of the animals. The administration of EGCG also reduced the content of FHP by 37.8% (p<0.001), the content of GAGs by 39.8% (p<0.001), and the content of NANA by 37.6% (p<0.001) in SG tissues compared with the relevant results of the second group of the animals.

Conclusions: The administration of EGCG under modeled systemic inflammation is an effective means of preventing and correcting the disruption of connective tissue of periodontium and submandibular salivary glands in rats: it reduces collagenolysis and depolymerization of proteoglycans and glycoproteins.

 

Wiad Lek 2018, 71, 4, 869-873

 

INTRODUCTION

The connective tissue play critical roles in the morphogenesis and differentiation of developing periodontium and salivary glands (SG), provides their strength and rigidity, in addition to multiple other functions [1, 2], but at the same time is very sensitive to impacts caused by endo- and exogenous pathogenic factors [3].

The extracellular matrix (ECM) composed of networks of fibrillar proteins (collagen, elastin), accessory proteins, hydrophilic heteropolysaccharides (glycosaminoglycans – GAGs) that are either not attached (e.g. hyaluronan) or are attached to proteins (proteoglycans), growth regulatory proteins that are sequestered in the network, and proteases and their inhibitors that regulate cleavage of ECM and associated proteins [2]. In SG and periodontium ECM directs cellular responses, including cell differentiation, migration, and polarization. The GAGs chains can act as reservoirs for various growth factors, e.g.  fibroblast growth factor receptors – FGF/FGF2, FGF7 [2].

The connective tissue of periodontium mainly contains sulphated GAGs – chondroitin-4/6-sulphate, dermatan, etc. [4, 5]. Their role consists in contributing to the formation of collagen and elastane fibers, maintaining water-electrolytic metabolism, providing selective permeability.

Connective tissue remodeling is essential for periodontal and SG pathology. Complex pathogenic pathways control the balance ECM synthesis / degradation. This applies, in particular, to inflammatory and dystrophic diseases of periodontium and SG against metabolic disorders, the leading pathogenesis link of which is systemic inflammation (SI) [6, 7]. The development of connective tissue disruption is considered as a consequence of the long activation of certain redox-sensitive transcription factors (NF-κB, AP-1) [8, 9]. This causes the expression of genes of inflammatory cytokines, metalloproteinases (MMPs), inducible nitric oxide synthase, etc., which can induce proteolytic and oxidative-nitrosative stress [10, 11].

Recently there has been demonstrated the number of pharmacological effects of green tea (Camellia sinensis) such as antioxidant, anti-inflammatory, anti-aging, antibacterial, antiviral and DNA-protective effects, associated with the presence of epigallocatechin-3-gallate (EGCG) in its chemical composition [12]. The experimental studied have proven the action mechanism of this polyphenol is implemented through the activation of Nrf2 due to proteolysis of an inhibitory protein Keap1 [13, 14]. This way enhances antioxidant activity of a number of enzymes through cis-acting enhancer sequence, known as antioxidant response element (ARE) [15, 16]. A particular role of ARE is determined by the fact that the condition of NF-κB- and АР-1-associated signaling pathways depends on its activity [17, 18].

However, the EGCG effects on the components of soft connective tissues of periodontium and SG are still unclear. Solving this problem will enable to evaluate the role of this polyphenol as a potential means of pathogenetic therapy of inflammatory and dystrophic diseases of periodontal tissues and salivary glands.

THE AIM

The aim of the present study was to investigate the effect of an inducer of the Keap1 / Nrf2 / ARE epigallocatechin-3-gallate on markers of disruption of periodontal and submandibular SG connective tissues in rats during the conditions of experimental SI.

MATERIALS AND METHODS

The studies were conducted on 30 white rats of the Wistar line weighing 180-220 g, divided into 3 groups: the 1st included intact animals, the 2nd was made up of animals after induced SI, and the 3rd included animals, which were injected EGCG (production of Sigma-Aldrich, Inc., USA) intraperitoneally in a dose of 21.1 mg / kg 3 times a week, starting on the 30th day of SI induction.

SI was induced by intraperitoneal administration of lipopolysaccharide (LPS) Salmonella typhi (pyrogenalum, “Medgamal”, Russia) in a dose that stimulated rise in temperature by 1.5 °C according to the scheme [19]: during the first week, 4 minimum pyrogenic doses (MPD) of 0.4 μg / kg of rat mass were administered 3 times a week. During the following seven weeks of the experiment, rats were given 4 MPD / kg of body weight once a week.

The research was conducted in compliance with the standards of the Convention on Bioethics of the Council of Europe’s ‘European convention for the protection of vertebrate animals used for experimental and other scientific purposes’ (Strasbourg, 18.III.1986). The animals were decapitated with ethereal anesthesia. Soft (gingiva and periodontal ligament) and calcified (alveolar bone) components of periodontium, as well as homogenate of submandibular SG were the objects of the study.

The level of collagenolysis was assessed by the content of free hydroxyproline (FHP) [20]. The process of depolymerization of proteoglycans and sialoglycoproteins was evaluated by determining their monomers – GAGs [21] and N-acetylneuraminic acid (NANA) [22] respectively.

Using a light microscope with an eyepiece micrometer, we estimated the distance from the dental alveolus edge to the lower edge of the crown of the third molar (L0) and the distance from its alveolus edge to the upper edge of the dental crown (L1) with the subsequent calculation of the molar root exposure index (MREI) by the formula: MREI = L0 / L1.

The findings obtained were statistically processed. To verify the normality distribution, the Shapiro-Wilk test was applied. If they corresponded to the normal distribution, then the Student’s t-test was used to compare independent samples. When the results ranges were not subject to normal distribution, statistical processing was performed using a nonparametric method, the Mann-Whitney test. Statistical calculations were performed using the “StatisticSoft 6.0” program.

RESULTS AND DISCUSSION

SI modeling resulted in the changes in biochemical markers of depolimerization of collagens, proteoglycans and glycoproteins in the tissues of periodontium (Table I). Thus, the FHP content went up by 66.2% (p<0.01), GAGs – by 66.8% (p<0.05), NANA – by 62.9% (p<0.001) in the soft periodontal tissues.

Concentration of these compounds in the calcified components of periodontium (alveolar bone) increased as well: FHP – by 69.9% (p<0.001), GAGs – by 72.4% (p<0.02), NANA – by 2.2 times (p<0.01).

These results are the evidence for the activation of the processes of collagenolysis and depolymerization of proteoglycans and sialoglycoproteins in both soft and hard periodontal tissues under modeled SI.

Resent studies have demonstrated the ability of Keap1 / Nrf2 / ARE system to control other redox-sensitive elements, including NF-κB і АР-1 [17, 18]. It has been shown the activation of NF-κB and AP-1 enhances collagenase-3 (MMP-13) gene expression [23]. At that NF-κB expression correlates better than AP-1 with MMP expression. It has been shown the activation of NF-κB is an important link in the pathogenesis of collagenolysis and proteoglycans depolymerization in the periodontal tissues under experimental metabolic syndrome. Introduction of an inhibitor of the nuclear translocation of NF-κB 4-methyl-N-(3–phenylpropyl) benzene-1,2-diamine in these conditions reduces the amount of FHP and GAGs in periodontal tissues [24]. It has been demonstrated the ability of neuronal nitric oxide synthase to limit the disruption of periodontal connective tissue associated with NF-κB down-regulation [11].

The connective tissues disruption is regarded as a critical mechanism in damaging to periodontium during the action of systemic (emotional and pain stress) [3] and local factors of periodontitis [7], and it impedes periodontal regenerative therapies and tissue engineering in periodontology [25].

Figure 1 illustrates the changes in MREI, which characterize the degree of alveolar process resorption. In rats exposed to modeled SI, MREI increased up to 37.5±2.2 (25.0±1.4 in intact animals), i.e. by 50.0% (p<0.01).

Administering EGCG reduced the content of FHP by 33.3 % (p<0.01), the content of GAGs by 39.4% (p<0.02), and content of NANA by 34.3% (p<0.001) in the soft periodontal tissues compared with the relevant findings in the second group of the animals.

In this condition the concentration of these compounds in the calcified components of periodontium (alveolar bone) lowered as well: FHP – by 41.9% (p<0.001), GAGs – by 41.0% (p<0.001), NANA – by 53.3% (p<0.001), MREI reduced to 27.1+1.6, i.e. by 27.7% (p<0.01) compared with the relevant findings in the second group of the animals.

The results indicate the activation of the processes of collagenolysis and depolimerization of proteoglycans and sialoglycoproteins in soft and hard periodontal tissues during modeled SI.

Taking into account that all of the above markers of connective tissue disruption depend on the NF-κB activation, we can assume that the result of the positive effect of EGCG as an inductor of the Keap1 / Nrf2 / ARE system is related to the NF-κB signaling limits. Previously it was shown that LPS induced NF-κB activation could be attenuated by diverse Nrf2 activators, such as phenethyl isothiocyanate, sulforaphane and curcumin [17]. The administration of the compounds significantly inhibited phosphorylation in the site of IκB kinase (IKK) / NF-κB inhibitor (IκB) and p65 NFκB subunit nuclear translocation, consequently alleviating NF-κB signaling [26]. It was also reported on the possibility to reduce IKKβ activity [27] and caspase-mediated proteolysis of NF-κB/p65 [28] by using EGCG.

SI modeling resulted in the changes in biochemical markers of depolymerization of collagen, proteoglycans and glycoproteins in the tissues of SG (Table II). Thus, the content of FHP grew up by 64.8% (p<0.001), the content of GAGs by 83.7% (p<0.001), and the content of NANA by 59.8% (p<0.001).

Early studies documented the importance of the interstitial ECM and epithelial-cell associated basement membrane in maintaining the structure of the epithelial lobules and implied that the SG connective tissue participates in regulating the process of branching morphogenesis and in facilitating exocrine secretion by the acinar structures [2]. The ECM and basement membrane is a dynamic structure that undergoes remodeling during SG morphogenesis and differentiation and participates in mediating changes in tissue shape [2].

The connective tissues disruption is considered as a critical link in the pathogenesis in structural failure and dysfunction of different organs, including SG during the systemic pathology [29]. The role of NF-κB activation in the mechanisms of these processes has been already proven [30].

The administration of EGCG reduced the content of FHP by 37.8% (p<0.001), the content of GAGs by 39.8% (p<0.001), and the content of NANA by 37.6% (p<0.001) in SG tissues compared with the relevant results of the second group of the animals.

The ability of EGCG to suppress the NF-κB activation is an important feature of this polyphenol that creates the preconditions for the successful implementation of the cytoprotective properties of Keap1 / Nrf2 / ARE signaling pathway.

Non-toxicity of EGCG is a benefit, which distinguishes it from most NF-κB activation inhibitors, the use of which is accompanied by a number of side effects [31].

CONCLUSIONS

Thus, the administration of epigallocatechin-3-gallate under modeled systemic inflammation is an effective means of preventing and correcting the disruption of connective tissue of periodontium and submandibular salivary glands in rats: it reduces collagenolysis and depolymerization of proteoglycans and glycoproteins.

REFERENCES

1. Duan X, Ji M, Deng F et al. Effects of connective tissue growth factor on human periodontal ligament fibroblasts. Arch Oral Biol. 2017 Dec;84:37-44.

2. Sequeira SJ, Larsen M, DeVine T. Extracellular matrix and growth factors in salivary gland development. Front Oral Biol. 2010;14:48-77.

3. Tarasenko LM, Neporada KS, Klusha V. Stress-protective effect of glutapyrone belonging to a new type of amino acid-containing 1, 4-dihydropyridines on periodontal tissues and stomach in rats with different resistance to stress. Bull Exp Biol Med. 2002 Apr;133(4):369-71.

4. Erlinger R, Willershausen-Zönnchen B, Welsch U. Ultrastructural localization of glycosaminoglycans in human gingival connective tissue using cupromeronic blue. J Periodontal Res. 1995 Mar;30(2):108-15.

5. Kirkham J, Robinson C, Smith AJ, Spence JA. The effect of periodontal disease on sulphated glycosylaminoglycan distribution in the sheep periodontium. Arch Oral Biol. 1992 Dec;37(12):1031-7.

6. Afanasiev VV, Stryuk RI, Arutyunyan SE et al. The state of salivary glands in the patients presenting with metabolic syndrome. Ross Stomatol Zh 2011;(3):17-9. (In Russian).

7. Pathogenesis of Periodontal Diseases: Biological Concepts for Clinicians; Nagihan Bostanci, Georgios N. Belibasakis (Eds). Springer Int Publ AG, 2018. 114 p.

8. Kaidashev I.P. NF-kB activation as a molecular basis of pathological process by metabolic syndrome. Fiziol Zh. 2012;58(1):93-101. (In Ukrainian).

9. Rasin MS, Kaidashev IP. The role of nuclear transcription factors in modern syntropy internal pathology. Ukr Med Chasop. 2014;(1):17-21. (In Russian).

10. Liacini A, Sylvester J, Li WQ, Zafarullah M. Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes. Matrix Biol. 2002 Apr;21(3):251-62.

11. Ljashenko LI, Kostenko VA. The role of NF-κB-mediated action of NO-synthases in disorganization of periodontal connective tissue under modeled metabolic syndrome. Zahal’na Patolohiya ta Patolohichna Fiziolohiya. 2013;8(3):53-7. (In Ukrainian).

12. Kim HS, Quon MJ, Kim JA. New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate. Redox Biol. 2014 Jan 10;2:187-95.

13. Kanlaya R, Khamchun S, Kapincharanon C, Thongboonkerd V. Protective effect of epigallocatechin-3-gallate (EGCG) via Nrf2 pathway against oxalate-induced epithelial mesenchymal transition (EMT) of renal tubular cells. Sci Rep. 2016 Jul 25;6:30233.

14. Sun W, Liu X, Zhang H et al. Epigallocatechin gallate upregulates NRF2 to prevent diabetic nephropathy via disabling KEAP1. Free Radic Biol Med. 2017 Jul;108:840-857.

15. Ma Q. Role of Nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. 2013;53:401-26.

16. Vomhof-Dekrey EE, Picklo MJ Sr. The Nrf2-antioxidant response element pathway: a target for regulating energy metabolism. J Nutr Biochem. 2012 Oct;23(10):1201-6.

17. Li W, Khor TO, Xu C et al. Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis. Biochem Pharmacol. 2008 Dec 1;76(11):1485-9.

18. Wardyn JD, Ponsford AH, Sanderson CM. Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochem Soc Trans. 2015 Aug;43(4):621-6.

19. Yelins’ka AM, Shvaykovs’ka OO, Kostenko VO. Sources of production of reactive oxygen and nitrogen species in tissues of periodontium and salivary glands of rats under modeled systemic inflammation. Problemy Ekologii ta Medytsyny. 2017;21(3-4):51-4.

20. Tetyanets SS. Method for the determination of free hydroxyproline in serum. Lab Delo. 1985;(1):61-6. (In Russian).

21. Sharayev PN. Method for the determination of glycosaminoglycans in biological fluids. Lab Delo. 1987;(5):530-2. (In Russian).

22. Methods of clinical and experimental research in medicine (Ed. IP Kaidashev). – Poltava, 2003. 320 p. (In Ukrainian).

23. Han Z, Boyle DL, Manning AM, Firestein GS. AP-1 and NF-kappaB regulation in rheumatoid arthritis and murine collagen-induced arthritis. Autoimmunity. 1998;28(4):197-208.

24. Ljashenko LI., Denisenko SV, Kostenko VA. Role of transcription nuclear factor κB in mechanisms of free radical processes impairment and connective tissue disorganization in periodontium under modeled metabolic syndrome. Aktual’ni Problemy Suchasnoyi Medytsyny: Visn Ukrayins’koyi Med Stomatol Akademiyi. 2014; 14(1):97-100. (In Ukrainian).

25. Chen FM, Jin Y. Periodontal tissue engineering and regeneration: current approaches and expanding opportunities. Tissue Eng Part B Rev. 2010 Apr;16(2):219-55.

26. Xu C, Shen G, Chen C et al. Suppression of NF-kappaB and NF-kappaB-regulated gene expression by sulforaphane and PEITC through IkappaBalpha, IKK pathway in human prostate cancer PC-3 cells. Oncogene. 2005 Jun 30;24(28):4486-95.

27. Yang F, Oz HS, Barve S et al. The green tea polyphenol (-)-epigallocatechin-3-gallate blocks nuclear factor-kappa B activation by inhibiting I kappa B kinase activity in the intestinal epithelial cell line IEC-6. Mol Pharmacol. 2001 Sep;60(3):528-33.

28. Gupta S, Hastak K, Afaq F et al. Essential role of caspases in epigallocatechin-3-gallate-mediated inhibition of nuclear factor kappa B and induction of apoptosis. Oncogene. 2004 Apr 1;23(14):2507-22.

29. Bonnans C., Chou J, Werb Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol. 2014 Dec; 15(12): 786–801.

30. Mozaffari MS, Abdelsayed R, Zakhary I et al. Submandibular gland and caries susceptibility in the obese Zucker rat. J Oral Pathol Med. 2011 Feb; 40(2): 194–200.

31. Lin Y, Bai L, Chen W, Xu S. The NF-kappaB activation pathways, emerging molecular targets for cancer prevention and therapy. Expert Opin Ther Targets. 2010 Jan;14(1):45-55.

 

Conflict of interest:

The Authors declare no conflict of interest

ADDRESS FOR CORRESPONDENCE

Vitalii O. Kostenko

Department of Pathophysiology,

Ukrainian Medical Stomatological Academy,

Shevchenko St., 23, 36000, Poltava, Ukraine

tel: +380532226966

e-mail: patofiziolog@umsa.edu.ua

Received: 06.02.2018

Accepted: 20.05.2018

Table I. Effect of EGCG on indices of periodontal connective tissue disruption during SI (M+m, n=30)

Groups of the animals

Soft components

Calcified components

(alveolar bone)

FHP,

µmol/g

GAGs, µmol/g

NANA, µmol/g

FHP,

µmol/g

GAGs,

µmol/g

NANA, µmol/g

Intact animals

4.08±0.48

1.93±0.34

4.56±0.17

3.06±0.28

1.70±0.30

2.01±0.35

Animals with SI

6.78±0.35 *

3.22±0.34 *

7.43±0.33 *

5.20±0.19 *

2.93±0.22 **

4.33±0.37 *

SI modeling + EGCG

4.52±0.31 **

1.95±0.19 **

4.88±0.36 **

3.02±0.24 **

1.73±0.04 **

2.02±0.23 **

Note (in table I-II): * – Р<0.05 compared with values of intact rats,

** – р<0.05 compared with values of the second group.

Table II. Effect of EGCG on indices of SG connective tissue disruption during SI (M+m, n=30)

Groups of the animals

FHP, µmol/g

GAGs, µmol/g

NANA, µmol/g

Intact animals

3.21±0.25

1.41±0.16

3.51±0.07

Animals with SI

5.29±0.12 *

2.59±0.10 *

5.61±0.28 *

SI modeling + EGCG

3.29±0.18 **

1.56±0.05 **

3.50±0.23 **

Fig. 1. MREI (M+m, n=30) in the intact animals (1), under the exposure to modeled SI (2), under modeled SI and EGCG administration (3); * – Р<0.05 compared with values of intact rats, ** – р<0.05 compared with values of the second group.