Study of anti-inflammatory, antioxidant, antimicrobial and mineralizing effects of an N-isopropenylimidazole zinc metal complex derivative in experimental endodontic-periodontal lesions in rats

Pavel A. Galenko-Yaroshevsky¹, Ivan I. Pavlyuchenko¹, Olga V. Shelemekh², Boris A. Trofimov³, Lidiya N. Parshina³, Viktor L. Popkov¹, Svetlana A. Demyanenko⁴, Oleg V. Tsymbalov¹, Andrey V. Zadorozhniy², Anait V. Zelenskaya¹, Irina B. Nektarevskaya², Alina V. Sergeeva¹, Nikolay E. Korovaykin¹, Svetlana A. Lebedeva^5,6

1 Kuban State Medical University, 4 Mitrofan Sedin St., Krasnodar 350063 Russia

2 Rostov State Medical University, 29 Nakhichevanskiy Ave., Rostov-on-Don 344022 Russia

3 AE Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, 1 Favorskyy St., Irkutsk 664033 Russia

4 Medical Institute named after S.I. Georgievsky (winner of the Red Banner of Labor Award), V.I. Vernadsky Crimean Federal University, 5/7 Lenin Blvd, Simferopol 295001 Russia

5 I.M. Sechenov First Moscow State Medical University, 8 Trubetskaya St., Bldg 2, Mosсow 119991  Russia

6 RUDN University, 6 Miklukho-Maklaya St., Moscow 117198 Russian Federation

Corresponding author: Pavel A. Galenko-Yaroshevsky (Galenko.Yarochevsky@gmail.com)

Abstract

Introduction: The combination treatment of patients with dentition problems including endodontic-periodontal lesions (EPL) is known to widely involve anti-inflammatory drugs, including non-steroidal anti-inflammatory agents, and antimicrobial drugs, in particular antibiotics and antiseptics, from which the former often lead to the development of gastro- and neuropathy, lesions of kidneys and liver, as well as the cardiovascular system, and the latter, in addition to serious side effects, lead to the development of steady resistance of pathogenic microorganisms and dysbacteriosis. This necessitates search for and development of new molecules with a wide range of therapeutic effects aimed to block the infectious and inflammatory process and to restore the functional ability of teeth in EPLs as much as possible (depending on a lesion severity). The aim of the study was to examine the anti-inflammatory, antioxidant, antimicrobial and mineralizing effects of an N-isopropenylimidazole zinc metal complex derivative in experimental endodontic-periodontal lesions in rats.

Materials and Methods: EPL simulation in experiments on the anesthetized rats was conducted in the following way: first, the pulp cavity of lower incisors was opened and left exposed to infection; then periodontal inflammation was induced using a ligature. Thirty days later, EPL developed in the animals. An N-isopropenylimidazole zinc metal complex derivative (laboratory code Pilim-1, prepared in form of gel), reference drug Metrogyl Denta® (M-D, dental gel), as well as the combination Pilim-1 + M-D were applied daily for 14 days on the lower incisors and appropriate gums, and also placed in the gingival pockets and pulp cavities, with pulp partially removed beforehands. After euthanasia of the animals on the 15^th day of the experiments, gingival homogenates were assayed for the activity of elastase, urease, catalase, the level of malondialdehyde, and the antioxidant-prooxidant index was calculated, while pulp homogenates were assayed for the activity of elastase, urease, alkaline and acidic phosphatases, and the mineralizing index was calculated.

Results and Discussion: In the gum in EPL, Pilim-1 and, especially, the combination Pilim-1+M-D more significantly than M-D have a pronounced anti-inflammatory, antioxidant and antimicrobial (reduce a microbial content level) effects. In the pulp in EPL, Pilim-1 and, to a greater extent, the combination Pilim-1 + M-D in a more pronounced way than M-D have anti-inflammatory, antimicrobial (reduce a microbial content level) and mineralizing effects. In the therapeutic action mechanisms of Pilim-1 and, especially, the combination Pilim-1 + M-D in EPL conditions, their ability to have an antihypoxic effect and an inhibitory effect on cyclooxygenase and 5-lipoxygenase is likely to play a very important role, in addition to their identified effect on the examined metabolic processes. Besides, a certain role may be played by the presence of zinc and imidazole in the structure of Pilim-1, as they have a wide range of biological activity.

Conclusion: The presented results of studying Pilim-1 and its combination with M-D indicate their high therapeutic efficiency, especially when using the latter in the experimental EPL in rats, which makes it possible to consider Pilim-1 and Pilim-1 + M-D as potential therapeutic agents to treat EPL.

Graphical Abstract

Keywords: antioxidant effect, N-isopropenylimidazole zinc metal complex derivative, microbial content, mineralizing effect, anti-inflammatory effect, endodontic-periodontal lesion

Introduction

The combination treatment of patients with dentition problems including endodontic-periodontal lesions (EPL) is known to widely involve anti-inflammatory drugs, including non-steroidal anti-inflammatory agents, and antimicrobial drugs, in particular antibiotics and antiseptics, from which the former often lead to the development of gastro- and neuropathy, lesions of kidneys and liver, as well as the cardiovascular system (Кarateev et al. 2018; Khoroshun and Lazarevа 2022; Kamchatnov et al. 2023; Minhas et al. 2023), and the latter, in addition to serious side effects, lead to the development of steady resistance of pathogenic microorganisms and dysbacteriosis (Bakhit et al. 2018; Kuzmina et al. 2019; Orekhova et al. 2020). It is generally believed that in the development and course of EPL, an important role is played by free-radical oxidation, disturbance of which leads to the imbalance of antioxidant and pro-oxidant systems, which causes a significant increase in the level of free radicals and the formation of “oxidative stress”, resulting in damage to cells and an increase in vascular and tissue permeability (Grebenchikov et al. 2016; Men’shchikova and Zenkov 2016; Atabay et al. 2017; Chen et al. 2019). All this necessitates search for and development of new molecules with a wide range of therapeutic effects aimed to block the infectious and inflammatory process and to restore the functional ability of teeth in EPL as much as possible (depending on a lesion severity). A very significant problem of EPL should be solved in a comprehensive and targeted manner by using drugs against which microorganisms develop no resistance and which can exert high anti-inflammatory, antimicrobial, detoxification, antioxidant, and regenerative effects (Tsepov et al. 2018; Ushakov and Tsaryov 2019).

In view of the above-mentioned data, our attention settled on a N-isopropenylimidazole zinc metal complex derivative with laboratory code Pilim-1, which, according to our earlier studies, has polyfunctional properties – anti-inflammatory, analgetic, antimicrobial, antioxidant, antihypoxic and wound-healing (Shakhmardanova and Galenko-Yaroshevsky 2015; Lebedeva et al. 2021, 2022, 2022а, 2023, 2023a; Galenko-Yaroshevsky et al. 2023, 2024) and can be of interest as a potentially promising agent in an EPL combination therapy.

The aim of the study was to examine the anti-inflammatory, antioxidant, antimicrobial, and mineralizing effects of an N-isopropenylimidazole zinc metal complex derivative in experimental endodontic-periodontal lesions in rats.

Materials and Methods

Test compound and reference drug

The substance of N-isopropenylimidazole zinc diacetate with laboratory code Pilim-1 (AE Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, Russia) as 1% gel based on sodium carboxymethylcellulose (sodium CMC; RossPolimer LLC, Russia); dental gel Metrogyl Denta® – M-D (Unique Pharmaceutical Laboratories, India), which contains metronidazole, chlorhexidine digluconate, propylene glycol, carbomer-940, edetate disodium, sodium saccharin, levomenthol, sodium hydroxide and water.

Animals

The studies were performed on 400 white Wistar rats weighing 210-280 g, kept in the vivarium standard conditions on a standard diet in accordance with the rules of good laboratory practice (GOST 33216-2014). The studies were conducted in compliance with the European Convention on the Protection of Vertebrates Used for Experiments or Other Scientific Purposes (as amended in Strasbourg, 2006); Law of the Russian Federation “On the Protection of Animals from Cruel Treatment” dated June 24, 1998; Good Laboratory Practice for Conducting Preclinical Trials in the Russian Federation (GOST 3 51000.3-96 and GOST P 53434-2009), Helsinki Declaration designed by the World Medical Association on Humane Treatment of Laboratory Animals (Report of the AVMA Panel on Euthanasia JAVMA, 2001); Guidelines on Maintenance and Care of Laboratory Animals (interstate standard GOST 33216-2014 dated July 1, 2016); Guidelines for Conducting Preclinical Trials of Drugs (Mironov 2012) and Guidelines for Working with Laboratory (Experimental) Animals in Preclinical (Non-clinical) Trials (Recommendations by the board of the Eurasian Economic Commission No. 33 of November 14, 2023, Moscow). The experiments were approved by the Ethical Committee of Rostov State Medical University of the Ministry of Health of Russia (Minutes No. 16/19 of September 17, 2019).

Experiment design

To study Pilim-1, reference drug M-D and the combination Pilim-1 + M-D, according to the aim, five groups of animals were formed:

1. Gingival homogenates were assayed for:

- elastase activity – 5 groups of 10 rats: Group 1 – intact gums (IG), Group 2 – experimental endodontic-periodontal lesion (E-EPL) without treatment (with applying only gel based on sodium-CMC), Group 3 – E-EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 – E-EPL + Pilim-1 + M-D (at a ratio of 1:1; 0.3 mL on teeth and gums);

- level of malondialdehyde (MDA) – 5 groups of 10 rats: Group 1 – IG, Group 2 – E-EPL without treatment, Group 3 – E-EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 – E-EPL + Pilim-1 + M-D (at a ration of 1:1; 0.3 mL on teeth and gums);

- urease activity – 5 groups of 10 rats: Group 1 – IG, Group 2 – E-EPL without treatment, Group 3 – E-EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 – E-EPL + Pilim-1 + M-D (at a ratio of 1:1; 0.3 mL to teeth and gums);

- catalase activity – 5 groups of 10 rats: Group 1 – IG, Group 2 – E-EPL without treatment, Group 3 – EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 – E-EPL + Pilim-1 + M-D (at a ratio of 1:1; 0.3 mL to teeth and gums).

2. Pulp homogenates were assayed for:

- elastase activity – 5 groups of 10 rats: Group 1 – intact pulp (IP), Group 2 – E-EPL without treatment, Group 3 –E-EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 –E-EPL + Pilim-1 + M-D (at a ratio of 1:1; 0.3 mL on teeth and gums);

- urease activity – 5 groups of 10 rats: Group 1 – IP, Group 2 – E-EPL without treatment, Group 3 – E-EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 –E-EPL + Pilim-1 + M-D (at a ratio of 1:1; 0.3 mL on teeth and gums);

- activity of alkaline phosphatase (APP) – 5 groups of 10 rats: Group 1 – IP, Group 2 – E-EPL without treatment, Group 3 – E-EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 – E-EPL + Pilim-1 + M-D (at a ratio of 1:1; 0.3 mL on teeth and gums);

- activity of acidic phosphatase (AP) – 5 groups of 10 rats: Group 1 – IP, Group 2 – E-EPL without treatment, Group 3 – E-EPL + Pilim-1 gel (0.3 mL on teeth and gums), Group 4 – E-EPL + M-D (0.3 mL on teeth and gums), Group 5 – E-EPL + Pilim-1 + M-D (at a ratio of 1:1; 0.3 mL on teeth and gums) (Fig. 1).

Figure 1. Flowchart of research design.

Experimental research models

The animals were randomized for each studied indicator into 5 groups of 10 individuals each.

E-EPL was simulated under Telazol-Meditin anesthesia (Telazol (Zoetis, Spain) – 0.3 mg (intramuscularly), Meditin (API-SAN LLC, Russia) – 0.8 mg (intramuscularly) and 0.1 % Atropine sulfate solution (Federal state unitary enterprise “Moscow Endocrine Plant”, Russia) – 0.01 mL (subcutaneously), per 100 g of animal weight). The pulp cavity of lower incisors was opened and left exposed to infection, and then periodontal inflammation was induced using a ligature method described by Polson and Zander (1983), Keles et al. (2005), Popkov et al. (2022), which involves applying ligatures made of EvroLen 4/0 suture material (EuroType, Russia) on the lower incisor necks, with further ligature placement in the gingival sulcus. Inoculation of the root canal system, the apical region of the studied teeth and periodontal tissues in rats occurred naturally within 30 days.

For preparing gel, sodium-CMC was pre-mixed with glycerin at a ratio of 1:2. Then, water heated to 40°C was gradually poured into this mixture, while constantly stirring with a Brayer BR1303 mixer (Brayer, China) until a homogeneous gel was obtained. The prepared solution was left for 2 hours for complete dissolution of sodium-CMC.

Pilim-1, M-D and the combination Pilim-1 + M-D were applied daily at a dose of 0.3 mL for 14 days on the lower incisors and the corresponding gums and also placed into the gingival pockets and pulp cavities after partial removal of pulp. Before applying the studied substances, the lower incisors and gums had been treated with a 0.05% chlorhexidine bigluconate solution (Tula Pharmaceutical Factory, Russia) for 3-5 minutes. After the studied substances and their combination had been applied, the rats had limited access to the feed and water for 2 hours.

The animals were eliminated from the experiment on the 45^th day after its onset, i.e. on the 15^th day after the treatment, with the help of chloroform (in small doses, drops) under an airtight glass cover. Then, pulp and gums were sampled from the area of lower incisors and homogenates were prepared, which were placed in 0.05 M of Tris-HCl buffer at pH 7.6. The obtained homogenates were kept in the refrigerator for 30 min at t+2-3°C and then centrifuged at 3500 rpm and +4°C for 15 minutes. Supernatant was placed in clean vials for biochemical studies (Levitsky et al. 2018).

The elastase activity in the gums and pulp was assayed by the degree of hydrolysis of the synthetic substrate N-t-BOC-L-alanine-p-nitrophenyl ester (BOC) (Sigma, USA) according to the method described by Levitsky et al. (2018) and Visser and Blout (1972). The enzyme activity was calculated using formula (1):

A = [(E₅ – E₀) – E] x n x 1000 / 1.0 x C x 300 x m, µkat/kg          (1)

where Е₀ – extinction at the 0^th minute; E₅ – extinction at the 5^th minute; E – control for BOC; 1.0 – volume of saliva, mL; 1000 – to convert grams into kilograms; n – sample dilution; 300 – incubation time, sec; С – conversion factor for expressing the extinction value as the concentration of p-nitrophenol; m – mass of tissue in 1 mL of buffer, g.

The activity of elastase was expressed in microkatals per 1 kg of tissue.

The MDA level in gingival homogenates was determined according to the method described by Stal’naya and Garishvilly (1977) and Levitsky et al. (2018), using a reaction with thiobarbituric acid forming stained trimethine complex, with peak absorption at 532 nm. The amount of MDA was calculated using formula (2):

A = E x 10⁶ x 1000 / 1.56 x 10⁵ x 0.5 x m, µmol/kg          (2)

where E – extinction of the experimental sample; 0.5 – volume of the added sample, mL; 1.56 х 10⁵ – molar coefficient; 10⁶ – to convert moles into micromoles; 1000 – to convert grams into kilograms; m – mass of tissue in 1 mL of buffer, g.

The results were expressed in µmol/kg of tissue.

The urease levels in homogenates of gums and pulp were determined using the method described by Gavrikova and Segen’ (1996) and Levitsky et al. (2018). A urea solution was used as a substrate. Sample extinction was measured using a photoelectric colorimeter (APEL AP-101, Japan) at 440 nm. The enzyme activity was calculated using formula (3):

U = (Ets – Ec) x 1000 / C x t x 0.2 x m, µkat/kg          (3)

where E_ts  – extinction of a test sample; E_c – extinction of a control sample; С – conversion factor for expressing extinction as µmol NH₄; t – incubation time, sec.; 1000 –- calculation per 1 kg of tissue; m – mass of tissue, g.

The activity of urease was expressed in microkatals per 1 kg of tissue.

The catalase activity in gingival homogenates was assayed using the method described by Levitsky et al. (2010). The color intensity was measured on a spectrophotometer with a wavelength of 410 nm, the color intensity being opposite to the activity of catalase expressed in µkat/kg of tissue. The enzyme activity was calculated using formula (4):

A = (Eblank – (Ets – Est)) x 1000 / 22.2 x 0.1 x 600 x m, µkat/kg          (4)

where E_blank – extinction of a blank sample; E_TS – extinction of a test sample; E_st – extinction of stain control; 1000 – to convert grams into kilograms; 0.1 – volume of the added sample, mL; 600 – incubation time, sec; 22.2 (M^-1 cm^-1) – molar extinction coefficient of H₂O₂; m – mass of tissue in 1 mL of buffer, g.

The antioxidant-prooxidant index (API) was calculated according to Levitsky et al. (2010) using formula (5):

API = Acat x 10 / C_MDA, µkat/kg          (5)

where A_сat – catalase activity (mkat/kg); C_MDA – MDA concentration (μmol/kg).

The activity of alkaline and acidic phosphatases in pulp was determined according to Bessey’s method modified in Levitsky et al. (2018). The color intensity of each sample was determined on a spectrophotometer with a wavelength of λ 400 nm. The activity of enzymes was calculated using formula (6):

A = DE x n x 1000 / C x T x V x m, µkat/kg          (6)

where ΔE – difference in extinctions between the test sample and stain control; n – sample dilution; 1000 – to convert grams into kilograms; t – incubation time, sec; V (0.1) – volume of biomaterial, mL; С – conversion factor for expressing extinction as the concentration of p-nitrophenol, µmol, determined by the calibration curve; m – mass of tissue in 1 mL of buffer, g.

The activity of alkaline phosphatase (APP) and acidic phosphatase (AP) was expressed in microkatals per 1 kg of tissue (µkat/kg).

The mineralizing index (MI) was calculated according to Levitsky et al. (2018), using formula (7):

MI = APP / AP,          (7)

where APP – activity of alkaline phosphatase; AP – activity of acidic phosphatase.

Statistical analysis

The results (the calculation of arithmetic means – M and standard errors of the mean – m) were statistically processed using Statistica Version 6.0 program (StatSoft Inc., USA), as well as special IBM PC software programs developed at the Department of Pharmacology of Kuban State Medical University. The hypotheses about the means were checked using Student’s t-test. The differences were considered reliable at p ˂ 0.05.

Results

Influence of Pilim-1 on inflammatory markers – activity of elastase and the MDA level – in the gum in EPL (G-EPL)

Elastase activity

The activity of elastase in G-EPL without treatment was found to have increased 1.4 times when compared to that in IG.

The use of Pilim-1 and reference drug M-D, as well as the combination Pilim-1 + M-D caused a 1.3-, 1.2- and 1.5-time decrease, respectively, in the elastase activity when compared to that in G-EPL without treatment. It is worth mentioning that under the influence of Pilim-1 and even more significantly under the influence of the combination Pilim-1 + M-D, the activity of this enzyme was comparable to that in IG (Table 1, Fig. 2).

Table 1.	Download as	_XLSX_	_CSV_
Influence of Pilim-1, M-D and the combination Pilim-1 + M-D on the activity of elastase and urease, and MDA level in G-EPL in rats (M ± m, n = 10)

Indicators and units of measurement	Groups of animals
	IG [1]	G-EPL w/o treatment [2]	G-EPL + Pilim-1 [3]	G-EPL + M-D [4]	G-EPL+ Pilim-1+M-D [5]
Elastase, µkat/kg	42.8 ± 1.8 (38.6 ÷ 47.0)	60.2 ± 2.3 (55.1 ÷ 65.3) p1-2 < 0.001	46.0 ± 1.9 (41.6 ÷ 50.4) p1-3 > 0.05 p2-3 < 0.001	51.4± 1.7 (47.5 ÷ 55.3) p1-4 < 0.02 p2-4 < 0.01 p3-4 < 0.05	40.6 ± 1.4 (37.4 ÷ 43.8) p1-5 > 0.05 p2-5 < 0.001 p3-5 < 0.05 p4-5 < 0.001
MDA, µmol/kg	21.2 ± 1.1 (18.8 ÷ 23.6)	32.5 ± 1.3 (29.6 ÷ 35.4) p1-2 < 0.001	24.2 ± 0.9 (22.2 ÷ 26.,2) p1-3 < 0.05 p2-3 < 0.001	28.4 ± 1.2 (25.7 ÷ 31.1) p1-4 < 0.001 p2-4 < 0.05 p3-4 < 0.02	20.8 ± 1.0 (18.6 ÷ 23.0) p1-5 > 0.05 p2-5 < 0.001 p3-5 < 0.05 p4-5 < 0.001
Urease, µkat/kg	1.46 ± 0.02 (1.41 ÷ 1.51)	2.18±0.04 (2.08 ÷ 2.28) p1-2 < 0.001	1.67 ± 0.05 (1.57 ÷ 1.77) p1-3 < 0.001 p2-3 < 0.001	1.80 ±0.03 (1.73 ÷1.87) p1-4 < 0.001 p2-4 < 0.001 p3-4 < 0.05	1.54 ± 0.03 (1.47 ÷ 1.61) p1-5 > 0.05 p2-5 < 0.001 p3-5 < 0.05 p4-5 < 0.001

Note: 1. IG – intact gum, G-EPL – gum in endodontic-periodontal lesion, M-D – Metrogyl Denta^®. 2. In parentheses – confidenсe interval at p < 0.05, in square brackets – animal group number.

Figure 2. Comparative assessment of the influence of Pilim-1, M-D and the combination Pilim-1 + M-D on the activity of elastase in G-EPL in rats. Note: IG – intact gum, G-EPL – gum in endontodic-periodontal lesion, M-D – Metrogyl Denta^®.

MDA level

Similar changes (as described above for elastase) were observed when determining the MDA level in G-EPL. In G-EPL without treatment, the level of MDA, when compared to that in animals with the IG, increased 1.5 times, and, when applying Pilim-1, M-D and the combination Pilim-1 + M-D, it decreased 1.3, 1.1 and 1.6 times, respectively. It is noteworthy that the MDA level reaches the baseline (as in IG) only when using the combination Pilim-1 + M-D (Table 1, Fig. 3).

Figure 3. Comparative assessment of the influence of Pilim-1, M-D and combination Pilim-1 + M-D on the MDA level in G-EPL in rats. Note: IG – intact gum, G-EPL – gum in endontodic-periodontal lesion, MDA –  malondialdehyde, M-D – Metrogyl Denta^®.

Iinfluence of Pilim-1 on the microbial content marker – the activity of urease – in G-EPL

The activity of urease in G-EPL without treatment was found to have increased 1.5 times when compared to that in IG.

The use of Pilim-1, M-D and the combination Pilim-1 + M-D caused a decrease in the urease activity by 1.3, 1.2 and 1.4 times, respectively. It is noteworthy that the activity of urease was almost comparable with that in IG only when Pilim-1 + M-D was applied (Table 1, Fig. 4).

Figure 4. Comparative assessment of the influence of Pilim-1, M-D and the combination Pilim-1 + M-D on the urease activity in G-EPL in rats. Note: IG – intact gum, G-EPL – gum in endontodic-periodontal lesion, M-D- Metrogyl Denta^®.

Influence of Pilim-1 on the activity of antioxidant enzyme catalase and API in G-EPL in rats

Catalase activity

The activity of catalase in G-EPL without treatment was found to have decreased 1.5 times when compared to that in IG.

The use of Pilim-1, M-D and the combination Pilim-1 + M-D induced an increase in the catalase activity 1.3, 1.2 and 1.4 times, respectively in comparison with such in G-EPL without treatment. It is noteworthy that the activity of catalase was almost comparable to such only when using the combination Pilim-1 + M-D (Table 2, Fig. 5).

Table 2.	Download as	_XLSX_	_CSV_
Influence of Pilim-1, M-D and the combination Pilim-1 + M-D on the catalase activity in G-EPL and API in rats (M ± m, n = 10)

Indicators and units of measurement	Groups of animals
	IG [1]	G-EPL w/o treatment [2]	G-EPL + Pilim-1 [3]	G-EPL + M-D [4]	G-EPL+ Pilim-1+M-D [5]
Catalase, µkat/kg	9.6 ± 0.1 (9.4 ÷ 9.8)	6.5 ± 0.2 (6.0 ÷ 7.0) p1-2 < 0.001	8.7 ± 0.1 (8.5 ÷ 8.9) p1-3 < 0.001 p2-3 < 0.001	7.8 ± 0.1 (7.6 ÷ 8.0) p1-4 < 0.001 p2-4 < 0.001 p3-4 < 0.001	9.4 ± 0.2 (8.9 ÷ 9.9) p1-5 > 0.05 p2-5 < 0.001 p3-5 < 0.01 p4-5 < 0.001
API, RU	4.52 ± 0.16 (4.15 ÷ 4.89)	2.0 ± 0.08 (1.83 ÷ 2.17) p1-2 < 0.001	3.60 ± 0.11 (3.36 ÷ 3.84) p1-3 < 0.001 p2-3 < 0.001	2.75 ± 0.12 (2.48 ÷ 3.02) p1-4 < 0.001 p2-4 < 0.05 p3-4 < 0.001	4.52 ± 0.14 (4.20 ÷ 4.84) p1-5 > 0.05 p2-5 < 0.001 p3-5 < 0.001 p4-5 < 0.001

Note: 1. IG – intact gum, G-EPL – gum in endodontic-periodontal lesion, M-D – Metrogyl Denta^®, API – antioxidant-prooxidant index. 2. In parentheses – confidenсe interval at p < 0.05, in square brackets – animal group number.

Figure 5. Comparative assessment of the influence of Pilim-1, M-D and combination Pilim-1 + M-D on the catalase activity in G-EPL in rats. Note: IG – intact gum, G-EPL – gum in endontodic-periodontal lesion, M-D- Metrogyl Denta^®.

API

The calculation of API showed that in EPL without treatment, this index decreased 2.3 times when compared to that in IG.

With the use of Pilim-1, M-D and the combination Pilim-1 + M-D, API when compared to that in G-EPL without treatment increased 1.8, 1.4 and 2.3 times, respectively. And, like in the case of catalase, when applying the combination Pilim-1 + M-D, API was almost comparable to that in IG (Table 2, Fig. 6).

Figure 6. Comparative assessment of the influence of Pilim-1, M-D and combination Pilim-1 + M-D on API in G-EPL in rats. Note: IG – intact gum, G-EPL – gum in endontodic-periodontal lesion, API – antioxidant-prooxidant index, M-D- Metrogyl Denta^®.

Influence of Pilim-1 on the activity of elastase and urease in the pulp in EPL (P-EPL)

Elastase activity

The activity of elastase in P-EPL without treatment was found to have increased 1.5 times when compared to that in IP.

The use of Pilim-1 and reference drug M-D, as well as the combination Pilim-1 + M-D, caused a 1.5-, 1.4- and 1.7-time decrease in the elastase activity, respectively, when compared to that in P-EPL without treatment. These data indicate that Pilim-1, Pilim-1 + M-D, as well as reference drug M-D, reduce the activity of elastase to the level of that in IP. Nevertheless, Pilim-1 and especially the combination Pilim-1 + M-D are more preferable in terms of anti-inflammatory action than M-D, although only a trend was observed in this regard (no statistically significant differences were found). When comparing the indicators of elastase activity under the influence of Pilim-1 and M-D, no differences were found, but when comparing the activity of this enzyme under the influence of the combination Pilim-1 + M-D with reference drug M-D and Pilim-1, the first indicator statistically significantly exceeded the second and the third – 1.3 and 1.2 times, respectively (Table 3, Fig. 7).

Table 3.	Download as	_XLSX_	_CSV_
Influence of Pilim-1, M-D and the combination Pilim-1 + M-D on the activity of elastase and urease in P-EPL in rats (M ± m, n = 10)

Indicators and units of measurement	Groups of animals
	IP [1]	P-EPL w/o treatment [2]	P-EPL + Pilim-1 [3]	P-EPL + M-D [4]	P-EPL+ Pilim-1+M-D [5]
Elastase, µkat/kg	62.0 ± 2.4 (56.6 ÷67.4)	95.0 ± 3.4 (87.5 ÷ 102.5) p1-2 < 0.001	64.0 ± 2.6 (58.1 ÷ 69.9) p1-3 > 0.05 p2-3 < 0.001	70.0 ± 3.2 (62.7 ÷ 77.3) p1-4 > 0.05 p2-4 < 0.001 p3-4 > 0.05	55.0 ± 3.0 (48.1 ÷ 61.9) p1-5 > 0.05 p2-5 < 0.001 p3-5 < 0.05 p4-5 < 0.01
Urease, µkat/kg	0.057±0.007 (0.042 ÷ 0.072)	0.116±0.012 (0.086 ÷ 0.146) p1-2 < 0.001	0.067±0.009 (0.047 ÷ 0.087) p1-3 > 0.05 p2-3 < 0.001	0.078±0.007 (0.063 ÷ 0.093) p1-4 < 0.05 p2-4 < 0.002 p3-4 > 0.05	0.052±0.008 (0.035 ÷ 0.069) p1-5 > 0.05 p2-5 < 0.001 p3-5 > 0.05 p4-5 < 0.02

Note: 1. IP – intact pulp, P-EPL – pulp in endodontic-periodontal lesion, M-D – Metrogyl Denta^®, 2. In parentheses – confidenсe interval at p < 0.05, in square brackets – animal group number.

Figure 7. Comparative assessment of the influence of Pilim-1, M-D and combination Pilim-1 + M-D on the elastase activity in P-EPL in rats. Note: IP – intact pulp, P-EPL – pulp in endodontic-periodontal lesion, M-D – Metrogyl Denta^®.

Urease activity

The activity of urease in P-EPL without treatment was found to have increased 2 times when compared to that in IP, which indicates a significant microbial content in the pulp.

The use of Pilim-1, M-D and the combination Pilim-1 + M-D led to a 1.7-, 1.5- and 2.2-time decrease in urease activity, respectively. It is noteworthy that under the influence of Pilim-1 and, to a greater extent, the combination Pilim-1 + M-D, the activity of urease was almost comparable to that in IP, whereas the activity of this enzyme when using reference drug M-D was statistically significantly – 1.4 times – higher than that in IP. It is important to mention that no statistically significant differences between the urease activity when using the combination Pilim-1 + M-D and Pilim-1 were observed, although there was a tendency to its more pronounced decrease under the influence of the former (Table 3, Fig. 8).

Figure 8. Comparative assessment of the influence of Pilim-1, M-D and the combination Pilim-1 + M-D on the urease activity in P-EPL in rats. Note: IP – intact pulp, P-EPL – pulp in endodontic-periodontal lesion, M-D – Metrogyl Denta^®.

Influence of Pilim-1 on markers of osteoblast and osteoclast – the activity of APP and AP – and their correlation – MI in P-EPL

APP activity

The studies have shown that the APP activity in P-EPL without treatment reduced 1.4 times when compared to that in IP.

The use of Pilim-1, M-D and the combination Pilim-1 + M-D caused a 1.6-, 1.3- and 1.7-time increase in the APP activity, respectively, when compared to that in P-EPL without treatment. The results obtained indicate that Pilim-1, Pilim-1 + M-D, as well as reference drug M-D increase the APP activity to the level of that in IP. When comparing the indicators of the APP activity under the influence of Pilim-1 and M-D, no differences were found, but when comparing the activity of this enzyme under the influence of the combination Pilim-1 + M-D with reference drug M-D and Pilim-1, the first indicator statistically significantly – 1.3 times – exceeded the second indicator and was almost comparable with the third, with a tendency to a more pronounced increase in the APP activity (Table 4, Fig. 9).

Table 4.	Download as	_XLSX_	_CSV_
Influence of Pilim-1, M-D and combination Pilim-1 + M-D on the activity of APP and AP, MI in P-EPL in rats (m ± m, n = 10)

Indicators and units of measurement	Groups of animals
	IP [1]	P-EPL w/o treatment [2]	P-EPL + Pilim-1 [3]	P-EPL + M-D [4]	P-EPL+ Pilim-1+M-D [5]
APP, µkat/kg	1050 ± 90 (847 ÷ 1253)	750 ± 80 (569 ÷ 931) p1-2 < 0.05	1170 ± 90 (967 ÷ 1373) p1-3 > 0.05 p2-3 < 0.01	1010 ± 60 (875 ÷ 1145) p1-4 > 0.05 p2-4 < 0.02 p3-4 > 0.05	1280 ± 70 (1123 ÷ 1437) p1-5 > 0.05 p2-5 < 0.001 p3-5 > 0.05 p4-5 < 0.01
AP, µkat/kg	29.46 ± 1.52 (26.06 ÷ 32.86)	48.32 ± 3.24 (41.02 ÷ 55.62) p1-2 < 0.001	31.56 ± 1.24 (28.76 ÷ 34.36) p1-3 > 0.05 p2-3 < 0.001	35.76 ± 1.38 (32.66 ÷ 38.86) p1-4 < 0.01 p2-4 < 0.002 p3-4 < 0.05	30.05 ± 1.42 (26.85 ÷ 33.25) p1-5 > 0.05 p2-5 < 0.001 p3-5 > 0.05 p4-5 < 0.01
MI, RU	35.6 ± 2.4 (30.2 ÷ 41.0)	15.5 ± 2.8 (9.1 ÷ 21.9) p1-2 < 0.001	37.1 ± 2.2 (32.2 ÷ 42.0) p1-3 > 0.05 p2-3 < 0.001	28.2 ± 2.5 (22.6 ÷ 33.8) p1-4 < 0.05 p2-4 < 0.001 p3-4 < 0.02	42.6 ± 2.6 (36.7 ÷ 48.5) p1-5 > 0.05 p2-5 < 0.001 p3-5 < 0.05 p4-5 > 0.001

Note: 1. IP – intact pulp, P-EPL – pulp in endodontic-periodontal lesion, M-D – Metrogyl Denta^®, APP – alkaline phosphatase, AP – acidic phosphatase, MI – mineralizing index. 2. In parentheses – confidenсe interval at p <0.05, in square brackets – animal group number.

Figure 9. Comparative assessment of the influence of Pilim-1, M-D and combination Pilim-1 + M-D on APP activity in P-EPL in rats. Note: IP – intact pulp, P-EPL – pulp in endodontic-periodontal lesion, APP – alkaline phosphatase, M-D – Metrogyl Denta^®.

AP activity

AP activity in P-EPL without treatment was found to have increased 1.6 times when compared to that in IP.

The use of Pilim-1, M-D, as well as the combination Pilim-1 + M-D caused a 1.4-, 1.5- and 1.6-time decrease in the AP activity, respectively, when compared to that in P-EPL without treatment. It is noteworthy that the AP activity was almost comparable to that in IP only when using Pilim-1 and the combination Pilim-1 + M-D. In addition, it is worth mentioning that the AP activity when using Pilim-1 was comparable to that when using the combination Pilim-1 + M-D. It is also important to emphasize that Pilim-1 and, especially, the combination Pilim-1 + M-D statistically significantly – 1.1 and 1.2 times, respectively – exceeded the reference drug M-D in inhibiting AP. As for the influence of Pilim-1 and the combination Pilim-1 + M-D on the AP activity, no statistically significant differences were found between them (Table 4, Fig. 10).

Figure 10. Comparative assessment of the influence of Pilim-1, M-D and combination Pilim-1 + M-D on AP activity in P-EPL in rats. Note: IP – intact pulp, P-EPL – pulp in endodontic-periodontal lesion, AP – acidic phosphatase, M-D – Metrogyl Denta^®.

Mineralizing index (MI)

When calculating MI, this indicator in EPL without treatment was found to have decreased 2.3 times when compared to that in IP.

The use of Pilim-1, M-D, as well as the combination Pilim-1 + M-D induced a 2.4-, 1.8- and 2.7-time increase in MI, respectively, when compared to that in P-EPL without treatment. It is important to note that MI was almost comparable to that in IP when using Pilim-1 and the combination Pilim-1 + M-D. At the same time, when using Pilim-1 and the combination Pilim-1 + M-D, MI indicators were not significantly different. Besides, Pilim-1 and, especially, the combination Pilim-1 + M-D statistically significantly – 1.3 and 1.5 times, respectively – exceeded reference drug M-D in terms of MI (Table 4, Fig. 11).

Figure 11. Comparative assessment of the influence of Pilim-1, M-D and combination Pilim-1 + M-D on MI in P-EPL in rats. Note: IP – intact pulp, P-EPL – pulp in endodontic-periodontal lesion, MI – mineralizing index, M-D – Metrogyl Denta^®.

Discussion

Pronounced anti-inflammatory, antimicrobial (a decreased microbial content), antioxidant and mineralizing effects of Pilim-1 in D-EPL and P-EPL can be due to the inhibitory effect of this substance on cyclooxygenase and 5-lipoxygenase (Galenko-Yaroshevsky et al. 2024), as well as to Pilim-1 having antihypoxic, membrane-stabilizing, bacteriostatic and reparative properties resulting from the combined action of its structural components – N-isopropenylimidazole and zinc (Shakhmardanova and Galenko-Yaroshevsky 2015, 2016).

The synthetic derivatives of imidazole, including a number of drugs – metronidazole, naphthyzin, phentolamine, ethimizol, ketoconazole, mebendazole, mercazolil, mercaptopurine, etc. – are known to exert high anti-inflammatory, analgesic, antimicrobial and other types of activity (Fadhil and Qhanim 2020; Gas et al. 2020; Rajam et al. 2020; Valls et al. 2020; Vidhya and Malar 2020; Gurevich et al. 2021; Pham et al. 2021; Patel et al. 2022; Kaldybaeva et al. 2023).

Zinc, as a trace element, participates in the formation of antioxidant status and stabilization of cellular structures (Panasenko et al. 2018), has an anti-inflammatory and antibacterial effect, which involves the suppression of pro-inflammatory cytokines – TNF-D, IL-1β, IL-6, IL-8, IL-12 – by reducing the activation of NF-kB (nuclear factor-kappa B), which is involved in the regulation of genes expression affecting the functioning of the immune system and inflammatory reactions. Moreover, an important role in the mechanism of the anti-inflammatory action of zinc is played by its ability to increase the expression of zinc finger protein A20, known as tumor necrosis factor, alpha-induced protein 3 or A20, which is the central regulator of the NF-kB (Liu et al. 2017; Voelkl et al. 2018; Ho et al. 2020; Nakano et al. 2020; Tobita et al. 2020; Lebedeva et al. 2023).

It is worth mentioning that the suppression of the inflammatory process may be due to zinc inhibiting the activity of serine kinase IKKβ (inhibitory kappa B kinase beta) in the cytosol (Liu et al. 2013; Gammoh and Rink 2017; Notov et al. 2022).

The Pilim-1-caused reduction of impaired bone mineralization in EPL can be due to the fact that zinc contained in Pilim-1, along with copper, manganese, and calcium, through accumulating in the bone system (about 22%), participates in the bone-forming activity of osteoblasts (Persicov and Brodsky 2002; Zakharova et al. 2012; Trisvetova 2021).

The capacity of combination Pilim-1 + M-D exert a more pronounced anti-inflammatory, antimicrobial (a decrease in microbial content), antioxidant and mineralizing effects in the gums and pulp, compared to that of Pilim-1 and M-D, can be due with the synergetic effect of the components. But further in-depth studies are needed to confirm this assumption.

From the results of the studies, it can be concluded that Pilim-1 and, to a greater extent, the combination Pilim-1 + M-D have pronounced anti-inflammatory, antioxidant, antimicrobial (reduce microbial content) and mineralizing effects in D-EPL and P-EPL in rats, surpassing reference drug M-D in this respect.

Conclusion

The presented results of the study of Pilim-1 and its combination with M-D indicate their high therapeutic efficiency, especially when using the latter, in experimental EPL in rats, which makes it possible to view Pilim-1 and Pilim-1 + M-D as a potential therapeutic agent for treating EPL.

Conflict of interests

Authors declare no conflict of interests.

Data availability

All of the data that support the findings of this study are available in the main text.

References

·      Atabay VE, Lutfioğlu M, Avci B, Sakallioglu EE, Aydoğdu A (2017) Obesity and oxidative stress in patients with different periodontal status: a case-control study. Journal of Periodontal Research 52(1): 51–60. https://doi.org/10.1111/jre.12368  [PubMed]

·      Bakhit M, Hoffmann T, Scott AM, Beller E, Rathbone J, Del Mar C (2018) Resistance decay in individuals after antibiotic exposure in primary care: a systematic review and meta-analysis. BMC Medicine 16(1): 126. https://doi.org/10.1186/s12916-018-1109-4 [PubMed] [PMC]

·      Chen M, Cai W, Zhao S, Shi L, Chen Y, Li X, Sun X, Mao Y, He B, Hou Y, Zhou Y, Zhou Q, Ma J, Huang S (2019) Oxidative stress-related biomarkers in saliva and gingival crevicular fluid associated with chronic periodontitis: A systematic review and meta-analysis. Journal of Clinical Periodontology 46(6): 608–622. https://doi.org/10.1111/jcpe.13112 [PubMed]

·      Fadhil A, Qhanim H (2020) Antimicrobial studying of (Imidazole) derivative from pyrimidine. International Journal of Pharmaceutical Research 12: 913–917. https://doi.org/10.31838/ijpr/2020.12.04.128

·      Galenko-Yaroshevsky PA, Shelemekh OV, Popkov VL, Zadorozhniy AV, Nektarevskaya IB, Bunyatyan ND, Murashko RA, Lebedeva SA, Zelenskaya AV, Uvarov AV, Gulevskaya ON, Alukhanyan LO, Glechyan TR, Sergeeva AV, Kornetskaya AV, Korovaykin NE (2024) Study of the anti-inflammatory, analgesic, ulcerogenic and anti-ulcerogenic activity of N-isopropenylimidazole zinc complex derivative. Research Results in Pharmacology 10(1): 23–43. https://doi.org/10.18413/rrpharmacology.10.443

·       Galenko-Yaroshevsky PA, Slavinskiy AА, Todorov SS, Popkov VL, Shelemekh OV, Lebedeva SА, Zadorozhniy AV, Zelenskaya AV, Alukhanyan LO, Nektarevskaya IB, Bunyatyan ND, Verevkin AА (2023) The effect of zinc complex of N-isopropenylimidazole on the morphological characteristics of gum tissues in experimental endodontic-periodontal lesions in rats. Research Results in Pharmacology 9(4): 1–12. https://doi.org/10.18413/rrpharmacology.9.10040

·      Gammoh NZ, Rink L (2017) Zinc in infection and inflammation. Nutrients 9(6): 624. https://doi.org/10.3390/nu9060624 [PubMed] [PMC]

·      Gas SA, Boda FA, Roxana PR (2020) Imidazole derivatives and their antibacterial activity – a mini-review. Mini-Reviews in Medicinal Chemistry 20(11): 1380–1392. https://doi.org/10.2174/1389557520999201209213648 [PubMed]

·       Gavrikova LM, Segen IT (1996) Urease activity of oral fluid in patients with acute and odontogenic infection of the maxillofacial area. Dentistry [Stomatologiya] 75 (5): 49–50. [in Russian]

·       Grebenchikov OA, Zabelinf TS, Filippovskaya ZhS, Gerasimenko, OH, Plotnikov EYu, Lykhvanstev BB (2016) Molecular mechanisms of oxidative stress. Bulletin of Intensive Therapy named after A.I. Saltanova [Vestnik intensivnoi terapii imeni AI Saltanova] 3: 13–21. https://doi.org:10.1016/j.phytochem 2014.09.002 [in Russian]

·      Gurevich K, Urakov AL, Basantsev AV, Samorodov AV, Danilin AA, Purygin PP, Klenova NA, Bashirov II, Bashirova LI (2021) Synthesis of new N-mono- and N,N-dialkylated imidazole derivatives and their antiplatelet and anticoagulation activity. Pharmaceutical Chemistry Journal 55(2): 119–122. https://doi.org/10.1007/s11094-021-02395-z

·      Ho SY, Kwan YP, Qiu B, Tan A, Murray HL, Barathi VA, Tan NS, Cheung CMG, Wong TY, Wahli W, Wang X (2020) Investigating the role of PPARβ/δ in retinal vascular remodeling using Pparβ/ δ-deficient Mice. International Journal of Molecular Sciences 21(12): 4403. https://doi.org/10.3390/ijms21124403  [PubMed] [PMC]

·       Kaldybaeva AB, Praliev KD, Sergazy A, Malmakova AE,Yu VK (2023) The potential of practicality of imidazol-containing derivatives (review). Chemical Journal of Kazakhstan [Khimicheskii Zhurnal Kazakhstana] 2 (82): 58–78. https://doi.org/10.51580/2023-2.2710-1185.14 [in Russian]

·       Kamchatnov PR, Chugunov AB, Chipova DT, Kazakov AYu (2023) Non-steroidal anti-inflammatory drugs and risk of heart failure. Russian Medical Journal [Russkii Meditsinskii Zhurnal] 2: 88–95. [in Russian]

·       Karateev AE, Nasonov EL, Ivashkin VT, Martynov AI, Yakhno NN, Arutyunov GP, Alekseeva LI, Abuzarova GR, Evseev MA, Kukushkin ML, Kopenkin SS, Lila AM, Lapina TL, Novikova DS., Popkova TV, Rebrov AP, Skorobogatykh KV, Chichasova NV (2018) Rational use of non-steroidal anti-inflammatory drugs. Clinical recommendations. Scientific and Practical Rheumatology [Nauchno-prakticheskaya Travmatologiya] 56: 1–29. https://doi.org/10.14412/rjtao20180 [in Russian]

·      Keles GC, Acikgoz G, Ayas B, Sakallioglu E, Firatli E (2005) Determination of systemically and locally induced periodontal defects in rats. The Indian Journal of Medical Research 121(3): 176–184. [PubMed]

·      Khoroshun MS, Lazareva AA (2022). The purpose of non-steroidal anti -inflammatory drugs: benefits and risks. University Therapy Bulletin [Universitetskii Terapevticheskii Vestnik] 4(1): 4–10.

·       Kuzmina AV, Asetskaya IL, Polivanov VA, Zyryanov SK (2019) Medical errors when using beta-lactam antibiotics: analysis of the Russian base of unsolicited reports. Antibiotics and Chemotherapy (Antibiotiki i Khimioterapiya] 11-12(64): 48–53. https://doi/org/10.37489/0235-2990-2019-64-11-12-48-53  [in Russian]

·      Lebedeva SA, Galenko-Yaroshevsky PA (Jr.), Mel’nik SI, Kozin SV, Demura TA, Arshinov YaR, Gulevskaya ON, Galenko-Yaroshevsky PA (2021) Evaluation of the wound healing action of an N-isopropenylimidazole zinc metal complex derivative on a linear skin wound in rats. Pharmacy [Pharmatsiya] 70 (6): 49–55. https://doi.org/10.29296/25419218-2021-06-09 [in Russian]

·       Lebedeva SA, Galenko-Yaroshevsky PA (Jr.), Rychka VO, Zharov JuV, Zavorina DS, Kozin SV (2022) Molecular aspects of the wound healing action of zinc as an essential trace element. Microelements in Medicine [Mikroelementy v Meditsine] 23 (1): 14–23. https://doi.org/10.19112/2413-6174-2022-23-1-14-23  [in Russian]

·      Lebedeva SA, Galenko-Yaroshevsky PA (Jr..), Mel’nik SI, Kozin SV, Demura TA, Arshinov YaR, Svistakova MV, Grigorevskikh EM, Galenko-Yaroshevsky PA (2022a) Wound healing action of a zinc metal-organic complex on a planar skin wound in rats. Scientific Results of Biomedical Studies [Nauchnye Resul’taty Biomeditsinskikh Issledovanii] 8 (1): 71–81. https://doi.org/10.18413/2658-6533-2022-8-1-0-5 [in Russian]

·       Lebedeva SA, Galenko-Yaroshevsky PA, Fateeva TV, Pashin SS, Pashina NR, Nektarevskaya IB, Zadorozhniy AV, Shelemekh OV, Ravaeva MYu, Chuyan EN, Alukhanyan LO, Glechyan TR, Mutig K, Materenchuk MYu (2023) Effective wound healing agents based on N-alkenylimidazole zinc complexes derivatives: future prospects and opportunities. Research Results in Pharmacology 9(3): 27–39. https://doi.org/10.18413/rrpharmacology.9.10047

·       Lebedeva SA, Galenko-Yaroshevsky PA (Jr.), Samsonov MYu, Erlich AB, Margaryan AG, Materenchuk MYu, Arshinov IaR, Zharov YuV, Zelenskaya AV, Shelemekh OV, Lomsadze IG, Demura TA (2023а) Molecular mechanisms of wound healing: the role of zinc as an essential microelement. Research Results in Pharmacology 9(1): 25–39. https://doi.org/10.18413/rrpharmacology.9.10003

·       Levitsky AP, Den’ga OV, Makarenko OA, Demyanenko NA, Rossakhanova LN, Knava OE (2010) Biochemical Markers of Inflammation of Oral Cavity Tissues (Guidelines). Odessa City Printing House, Odessa, 16 p. [in Russian]

·      Levitsky AP, Makarenko OA, Demyanenko SA (2018) Methods of Experimental Dentistry. Tarpan Publishing House LLC, Simferopol, 78 p. [in Russian]

·      Liu MJ, Bao S, Gálvez-Peralta M, Pyle CJ, Rudawsky AC, Pavlovicz RE, Killilea DW, Li C, Nebert DW, Wewers MD, Knoell DL (2013) ZIP8 regulates host defense through zinc-mediated inhibition of NF-kappaB. Cell Reports 3(2): 386–400. https://doi.org/10.1016/j.celrep.2013.01.009 [PubMed] [PMC]

·      Liu T, Zhang L, Joo D, Sun SC (2017) Nf-kB signaling in inflammation. Signal Transduction Targeted Therapy 2: 17023. https://doi.org/10.1038/sigtrans.2017.23 [PubMed] [PMC]

·       Men’shchikova EB, Zenkov NK (2016) Modern approaches in the analysis of oxidative stress, or how to measure the immeasurable. Acta Biomedica Scientifica (East Siberian Biomedical Journal) 1(3(2)): 174–180. https://doi.org/10.12737/article_590823A565AA50.41723117 [in Russian]

·      Minhas D, Nidhaan A, Husni ME (2023) Recommendations for the use of nonsteroidal anti-inflammatory drugs and cardiovascular disease risk: decades later, any new lessons learned? Rheumatic Disease Clinics of North America 49(1): 179–191. https://doi.org/10.1016/j.rdc.2022.08.006 [PubMed]

·      Mironov AN (2012) Guidelines for the Preclinical Studies of Drugs. Grif and K., Moscow, 944 p. [in Russian]

·      Nakano Y, Arima T, Tobita Y, Uchiyama M, Shimizu A, Takahashi H (2020) Combination of peroxisome proliferator-activated receptor (PPAR) alpha and gamma agonists prevents corneal inflammation and neovascularization in a rat alkali burn model. International Journal of Molecular Sciences 21(14): 5093. https://doi.org/10.3390/ijms21145093 [PubMed] [PMC]

·       Notova SV., Kazakova TV, Marshinskaya OV, Shoshina OV (2022) Metal-ligand forms of iron and zinc in the body. Kazan Medical Journal [Kazansky Meditsinskii Zhurnal] 103(2): 259–268. https://doi.org/https://doi.org/10.17816/kmj2022-259 [in Russian]

·      Orekhova LYu, Loboda ES, Kosova EV, Vashneva VYu, Petrov AA (2020) Modern antibiotic therapy in periodontology. Periodontology [Parodontologiya] 25(3): 217–223. https://doi.org/10.33925/1683-3759-2020-25-3-217-223 [in Russian]

·       Panasenko LM, Kartseva TV, Nefedova ZhV, Zadorina-Khutornaya EV (2018) The role of basic minerals in the nutrition of children. Russian Bulletin of Perinatology and Pediatrics [Rossiiskii Vestnik Perinatologii i Pediatrii] 63 (1): 122–127. https://doi.org/10.21508/1027-4065-2018-63-1-122-127 [in Russian]

·      Patel JA, Patel NB, Maisuriya PK, Tiwari MR, Purohit AC (2022) Structure activity design, synthesis and biological activity of newer imidazole-triazine clubbed derivative as antimicrobial and antitubercular agents. Letters in Organic Chemistry 19(2): 126–134. https://doi.org/10.2174/1570178618666210521150011

·      Persicov АV, Brodsky B (2002) Unstable molecules form stable tissues. Proceedings of the National Academy of Sciences 99(3): 1101–1103. https://doi.org/10.1073/pnas.042707899 [PubMed] [PMC]

·      Pham CT, Nguyen V, Choi Y, Kim D, Jung O, Lee DJ, Kim HJ, Lee MW, Yoon J, Kim HM, Lee S (2021) Hypochlorite-activated fluorescence emission and antibacterial activities of imidazole derivatives for biological applications. Frontiers in Chemistry 9: 713078. https://doi.org/10.3389/fchem.2021.713078 [PubMed] [PMC]

·      Polson AM, Zander HA (1983) Effect of periodontal trauma upon intrabony pockets. Journal Periodontology 10(54): 586–591. https://doi.org/10.1902/jop.1983.54.10.586 [PubMed]

·       Popkov VL, Zadorozhniy MA, Kheighetyan AV, Galenko-Yaroshevsky PA (2022) The influence of the combination of Soderm®-Forte gel and metabolic energy corrector Сytoflavin on the course of the inflammatory process in the conditions of experimental periodontitis. Dentistry for all [Stomatologiya dla Vsekh] 2(99): 46–53. https://doi.org/10.35556/idr-2022-(99)46-53 [in Russian]

·      Rajam S, Dileepan B, Maruthamuthu B (2020) Spectral characterization and biological activity of newly synthesized imidazole derivative. World Journal of Pharmacy and Pharmaceutical Sciences 4(4): 887–897.

·      Shakhmardanova SA, Galenko-Yaroshevsky PA (2015) Metal Complex Derivatives of 1-alkenylimidazole. Antihypoxic Properties, Mechanisms of Action, Prospects for Clinical Use. Prosveshchenie-Yug, Krasnodar, 276 p. [in Russian]

·      Shakhmardanova SA, Galenko-Yaroshevsky PA (2016) Metal complex compounds of zinc with N-alkenylimidazoles: biological activity and application in medicine (review). Sechenov Bulletin [Sechenovskii Vestnik] 3(25): 84–90. [in Russian]

·       Stal’naya ID, Garishvilly TG (1977) Method for Determining Malonyldialdehyde Using Thiobarbituric Acid. Modern Methods in Biochemistry. Medicine, Moscow, 560 p. [in Russian]

·      Tobita Y, Arima T, Nakano Y, Uchiyama M, Shimizu A, Takahashi H (2020) Peroxisome proliferator-activated receptor beta/delta agonist suppresses inflammation and promotes neovascularization. International Journal of Molecular Sciences 21(15): 5296. https://doi.org/10.3390/ijms21155296 [PubMed] [PMC]

·       Trisvetova EL (2021) The role of zinc in human life. Medical News [Meditsinskie Novosti] 9 (324): 37–42. [in Russian]

·      Tsepov LM, Nikolaev AI, Petrova EV, Nesterova MM (2018) Pathogenetic rationale for the clinical use of drugs in combination therapy in inflammatory periodontal diseases (literature review). Periodontology [Parodontologiya] 23(2): 4–9. https://doi.org/10.25636/PMP.1.2018.2.1 [in Russian]

·       Ushakov RV, Tsarev VN (2019) Antimicrobial Therapy in Dentistry. Principles and Algorithms. Practical Medicine, Moscow, 240 p. [in Russian]

·      Valls A, Andreu JJ, Falomir E, Luis SV, Atrian-Blasco E, Mitchell SG (2020) Imidazole and imidazolium antibacterial drugs derived from amino acids. Pharmaceuticals 13: 482. https://doi.org/10.3390/ph13120482 [PubMed] [PMC]

·      Vidhya MR, Malar SJ (2020) Imidazole derivatives – potential compounds with antimicrobial and antioxidant efficacy. International Journal of Advanced Science and Technology 29(5): 183–189.

·      Visser L, Blout ER (1972) The use of p-nitrophenyl N-tertbutyloxycarbonyl-L-alaninate as substrate for elastase. Biochimica et Biophysica Acta 268(1): 257–260. https://doi.org/10.1016/0005-2744(72)90223-9

·      Voelkl J, Tuffaha R, Luong TT D, Zickler D, Masyout J, Feger M, Verheyen N, Blaschke F, Kuro-O M, Tomaschitz A, Pilz S, Pasch A, Eckardt KU, Scherberich JE, Lang F, Pieske B, Alesutan I (2018) Zinc inhibits phosphate-induced vascular calcification through TNFAIP3-mediated suppression of NF-κB. Journal of the American Society of Nephrology 29(6): 1636–1648. https://doi.org/10.1681/ASN.2017050492  [PubMed] [PMC]

·       Zakharova IN, Tvorogova TM, Vorobyeva AS, Kuznetsova OA (2012) Microelementosis as a factor in the formation of osteopathy in adolescents. Pediatrics [Pediatriya] 91 (1): 67–75. [in Russian]

Author contribution

Pavel A. Galenko-Yaroshevsky, Corresponding member of the Russian Academy of Sciences, Doctor Habil. of Medical Sciences, Professor, Head of the Department of Pharmacology, Kuban State Medical University, Krasnodar, Russia; e-mail: Galenko.Yaroсhevsky@gmail.com, ORCID ID: https://orcid.org/0000-0003-3190-1437. The author developed a concept, formed the idea and direction of research, developed and designed the methodology, and formulated the key goals and objectives.

Ivan I. Pavlyuchenko, Doctor Habil. of Medical Sciences, Professor, Head of the Department of Biology with the Course of Medical Genetics, Kuban State Medical University, Krasnodar, Russia; е-mail: inter-dekanat@mail.ru, ORCID ID: https://orcid.org/0000-0001-8019-9598. The author developed the key goals and objectives, conducted a critical review of the manuscript, analyzed it and provided valuable feedback.

Olga V. Shelemekh, graduate student of the Department of Dentistry No. 4, Rostov State Medical University, Rostov-on-Don, Russia; e-mail: lioli777@yandex.ru, ORCID ID: https://orcid.org/0000-0003-3488-9971. The author took part in conducting research, collection of primary data, their analysis, statistical processing and structuring the article.

Boris A. Trofimov, Doctor Habil. of Chemical Sciences, Professor, member of the Russian Academy of Sciences, Head of the Laboratory of Unsaturated Heteroatom Compounds, A.E. Favorsky Irkutsk Institute of Chemistry, Irkutsk, Russia; e-mail: boris_trofimov@irioch.irk.ru, ORCID ID: https://orcid.org/0000-0002-0430-3215. The author developed the key goals and objectives, conducted a critical review of the manuscript, analyzed it and provided valuable feedback.

Lidiya N. Parshina, Doctor Habil. of Chemical Sciences, senior specialist of the Laboratory of Unsaturated Heteroatom Compounds, A.E. Favorsky Irkutsk Institute of Chemistry, Irkutsk, Irkutsk, Russia; e-mail: parshina@irioch.irk.ru, ORCID ID: https://orcid.org/0000-0002-5516-6214. The author developed the key goals and objectives, conducted a critical review of the manuscript, analyzed it and provided valuable feedback.

Viktor L. Popkov, Doctor Habil. of Medical Sciences, Professor of the Department of Orthopedic Dentistry, Kuban State Medical University, Krasnodar, Russia; e-mail: vict.popkoff2015@yandex.ru, ORCID ID: https://orcid.org/0000-0001-7362-0073. The author developed the key goals and objectives, conducted a critical review of the manuscript, analyzed it and provided valuable feedback.

Svetlana A. Demyanenko, Doctor Habil. of Medical Sciences, Professor, Head of the Department of Dentistry and Orthodontics, Medical Institute named after S.I. Georgievsky (winner of the Red Banner of Labor Award), V.I. Vernadsky Crimean Federal University, Simferopol, Russia; e-mail: dc.kvalitet@gmail.com, ORCID ID: https://orcid.org/0000-0002-2743-498X. The author developed the key goals and objectives, conducted a critical review of the manuscript, analyzed it and provided valuable feedback.

Oleg V. Tsymbalov, Doctor Habil. of Medical Sciences, Professor, Professor of the Department of Surgical Dentistry and Maxillofacial Surgery, Kuban State Medical University, Krasnodar, Russia; e-mail: tsimbal_ov@mail.ru, ORCID ID: https://orcid.org/0000-0002-7970-323X. The author developed the key goals and objectives, conducted a critical review of the manuscript, analyzed it and provided valuable feedback.

Andrey V. Zadorozhniy, Candidate of Medical Sciences, Associate Professor, Head of the Department of Dentistry No. 4, Rostov State Medical University, Rostov-on-Don, Russia; e-mail: stomvr1@gmail.com, ORCID ID: https://orcid.org/0000-0001-9552-8542. The author edited and prepared the text of the manuscript, was engaged in developing research design, providing the resources for the research: consumables, animals, tools, computer equipment for analysis – and correction of the final draft of the manuscript.

Anait V. Zelenskaya, Candidate of Medical Sciences, Associate Professor of the Pharmacology Department, Kuban State Medical University, Krasnodar, Russia; e-mail: anait_06@mail.ru, ORCID ID: https://orcid.org/0000-0001-9512-2526. The author participated in research design, analyzed and interpreted the research results; prepared and edited the text of the manuscript.

Irina B. Nektarevskaya, Candidate of Medical Sciences, Associate Professor of the Department of Dentistry No. 4, Rostov State Medical University, Rostov-on-Don, Russia; e-mail: nektir4546@mail.ru, ORCID ID: https://orcid.org/0009-0009-6733-9977. The author was engaged in conducting information retrieval research, animal handling and analysis of the obtained results.

Alina V. Sergeeva, teaching assistant, Department of Pharmacology, Kuban State Medical University, Krasnodar, Russia; e-mail: alina_sergeeva_v@mail.ru, ORCID ID: https://orcid.org/0000-0003-4335-2156. The author was engaged in developing research design, conducting research; analysis and interpretation of the obtained data.

Nikolay E. Korovaykin, 3^rd-year student, Department of General Medicine, Kuban State Medical University, Krasnodar, Russia; e-mail: nikkoro@bk.ru, ORCID ID: https://orcid.org/0009-0004-9543-7906. The author was engaged in developing research design, conducting research; analysis and interpretation of the obtained data.

Svetlana A. Lebedeva, Doctor Habil. of Biological Sciences, Associate Professor, Professor of the Department of Pharmacology, A.P. Nelyubin Institute of Pharmacy, Sechenov First Moscow State Medical University, Moscow, Russia; Department of Medical Elementology, RUDN University, Moscow, Russia; e-mail: Lebedeva502@yandex.ru, ORCID ID: https://orcid.org/0000-0001-8769-1040. The author developed the key goals and objectives, conducted a critical review of the manuscript and provided valuable feedback.