Introduction. One of the directions of modern dentistry is the search for new effective drugs for treating inflammatory periodontal diseases. The leading role of periodontopatogenic bacteria in the provoking of the any forms of periodontitis and the lack of efficacy of currently using antibiotics make this problem especially important [1, 2, 3].

That’s why synthetic analogues of the natural peptide indolicidin, which have a wide range of antimicrobial effects, are of great interest to researchers [4, 5, 6]. Recently, we have shown the protective action of indolicidin-like peptides on morphological and biochemical changes in the periodontium in periodontal disease [7]. It’s known that the inflammatory processes, including periodontitis, are accompanied with the disorders of vasoactive substances secretion [8].

The aim of the study is the evaluation of the pharmacological effect of indolicidin-like peptides on the content of endothelin-1 and nitric oxide decay products in the animals with experimental periodontitis.

Materials and methods. Eighty Vistar male rats were obtained from the branch “Stolbovaya” of the Federal State Budgetary Institution of Science “Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency”. The naïve group consists of 8 animals. Experimental periodontitis was performed in accordance with the method proposed by A.I. Volozhin and S.I. Vinogradova [9]. The animals received the chloral hydrate intraperitoneally at a dose of 0.3 g / kg b.w. and 2% novocaine solution locally. The silk thread was put in the form of eight on the lower incisors after the dissection of interdental papilla. Then the silk ligature was immersed in the periodontal groove and it was fixed with additional nodes for two weeks. The animals were sacrificed at the first, second and third weeks after by an overdose of anesthesia.

The concentration of endothelin-1 in the gum tissue homogenate was measured by immune-enzyme assay using a set produced by Biomedica Medizinprodukte GmbH Co KG (Austria) and it was expressed in pmol/ml. Preliminary, the gum tissue (100 mg) was homogenized in the saline solution (pH=7.2) in the low temperature (+2-+3^oC) and then homogenate was centrifuged in 3000 rpm during 15 minutes.

Nitric oxide decay products content in the plasma was measured by a 546-nm spectrophotometer [10].

The rats with experimental periodontitis were divided into the following groups: control group (periodontitis and saline solution injections) – 24 rats, the first experimental group (periodontitis and indolicidin N7 injections) – 24 rats, the second experimental group (periodontitis and indolicidin N8 injections) – 24 rats.

The action of indolicidin-like peptides N7 (H – Ile – Leu – Pro – Trp – Lys – Lys – Pro – Trp – Lys – Pro – Trp – Arg – Arg – NH₂) and N8 (H – Ile – Lys – Pro – Trp – Lys – Trp – Pro – Trp -Lys – Pro – Trp – Arg – Arg - NH₂) (LLC “NPF“ Verta””, St. Petersburg, Russia)” on the periodontitis development was investigated. The rats in the control group were intraperitoneally injected with 0.2 ml of normal saline 7 days. The rats in experimental groups were injected under the same schedule with 0.2 ml of the solution of N7 or N8 peptide in normal saline. The dose of indolicidin-like peptides was 500 μg/kg of body weight. The solution was prepared ex tempore. These doses were chosen on the basis of the literature data and preliminary experiments [7]. 

The study was carried out in accordance with the regulations for ensuring the human treatment of animals declared in the Helsinki Declaration of the World Medical Association on the Humane Attitude to Laboratory Animals (2000), European Community Directives (86/609 EC) and the Rules of Proper Laboratory Practice in the Russian Federation (Order No.199n of the Ministry of Public Health of the Russian Federation dated 01.04.2016). The approval of the Regional Ethics Committee (REC) (protocol No. 3 of 10.27.2015) was received.

Experimental results were statistically processed by methods of variation statistics. The significance of discrepancies was defined according to Student’s t-criterion after the checking of normal distribution of the parameters. MS Excel and Statistica 10 software were used for statistical processing.

Results and discussion. Mean values of the endothelin-1 concentration in the gum homogenate and nitric oxide decay products in the plasma in the naïve, control and treated with indolicidin-like peptides N7 and N8 groups are shown in Table 1. The studied indices of the control group rats increased in comparison with those in the naïve animals during the entire observation. The arising in the content of nitric oxide decay products indicates to the reinforcement of nitric oxide turnover and a decrease in its concentration [8].

The long-term treatment with indolicidin-like peptides N7 and N8 revealed a significant decrease in the endothelin-1 and nitric oxide decay products in rats with periodontitis at the 2^nd week of the experiment in comparison with those in the control group: nitric oxide stable metabolites concentration decreased by 12.5% and 11.4% (p<0.05) and endothelin-1 content – by 25.8% and 25.0% (p<0.001) after the injection of indolicidin analogues N7 and N8 respectively.

This dynamics continues at the 3^rd week: endothelin-1 and nitric oxide decay products turned out to be significantly less than in the control rats (p<0.05-0.01).

The results of our study confirm the data about disturbances in the endothelin and nitric oxide content in periodontitis [11, 12]. The pathogenesis of endothelium injury in the inflammation is complicated. The direct damaging effect of periodontal microbes on the vascular endothelium is mainly developed due to their adhesion and invasion into endothelial cells [13]. Endothelial damage leads to intravascular changes: platelet aggregation and the activation of coagulation [13]. It has been also established that ET-1 was involved in the regulation of proinflammatory interleukins, especially interleukin-1b expression in gingival tissues [11].

The microcirculation system provides mainly metabolic homeostasis in the organs and tissues. Recently it was declared that periodontitis is characterized by a significant decrease in capillary perfusion in the periodontium [8]. A critical decrease in microcirculation and vasomotor activity of blood vessels in patients with periodontitis is followed by disturbances in the microcirculation adaptation to local metabolic needs [8, 13].

The long-term administration of indolicidin-like peptides N7 and N8 was followed by a protective pharmacological effect on the content of vasoconstrictor and vasodilator. These data confirm that indolicidin-like peptides are able to correct microcirculation in the affected periodontium. In our opinion, the effect could be related to the action of the peptides on the vasoactive substances synthesis by the macrophages and platelets, including interleukins, arachidonic acid cascade metabolites, growth factors, etc. The stimulation of macrophage adhesion property indicating to their functional activation under the influence of synthetic indolicidins has been already established [14].

Comparative investigation of the pharmacological effect of indolicidin-like peptides on endothelin-1 and nitric oxide decay products content in periodontitis didn’t reveal the significant difference. Further research is required for better understanding molecular mechanisms of vasoprotecting effect of synthetic indolicidins.

Conclusion

1. The increase in endothelin-1 content in gum tissue and nitric oxide decay concentration in plasma has been established in periodontitis.
2. The long-term administration of indolicidin-like peptides N7 and N8 had protective effect on the vasoactive substances content: endothelin-1 and nitric oxide.
3. There was no significant difference between the pharmacological effects of indolicidin analogues N7 and N8 on the vasoactive substances content in periodontitis.

Financial support

Supported by The Federal State Autonomic Educational Institution of Higher Education “Belgorod State National Research University”.