Peculiairities of mRNA TLR-2 , TLR4 expression of the oral cavity epithelium in children under conditions of chronic catarrhal gingivitis and diabetes mellitus

TLRs in epithelium of the oral cavity can be effected as due to the dental as general somatic pathology. TLR availability in the external membrane of neutrophils, macrophages, keratocytes was found to be a starting point in triggering inflammation in the periodontal tissues ensuring molecular reception of a pathogen with further involvement of important components in the inherited immunity. These effectors possess phagocytic and killer activities, ensure a number of signals activating and directing antigen-specific response by the cells of the adaptive immune system.

Особенности экспрессии мРНК TLR-2, TLR-4 эпителия ротовой полости детей, больных хроническим катаральным гингивитом, на фоне сахарного диабета The system of inherited immunity is formed in the process of evolution of the vertebrate animals and realized by means of cells-effectors participating in the first line of protection against all antigenic foreign compounds.The following types of cells are contained in it: epithelial cells, macrophages, dendritic cells, granulocytes, mast cells, NK-cells etc. Pattern recognition receptors (PRRs) play an important role among the factors of inherited immunity [1,4,7].Among PRRs toll-like receptors (TLRs) occupy a special position.These effectors possess phagocytic and killer activities, ensure a number of signals activating and directing antigen-specific response by the cells of the adaptive immune system.They serve as a bridge between pathogen-associated molecular structures (PAMSs) and antigen-specific cells of the adaptive immune response, and transmit the signals of specific hereditarily coded receptors in soluble mediators connected with T-and B-cells through the specific cytokine-chemokine receptors.After reaction with microbial PAMSs the majority of TLRs induce activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and cytokine production, mainly by MyD88-dependent way [2,5,6], stimulating more stages of inflammation and ensuring activation of different types of cells participating in maintenance and regulation of inflammation.Epithelium of the oral cavity and TLR containing in them are under the effect of changes both in dental and general somatic pathology [3,6].TLR availability in the external membrane of neutrophils, macrophages, keratocytes was found to be a starting point in triggering inflammation in the periodontal tissues ensuring molecular reception of a pathogen with further involvement of important components in the inherited immunity [3,4,8].In general, TLR is one of the strongest cellular modulators.
Nowadays the study of mRNA мРНК TLR-2, TLR-4 expression of the oral epithelium in children afflicted with diabetes mellitus under conditions of chronic inflammation in the periodontal tissue becomes especially topical.Awareness of their role in pathogenesis of many diseases enables not only to prognosticate the risk of development of pathology or severity of its course, but to choose specific therapy for every category of patients.
Objective of the investigation is to study mRNA TLR-2, TLR-4 in the epithelium of the oral cavity in children under conditions of chronic catarrhal gingivitis against diabetes mellitus considering metabolic disorders available.

Materials and methods
To solve the purpose we examined 12-year old children afflicted by type I diabetes mellitus (30 individuals) who were treated in-patiently at Endocrinology Department of the Regional Municipal Establishment "Regional Children Clinical Hospital", Chernivtsi (I group).II group (30 somatically healthy children with signs of chronic catarrhal gingivitis)and the group of comparison (30 somatically and dentally healthy children of the same age) were formed from the schoolchildren of Comprehensive Secondary School № 22 in the town of Chernivtsi.
According to the degree of severity of gingivitis among the children of I and II groups there was mild degree of severity of chronic catarrhal gingivitis found (10 and 18 children respectively), moderate degree of severity (17 and 8 children) and severe degree (3 and 4 children).
To analyze gene expression the method of polymerase chain reaction was applied with a reverse transcription in the regime of real time (RT-RRT).The object for molecular-genetic examinations by means of RT-RRT method was the buccal epithelium.
Isolation of total RNA was conducted using the set "Trizol RNA Prep 100" (Isogen Lab., LTD, RF).Preparation for the reactions and the reactions themselves were carried out according to the Protocol.
Statistical analysis was performed using the biostatistics methods of the licensed program Statistica 6.0 (StatSoft, USA).The mean value (M), mean error (m), reliability of statistical indices (p) have been taken for consideration.The data have been presented in the form of (M ± m) and were considered reliable by the level of statistical significance of p < 0.05.

Results of investigation and discussion
RT-RRT-analysis of the buccal epithelium showed an increased relative level of expression of mRNA TLR-2, TLR-4 in children afflicted by diabetes mellitus and somatically healthy children under conditions of chronic catarrhal gingivitis (CCG) (Table 1).
In children afflicted by diabetes mellitus the content of mRNA TLR-2 (90.0755) is in 5.5 times higher as compared to somatically healthy children under conditions of CCG -15.1505.Expression of mRNA TLR-4 in children of I group increased 6 times as compared to the children from II group.Such results are evidenced by certain literary data and are indicative of an infectious genesis of inflammatory process in the periodontal tissues.
Expression of mRNA TLR-2 in a considerable number of children of both experimental groups was high with a tendency to increase depending on the degree of severity of CCG in children afflicted by diabetes mellitus (Fig. 1).Under conditions of mild degree of CCG in children with comorbid pathology the index was 44.1761, in somatically healthy children -14.3251.Children with moderate degree of CCG severity were characterized by the following data: 112.9692 -in І group and 18.7071 -in ІІ group.The highest data were found in children against the ground of comorbid somatic pathology under conditions of severe degree of CCG (113.3434).
The level of mRNA expression of TLR-4 (Fig. 2) in children with the signs of mild degree of CCG against the ground of somatic pathology was 26.0951, and among somatically healthy children it was a little lower -9.8618.Under conditions of moderate and severe degrees of CCG expression of mRNA TLR-4 increased in children with comorbid pathology as compared to somatically healthy children, 99.7132 and 103.5418 and 19.2791 and 19.8159 respectively.

Conclusions
1.The conducted molecular-genetic study of a relative level of mRNA TLR-2 and TLR-4 in the epithelium of the oral cavity was indicative of the fact that in children against diabetes mellitus relative levels of mRNA TLR-2, TLR-4 are considerably higher.
2. The investigation conducted is of a great importance to improve the methods of diagnostics of inflammatory processes in the periodontal tissues by means of detection of the local immunity condition in the oral cavity and elaboration of therapeutic-preventive programs for children suffering from type I diabetes mellitus.Оригинальные исследования

Fig. 1 .Fig. 2 .
Fig. 1.Relative level of expression mRNA TLR-2 of the buccal epithelium in children afflicted by DM and somatically healthy children under conditions of CCG.

Table 1 .
Relative level expression of mRNA of TLR-2, TLR-4 genes of the buccal epithelium in children afflicted by DM and somatically healthy children under conditions of CCG in comparison with dentally and somatically healthy children (M ± m) : the probability of difference compared with Group 1 & Group 2; p 2 : the probability of difference compared with Group 1 & Group 3; p 3 : the probability of difference compared with Group 2 & Group 3.