Levels of Hsp60 in periodontal tissue of rats: influence of injections of hyaluronic acid

. Levels of Hsp60 in periodontal tissue of rats: influence of injections of hyaluronic acid. Heat shock protein 60 (Hsp60) is considered as one of the possible autoantigens involved in the pathogenesis of a number of chronic diseases including periodontal diseases. The application of hyaluronic acid or hyaluronan (HA) in the treatment of periodontitis has been evaluated in several clinical trials, however, the effect of hyaluronic acid on heat shock protein 60 level in periodontal soft tissues has not been studied. The aim of this work was to evaluate the effect of HA injections on levels of Hsp60 in periodontal tissue of the rats. Samples of periodontal tissue of mandibular incisors of adult male Wistar rats at 10-12 months of age were investigated. Rats were distributed into the control group and the periodontitis group. Visual manifestations of hyperemia of the gums around the incisors were the criterion for selecting animals into the periodontitis group. There were two subgroups in the control group: intact rats (I); intact rats after HA “hyaDENT BG” 1.0 MDa (BioScience GmbH, Germany) treatment (I+“G-1.0”). There were four subgroups in the periodontitis group: rats with periodontitis (P); rats with periodontitis after HA “hyaDENT BG” 1.0 MDa (BioScience GmbH, Germany) treatment (P+“G-1.0”); rats with periodontitis after HA “SERTOBEC” 2.4 MDa (S.C. Rompharm Company S.R.L., Romania) treatment (P+“S-2.4”); rats with periodontitis after HA “SERTOBEC Tendon” 2.4 М D а (S.C. Rompharm Company S.R.L., Romania) treatment (P+“ST-2.4”). in total lysates of periodontal tissue were tested by Western blotting method before and after the treatment with HA (one month after the last injection). There was no significant difference between levels of Hsp60 in total lysates of periodontal tissue of intact rats and rats with periodontitis before treatment (p>0.05). Rats with periodontitis showed decreased inflammation in the periodontal tissue after treatment with HA with different molecular weight. Intact rats and rats with periodontitis which were treated with HA “hyaDENT BG” 1.0 MDa showed reduced levels of Hsp60 in total lysates of periodontal tissue comparatively with levels of Hsp60 before treatment (by 15.4 and 10.7 times respectively, p<0.001). Rats with periodontitis which were treated with HA “SERTOBEC” 2.4 MDa or HA “SERTOBEC Tendon” 2.4 MDa also showed reduced levels of Hsp60 in total lysates of periodontal tissue comparatively with levels of Hsp60 before treatment (by 21.3 and 16.4 times respectively, p<0.001). Rats with periodontitis showed the decrease in inflammation in periodontal tissue after treatment with HA with different molecular weight. Injections of HA has contributed to reduce levels of Hsp60 in periodontal tissue of intact rats and rats with periodontitis.

in total lysates of periodontal tissue were tested by Western blotting method before and after the treatment with HA (one month after the last injection). There was no significant difference between levels of Hsp60 in total lysates of periodontal tissue of intact rats and rats with periodontitis before treatment (p>0.05). Rats with periodontitis showed decreased inflammation in the periodontal tissue after treatment with HA with different molecular weight. Intact rats and rats with periodontitis which were treated with HA "hyaDENT BG" 1.0 MDa showed reduced levels of Hsp60 in total lysates of periodontal tissue comparatively with levels of Hsp60 before treatment (by 15.4 and 10.7 times respectively, p<0.001). Rats with periodontitis which were treated with HA "SERTOBEC" 2.4 MDa or HA "SERTOBEC Tendon" 2. 4 MDa also showed reduced levels of Hsp60 in total lysates of periodontal tissue comparatively with levels of Hsp60 before treatment (by 21 Heat shock protein 60 (Hsp60) is considered as one of the possible autoantigens involved in the pathogenesis of a number of chronic diseases including periodontal diseases [9]. Hsp60 belongs to a family of highly concerved stress proteins. The production of these proteins is evaluated under different stressors factors (cellular insult, environmental changes, temperature, infections, tumors etc.). Despite being highly homologous between eukaryotic and prokaryotic, Hsp60 is strongly immunogenic and immune responses to bacterial Hsp60 are speculated to initiate chronic inflammatory diseases in humans.
Periodontitis is defined as the chronic inflammation of periodontal supporting tissue caused by specific pathogenic microorganisms, resulting in the advanced destruction of the periodontal ligament and the alveolar bone and subsequently leading to the formation of a gingival recession and a periodontal pocket [19]. The precise mechanisms of tissue destruction, however, have not been fully elucidated.
Human Hsp60 is expressed abundantly in periodontitis lesions and can be an antigen-target for cross-reactive antibodies against bacterial Hsp60 [3,8,9]. Similar to bacterial lipopolysaccharide, Hsp60 is able to stimulate tumour necrosis factor (TNF)-α production from macrophages [3]. Thus, immune response to Hsp60 derived from either inflammatory tissue or bacteria was thought to play an important role in the development of autoimmune processes in periodontal tissue.
На умовах ліцензії CC BY 4.0 HA is an ubiquitous non-sulphated glycosaminoglycan with a high molecular weight. It is one of the components of the extracellular matrices of the connective tissue. HA has been identified in all periodontal tissues, being particularly prominent in the non-mineralized tissues such as gingiva and periodontal ligament and in only low quantities in mineralized tissues such as cementum and alveolar bone [14]. Fibroblasts, osteoblasts, and cementoblasts are capable of synthesizing HA in their cell membrane and secreting it to the exterior [13]. HA has anti-inflammatory, anti-edematous effect, promotes wound healing and also connective tissue angiogenesis [14]. There are no reports describing the effect of HA on levels of Hsp60 in periodontal tissue.
The aim of this work was to evaluate the effect of HA injections on levels of Hsp60 in the periodontal tissue of rats.

MATERIALS AND METHODS OF RESEARCH
Samples of periodontal tissue of mandibular incisors of adult male Wistar rats were investigated. Rats, 10-12 months of age, weighing 200-220 g were from the vivarium of the Bogomolets National Medical University. Animals were caged in a room with controlled temperature (18° C) and humidity (60.5%). The protocol of this study was approved by the Ethics Committee of the Bogomolets National Medical University. In experimental studies on animals adhered to the "European Convention for the protection of vertebrate animals used for experimental and other scientific purposes" (Strasbourg, 1986), "General ethical principles of animal experiments", approved by the First National Con Rats were distributed into the control group and the periodontitis group. Visual manifestations of hyperemia of the gums around the incisors were the criterion for selecting animals into the periodontitis group. There were two subgroups in the control group: intact rats (I); intact rats after HA "hyaDENT BG" 1.0 MDa (BioScience GmbH, Germany) treatment (I+"G-1.0"). There were four subgroups in the periodontitis group: rats with periodontitis (P); rats with periodontitis after HA "hyaDENT BG"1.0 MDa (BioScience GmbH, Germany) treatment (P+"G-1.0"); rats with periodontitis after HA "SERTOBEC" 2.4 MDa (S.C. Rompharm Company S.R.L., Romania), treatment (P+"S-2.4"); rats with periodontitis after HA "SERTOBEC Tendon" 2.4 МDа (S.C. Rompharm Company S.R.L., Romania) treatment (P+"ST-2.4"). There were three animals in each subgroup. Rats were injected 0.05 ml HA in the area of alveolar ridge of central incisors once a week, three times. Levels of Hsp60 in total lysates of periodontal tissue were tested by Western blotting before and after the treatment with HA of different molecular weight (1 month after the last injection).
Samples of periodontal tissue were prepared by homogenization in ice-cold RIPA buffer (20mM Tris-HCl, pH 7.5; 0.15 M NaCl; 1mM EDTA; 1% sodium deoxycholate; 0.1% SDS and 0.1% protease inhibitor cocktail). After 30 a minutes' extraction at +4°C, the samples were centrifuged at 10 000 x g for 20 min. at +4°C. The supernatants were collected and the protein concentration was determined by Nanodrop. Protein samples (100 μg) were electrophoresed in 12% SDS polyacrylamide gel. The wet electrotransfer of proteins to the nitrocellulose membranes was held at 90 V for 40 min. The membranes were blocked for 1 h. at a room temperature with blocking buffer (5% non-fat milk in PBS-tween buffer) and then probed with specific primary antibodies for 18 h. at +4°C. After washing in PBS tween buffer (10 min x 3) the membranes were incubated with peroxidase-conjugated secondary anti-rabbit (in case of Hsp60) or anti-mouse (in case of GAPDH) antibodies for 1 h. at a room temperature. The polyclonal anti-GroEL antibody produced in rabbit and polyclonal anti-GAPDH antibody produced in mouse were developed in IMBG NAS of Ukraine. Immunologic crossreactivity of GroEL with Hsp60 was demonstrated previously [17]. The treatment of the membranes with secondary antibodies was followed by washing in PBS tween buffer (10 min x 3) and chemiluminescence detection according to manufacturer's instructions by ChemiDoc. All samples of periodontal tissue were examined for levels of Hsp60 and GAPDH in six reproducible repetitions. The results of the first one are presented in the article.
Statistical analysis was performed using software environments STATISTICA 8.0 (StatSoft Inc. 2007, USA) and MS Excel 2010 (license number K9366093I 2016). Data were analyzed using Student's t-test, a p<0.001 was considered significant.

RESULTS AND DISCUSSION
Rats with periodontitis showed decreased inflammation in the periodontal tissue after treatment with HA of different molecular weight. There were a decrease in gum hyperemia, an increase in soft tissue volume and an increase in vascularization in parodont of rats. 21/ Том XXVI / 2 Results of the investigation of the effect of HA injections on levels of Hsp60 in the total lysates of periodontal tissue are presented in the Figure. There was no significant difference between levels of Hsp60 in total lysates of periodontal tissue of intact rats and rats with periodontitis before treatment (p>0.05).
Intact rats and rats with periodontitis which were treated with HA "hyaDENT BG" 1.0 MDa showed reduced levels of Hsp60 in total lysates of perio-dontal tissue comparatively with levels of Hsp60 before treatment (by 15.4 and 10.7 times respectively, р<0.001). Rats with periodontitis which were treated with HA "SERTOBEC" 2.4 MDa or HA "SERTOBEC Tendon" 2.4 МDа also showed reduced levels of Hsp60 in total lysates of periodontal tissue comparatively with levels of Hsp60 before treatment (by 21.3 and 16.4 times respectively, p<0.001).

Influence of injections of hyaluronic acid of different molecular weight on levels of Hsp60 in periodontal tissue of rats
На умовах ліцензії CC BY 4.0 The integrity of periodontal tissues provides an effective barrier against microbial invasion and preventes the destruction of tissues by bacterial toxins and enzymes. This integrity is lost during chronic inflammation associated with periodontal diseases [11].
It was shown, the endogenous HA component results to be lacking within the epithelium and the gingival connective tissue with a consequent structural failure and loss of normal features of the gingiva. In patients with chronic periodontitis, there is a rapid loss of HA high molecular weight due to enzymatic digestive processes. Hyaluronidase, an enzyme released by microorganisms of bacterial plaque, plays an essential role in such mechanism [10].
Because of its properties and the non-immunogenicity of the highly purified form, HA has already found medical applications for many years. The application of HA in nonsurgical periodontal treatment has been evaluated in several clinical trials, but the current evidence is insufficient to support the routine clinical use [2,5,6,12,15,16].
The aim of this pilot study was to investigate the influence of injection of HA on levels of Hsp60 in periodontal tissue. Significant difference between levels of Hsp60 in total lysates of the periodontal tissue of intact rats and rats with parodontitis before treatment was not demonstrated. The treatment with HA of different molecular weight has contributed to reduce levels of Hsp60 in total lisates of periodontal tissue of intact rats and rats with periodontitis.
Our clinical observations revealed a decrease in inflammation processes in parodontal tissue of rats with parodontitis after treatment with HA of different molecular weight.
Different mechanisms have been proposed to explain the influence of HA on the inflammatory process.
HA may regulate the inflammatory response, acting as an antioxidant by scavenging reactive oxygen species [6].
It was considered that HA produces a physical barrier against bacteria and their products in extracellular matrix. At physiological concentrations, HA molecules entangle and form a random network of chains. Such network interacts with other macromolecular components. HA excludes other macromolecules especially large ones that cannot find space in the network. It also retards the diffusion of other substances that can not penetrate the network [10].
HA has been shown to be bacteriostatic with respect to periodontal pathogens particularly to Actinobacillus actinomycetemcomitans, Prevotella oris, Staphylococcus aureus, and Propionibacterium acnes strains. Among the bacterial strains studied, HA was found to have no bactericidal effects [4].
HA may also prevent periodontal pathogen colonization by directly preventing microbial proliferation [6].
We were determined levels of Hsp60 in total lysates of periodontal tissue. Many different bacteria are present on the tissue surfaces and form the film. Protein that we determined in total lysates of periodontal tissue may be also bacterial Hsp60. Injections of HA of different molecular weight contributed to reduce the inflammation and possibly contributed to decline of the periodontal pathogens quantity. Thus, we observed decreased levels of Hsp60 in total lysates of periodontal tissue of rats.

CONCLUSION.
Rats with periodontitis showed decreased inflammation in periodontal tissue after treatment with HA of different molecular weight. Injections of HA contributed to reduce levels of Hsp60 in periodontal tissue of intact rats and rats with periodontitis.
Conflict of interests. The authors declare no conflict of interest.