Background
Periodontitis is a highly prevalent chronic inflammatory disease with social, psychological and physiological impacts. The disease is characterized by progressive and irreversible destruction of the tooth-supporting tissues, i.e. the periodontium, which comprises the gingiva, periodontal ligament (PDL), root cementum and alveolar bone [
1,
2]. Microorganisms of the subgingival biofilm, such as
Fusobacterium nucleatum, are essential for the initiation and progression of periodontitis [
3‐
5]. The periodontopathogenic microorganisms, their constituents and products evoke an inflammatory host response, which leads to increased levels of inflammatory mediators, such as interleukin (IL)-1β, in gingival tissues and crevicular fluid (GCF). The inflammatory processes cause the release of matrix-degrading proteases and osteoclast-activating factors, which can ultimately result in attachment and tooth loss [
3,
4,
6].
Periodontitis has been linked to a plethora of systemic diseases and conditions, such as type II diabetes, obesity and metabolic syndrome [
7‐
10]. Although the exact underlying mechanisms for the interactions between periodontitis and these systemic diseases are only partially known, it has been suggested that adipokines might be a critical pathomechanistic link [
11‐
13]. Obese patients are characterized by an increased amount of adipose tissue, which represents a complex endocrine and highly active metabolic organ. In the adipose tissue, adipocytes and other cells, such as leukocytes, secrete numerous bioactive molecules, which are collectively called adipokines [
14‐
16]. Besides their function in the metabolic regulation, adipokines also regulate inflammatory and wound healing processes. Leptin and visfatin are adipokines with proinflammatory characteristics. Since the leptin secretion is proportional to the size and number of adipocytes, the plasma leptin level is increased in obese patients and decreased after weight loss [
17]. As for leptin, the plasma level of visfatin, which is predominantly secreted by adipocytes and macrophages, is also enhanced in obese individuals [
18,
19]. These adipokines can also be measured in the gingival tissues and GCF. Interestingly, their gingival levels seem to be altered in the presence of periodontitis, suggesting a local production and role of these adipokines in periodontal diseases [
20‐
25].
The polypeptide hormone somatostatin (SST) antagonizes growth hormone (GH) and exerts antiproliferative, antiangiogenetic, proapoptotic, anti-nociceptive and other effects [
26,
27]. Although very little is known about the presence and actions of SST in periodontal tissues, a few studies have detected SST-producing dendritic cells in the gingival epithelium and the subepithelial connective tissue [
28‐
30]. SST can bind to five ubiquitously distributed G protein-coupled receptors (SSTR1–5), which mediate the aforementioned effects of SST [
26]. Activation of SSTR2, which is one of the 5 receptors, results in a reduced expression of vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and their receptors, which all have been shown to be critical in periodontal health and repair [
31‐
34]. Furthermore, SSTR2 could be a potential target for diagnostic strategies in periodontal bone infection and inflammation [
35]. Deciphering the regulation of SSTR2 might be important for a better understanding of the aetiopathogenesis of periodontal diseases. Therefore, the objective of the present study was to examine the regulation of SSTR2 in periodontal cells and tissues under inflammatory, microbial and obesity-related conditions.
Material und methods
Culture and treatment of cells
PDL fibroblasts of caries-free and periodontal healthy teeth extracted for orthodontic reasons were harvested from 4 patients (mean age: 14.5 years, min-max: 11–19 years; gender: 3 male/1 female). Approval of the Ethics Committee of the University of Bonn (#117/15) as well as written and informed consent of the patients or legal guardians were given. Cells were cultured as previously described. Briefly, PDL tissue explants from the middle third of the roots were grown in Dulbecco’s minimal essential medium (DMEM, Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 units penicillin and 100 μg/ml streptomycin (PenStrep Invitrogen) at 37 °C in a humidified atmosphere of 5% CO
2 [
36,
37]. For the experiments, PDL fibroblasts of passages between 3 and 5 were used at 80% confluence. One day prior to the experiments, the FBS concentration was reduced to 1%. Physiological concentrations of IL-1β (0.1–10 ng/ml; Calbiochem, San Diego, CA, USA), and
F. nucleatum (ATCC 25586; optical density at wave length of 660 nm: 0.0125–0.05), leptin (1–10 ng/ml; R&D Systems, Minneapolis, MN, USA), and visfatin (30–300 ng/ml; Biomol, Hamburg, Germany) were applied as in our previous studies [
21‐
23,
38‐
43].
F. nucleatum was inactivated via suspension in PBS (optical density at wave length of 660 nm = 1, equivalent to 1.2 × 10
9 bacterial cells/ml) and ultrasonication of 160 W twice for 15 min. Inactivation of bacteria was checked by subcultivation on Schaedler agar plates in anaerobic conditions. PDL fibroblasts were exposed to the different stimulants for 1 d. Untreated cells served as control.
Analysis of gene expression
The SSTR2 gene expression was analyzed via real-time polymerase chain reaction (RT-PCR) by using an iCycler iQ5 Detection System (Bio-Rad Laboratories, Munich, Germany). Briefly, RNA was extracted with a RNA extraction kit (RNeasy Protect Mini Kit, Qiagen, Hilden, Germany) and transcribed to cDNA with the iScript Select cDNA Synthesis Kit (Bio-Rad Laboratories). Afterwards, a 25 μl reaction mixture was prepared, containing 2.5 μl QuantiTect Primer Assay (Qiagen), 12.5 μl QuantiTect SYBR Green Master Mix (Qiagen) and 9 μl of nuclease free water as well as 1 μl of cDNA. The manufacturer’s protocol comprised a heating phase at 95 °C for 5 min for enzyme activation as well as 40 cycles of a denaturation step at 95 °C for 10 s and a combined annealing/extension step at 60 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.
Analysis of intracellular protein levels
PDL fibroblasts were cultured on glass coverslips in the presence and absence of IL-1β, F. nucleatum, leptin or visfatin (Carl Roth, Karlsruhe, Germany) for 24 h. Afterwards, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) at pH 7.4 and room temperature for 10 min, washed with PBS (Sigma-Aldrich), and treated with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min. Background staining was prevented by the application of serum block for 20 min (Dako, Hamburg Germany). After washing, cells were incubated with a rabbit polyclonal anti-SSTR2 antibody (10 μg/ml; R&D Systems) at 4 °C overnight. Goat anti-rabbit IgG-HRP (Dako) was applied as a secondary antibody for 45 min. Antibody binding was visualized by DAB chromogen (Thermo Fisher Scientific, Waltham, MA, USA) staining for 10 min at room temperature. Two washing steps with PBS (Sigma-Aldrich) were always performed between the incubation steps. Finally, cells were counterstained with Mayer’s Hematoxylin (Merck Eurolab, Dietikon, Switzerland) for 1 min and coverslipped with DePex mounting medium (Serva Electrophoresis, Heidelberg, Germany). Standardized photomicrographs were taken with an Axioskop 2 microscope (Carl Zeiss, Jena, Germany) equipped with an AxioCam MRc camera (Carl Zeiss) and the AxioVision 4.7 software (Carl Zeiss).
Human biopsies
Gingival biopsies of 7 periodontally healthy donors (mean age: 22.1 years, min–max: 18–26 years; gender: 2 male/5 female) and 7 periodontally-diseased patients (mean age: 58.4 years, min–max: 29–81 years; gender: 5 male/2 female) were used for our experiments. These biopsies were obtained during wisdom tooth extraction and extractions for orthodontic or periodontal reasons in the Department of Oral Surgery of the University of Bonn [
44]. Written and informed consent by the subjects and approval by the Ethics Committee of the University of Bonn (#043/11) were provided. Periodontal health or disease were assessed by gingival index (GI), probing pocket depth (PD), clinical attachment level (CAL), and radiographic bone loss. No clinical inflammation (GI = 0), a PD below 3 mm and neither clinical nor radiographic bone loss were categorized as periodontally healthy. Periodontitis patients were identified by sites with a GI > 1 and PD ≥ 5 mm and clinical as well as radiographic bone loss ≥3 mm, as previously described [
21,
44,
45]. Smokers as well as patients taking any medication were excluded from the study.
The gingival biopsies were harvested as described above and then further analyzed by RT-PCR. In addition, the SSTR2 protein levels of the biopsies were studied by immunohistochemistry. Briefly, the gingival tissues were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 2 d. Subsequently, the tissues were hydrated, then dehydrated in an ascending ethanol series (AppliChem, Darmstadt, Germany) and, subsequently, embedded in paraffin (McCormick Scientific, Richmond, IL, USA). Tissue slices of 3 μm thickness were cut and mounted on glass slides (Carl Roth). Finally, the slices were dried overnight, deparaffinized, rehydrated, and rinsed in TBS (TRIS und NaCl, MP Biomedicals, Illkirch, France; Merck KGaA, Darmstadt, Germany) for 10 min. Endogenous peroxidase was blocked with 0.3% methanol (AppliChem)/H2O2 (Merck Eurolab) solution for 10 min. Afterwards, an additional blocking step with serum block (Dako) for 20 min was carried out. Finally, the sections were incubated with a rabbit polyclonal anti-SSTR2 antibody (2.5 μg/ml; Research and Diagnostic Systems) in a humid atmosphere at 4 °C overnight. Goat anti-rabbit IgG-HRP (Dako) was used as a secondary antibody for 30 min, and the antibody binding was visualized by DAB chromogen (Thermo Fisher Scientific). Afterwards, the sections were counterstained with Mayer’s Hematoxylin (Merck Eurolab), and then imaged with an Axioskop 2 microscope (Carl Zeiss) equipped with an AxioCam MRc camera (Carl Zeiss) and the AxioVision 4.7 software (Carl Zeiss), as described above.
Experimental periodontitis model
An experimental periodontits model was applied to analyze the expression of SSTR2 in gingival biopsies during the development of periodontal disease [
46]. Thirty-two male adult Holtzman rats (average weight: 300 g) were assigned randomly to two experimental groups: control (sham-operated) and experimental periodontitis. Periodontitis was induced through cotton ligatures at the cervical area of the upper first molars. The ligatures were placed around the teeth, knotted mesially and left there for up to 20 d. Protocol followed the ARRIVE guidelines, and approval of the Ethical Committee on Animal Experimentation (protocol number: 23/2012) from the School of Dentistry at Araraquara, São Paulo State University – UNESP, was obtained. Animal housing was provided at the animal facilities of the School of Dentistry at Araraquara under controlled conditions (22–25 °C, 12 h light/dark cycle, standard laboratory diet and water ad libitum). Ten % ketamine hydrochloride (0.08 ml/100 g body weight) and 2% xylazine hydrochloride (0.04 ml/100 g body weight) were injected intramuscularly. Following 6 d, 8 d, 12 d, and 20 d, 4 animals per group were sacrificed by an anesthetic overdose. The maxillary jaws were harvested and, subsequently, the gingival tissues around the maxillary first molars were dissected for RNA extraction and RT-PCR (see above).
Diet-induced obesity model
Ten male Wistar rats purchased from Charles River (Sulzfeld, Germany) were kept in a temperature (21 °C) and humidity (35%) controlled environment with a 12-h dark-light cycle. All animals received food and water ad libitum. All procedures were conducted according to the ethical standards of the University of Bonn (Az 87–51.04.2010.A394). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The rats were equally divided into two groups. The animals of the test group were fed a normal diet until week 4 and, subsequently, a high-fat (25.1% fat), high sucrose (28.8% sucrose) diet (HFSD, sniff, Germany) until week 15. The animals of the control group were provided a normal diet for the entire 15 weeks. Animal weight was determined and documented weekly [
47]. At week 15, the rats of the two groups were sacrificed for further analyses. The jaws were harvested and, subsequently, the gingival tissues were dissected for RNA extraction and RT-PCR (see above).
Statistical analysis
Statistical analysis was carried out with the IBM SPSS Statistics software (Version 22, IBM SPSS, Chicago, IL, USA). Mean values and standard errors of the mean (SEM) were calculated for quantitative data. Experiments were done in triplicate and repeated at least twice. For statistical comparisons, the t-test, Mann-Whitney-U test or ANOVA followed by the post-hoc Dunnett’s test were used. Differences between groups were considered significant at p < 0.05.
Discussion
The present study provides novel insights into the regulation of SSTR2 in periodontal cells and tissues. Our in-vitro and in-vivo experiments show that proinflammatory, microbial and obesity-associated molecules cause a strong upregulation of SSTR2. Since SST has antiproliferative, antiangiogenetic, and proapoptotic effects, our findings suggest that SSTR2 might play a critical role in periodontal diseases.
There are only few studies, which have reported on SST and its receptors in periodontal tissues [
28‐
30]. However, the role of the SST/SSTR system in periodontal health and disease has yet to be elucidated. We therefore exposed periodontal fibroblasts to IL-1β, which has been shown to be increased at periodontally inflamed sites, and to the periodontopathogen
F. nucleatum, which is a gram-negative anaerobic microorganism associated with gingivitis and periodontitis [
5,
6]. Our experiments revealed that the SSTR2 expression was enhanced under these inflammatory and microbial conditions, indicating that the SST/SSTR system might play an important role in periodontal inflammation and infection. Interestingly, a study on human corneal epithelial cells and an immortalized epithelial cell line from human conjunctiva also demonstrated increased SSTR2 expressions in response to proinflammatory cytokines, such as IL-1ß, and bacterial components, such as lipopolysaccharides or peptidoglycans [
48]. These findings concur well our data and emphasize the potential role of the SST/SSTR2 system in human physiology and pathophysiology. Moreover, since periodontitis is linked with obesity, we also incubated periodontal fibroblasts with the proinflammatory adipokines leptin and visfatin, whose plasma levels are increased in obese individuals [
17,
19]. These adipokines are also produced locally in periodontal tissues and altered at periodontally inflamed sites, as studies by our group and other investigators have shown [
11,
21‐
23,
49]. Like IL-1β and
F. nucleatum, these adipokines also induced an upregulation of SSTR2, underlining their proinflammatory characteristics and indicating a potential mechanism whereby adipokines could contribute to periodontal destruction. To our best knowledge, our study is the first one which links visfatin with SSTR2. The stimulatory effects of IL-1β,
F. nucleatum, leptin and visfatin on SSTR2 were also observed at protein level, indicating that our transcriptional findings are physiologically relevant. In our experiments, the concentrations of the stimulants were in the physiological range and consistent with the doses of previous studies [
44,
45,
49‐
51].
In order to validate our in-vitro findings in a more complex environment, we also studied the SSTR2 expression in human biopsies from periodontally healthy subjects and from periodontitis patients. As demonstrated by RT-PCR and immunohistochemistry, SSTR2 levels were strongly enhanced at sites of periodontitis as compared to control sites, thereby confirming the findings of our in-vitro study, where IL-1β was used to mimic periodontal inflammation and
F. nucleatum was applied to simulate periodontal infection. In a rat experimental periodontitis model, we also investigated the time course of SSTR2 expression in gingival biopsies. In this model, ligatures were used to induce periodontitis, which resulted in significant alveolar bone resorption, as presented in our previous study [
46]. The in-vivo study supported our in-vitro data and the findings from the human biopsies by demonstrating that microbial plaque accumulation finally results in an upregulation of SSTR2. Interestingly, a recent study focused on the expression of SSTRs in the jejunums of
Cryptosporidium parvum-infected rats [
52]. The investigators found a significant increase in the SSTR2 expression in the inflamed jejunum following infection, indicating that SSTRs may regulate inflammatory pathways in rat intestine. These findings are in accordance with our results showing increased SSTR2 levels at inflamed sites. By contrast, SSTR2 was found to be downregulated in a rat lipopolysaccharide-induced periodontitis model [
53]. However, in this model, periodontitis was caused by injection of
Escherichia coli lipopolysaccharides into the palatal gingival sulci. In our model, periodontitis was established through cotton ligatures at the cervical area of the upper first molars, which usually results in the accumulation of a complex multispecies biofilm. We think that this model better simulates the plaque-induced development of periodontitis, as observed in humans, and the differences in the applied models might explain the opposite results. Moreover, our in-vivo findings are in line with our in-vitro data, thereby supporting our in-vivo observation. Taken together, our in-vitro and in-vivo results suggest a potential role of SSTR2 in the aetiopathogenesis of periodontitis.
Furthermore, we studied possible effects of obesity on the SSTR2 expression in gingival biopsies in a rat diet-induced obesity model [
47]. This in-vivo study revealed that obesity leads to an enhanced SSTR2 expression in gingival tissues, thereby supporting our in-vitro experiments with adipokines. Interestingly, acute central leptin infusion on the SSTR-effector system in the brain of rats also resulted in increased SSTR2 gene and protein levels [
54], indicating a regulatory role of leptin on this receptor.
Although the present study suggests a potential role of SSTR2 in the aetiopathogenesis of periodontal disease, we have not examined the actions of SST itself and the regulation of other SSTRs. Therefore, future studies should also focus on the actions of SST and on the roles of other SSTRs in periodontal cells and tissues. Additionally, further studies should analyze the role of the SST/SSTR system in different periodontal cells like gingival fibroblasts.
In the present study, cells were exposed to the periodontopathogen
F. nucleatum. However, periodontitis is a chronic inflammatory disease caused by a highly complex biofilm [
5]. Therefore, further studies should also examine the SSTR2 regulation by other microorganisms and their combinations. In addition, like the microbial biofilm, the inflammatory processes are also complex and involve a plethora of pro- and antiinflammatory mediators [
6]. Whether these molecules exert the same or opposite effects, as compared to IL-1β, has yet to be determined.
It has been suggested that adipokines, such as leptin and visfatin, could represent a pathomechanistic link in the interactions between periodontitis and obesity [
11]. It is possible that adipokines contribute to the aetiopathogenesis of periodontitis, as a lot of studies suggest [
11‐
13]. However, adipokines are also produced locally by periodontal cells and tissues [
21‐
23]. Interestingly, the local production of these adipokines is regulated by inflammatory and microbial signals, suggesting that adipokines also play a role in periodontal inflammation and destruction in the absence of obesity. Future studies should also examine the actions of other adipokines, such as resistin or apelin, on the SST/SSTR2 system in the periodontium. Moreover, further studies should be dedicated to the intracellular signaling pathways, which are involved in the observed SSTR2 upregulation.