PPAR, RXR, and VDR Expressions in Diabetes
Condition: Diabete Mellitus; Periodontitis
MeSH terms: Periodontitis; Diabetes Mellitus
Study type: Observational

Summary: 
      Objective: Diabetes and periodontitis are two chronic inflammatory diseases sharing specific
      etiopathogenetic mechanisms, and both cause severe inflammation and destruction. The aim of
      the present study was to determine the receptor expressions of Peroxisome
      proliferative-activated receptor (PPAR)-γ, Retinoid X receptor (RXR)-α, Vitamin D receptor
      (VDR), and nuclear factor kappa B (NF-κB) expressions in healthy gingiva and diseased
      gingival samples with diabetes.

      Methods: 45 participants as 1; healthy controls (C), 2; periodontitis group (P), and 3;
      diabetes and periodontitis group (DP) were enrolled. Plaque index (PI), gingival index (GI)
      and clinical attachment levels (CAL) were recorded in all participants. Two gingival biopsies
      from each participant were obtained, and one underwent histological tissue processing while
      the other underwent RT-PCR analysis of nuclear receptors. Inflammatory and fibroblast cell
      counts, PPAR-γ, RXR-α, and VDR were evaluated.

      Results: Fibroblast cells were lowest in the DP group and highest in the healthy group.
      PPAR-γ, VDR, RXR, and NF-κB expressions were higher in the healthy controls in the RT-PCR
      analysis and similar in the other groups. Immunohistochemistry analysis also showed similar
      results.

      Conclusion: Results concluded that healthy gingival samples had higher PPAR-γ, RXR, VDR, and
      NF-κB expressions, and the immunohistochemistry results also supported this finding. The
      healthy gingiva contained higher fibroblast cells and lower inflammatory cells.
    

Detailed description: 
      The present study protocol was approved by the ethical committee of the Medical Ethics
      Committee of Tokat Gaziosmanpasa University. The study was designed as a cross-sectional
      clinical study and conducted at Tokat Gaziosmanpaşa University Faculty of Dentistry
      Department of Periodontology. Informed consent was obtained from all participants, and
      detailed oral and radiological examination was performed by an experienced clinician
      (H.B.Y.).

      Forty-two participants, 14 participants in each group, were enrolled in the study. The study
      groups were as follows.

      Healthy controls (C, mean age 41.05±1.80, seven men, seven women) Periodontitis patients with
      stage 3 grade B, (P, mean age 42.35±1.92, seven men, seven women) Patients with diabetes and
      periodontitis with stage 3 grade C, (DP, mean age 41.09±1.28, seven men, seven women)

      All patients and healthy individuals went through a detailed oral examination, and
      periodontal health and periodontitis diagnosis were based on the criteria defined by the
      recent classification of periodontal health and diseases in the 2017 International World
      Workshop for a Classification of Periodontal Diseases and Conditions [21].

      All participants were never smokers. Exclusion criteria were conditions which might affect
      the inflammatory state, the existence of less than ten or over missing teeth, tobacco or any
      drug use, presence of pregnancy or lactation, and existence of antibiotic or periodontal
      therapy within previous six months. Diabetes patients in the present study had HbA1c values
      greater than 7.0 even though they were under diabetic treatment. And the diabetic patients
      who had HbA1c values below 7.0 were not included.

      Clinical measurements Clinical parameters were plaque index (PI), gingival index (GI) and
      clinical attachment levels (CAL), and all analyses were performed before the biopsy
      procedure. Full mouth measurements and the measurements of the relevant teeth were recorded
      separately. CAL was measured from the cement-enamel junction to the bottom of the periodontal
      pocket and Williams's type periodontal probe was used for measurements (Hu-Friedy Co.,
      Chicago, IL, USA). PI and GI measurements were performed via an explorer (Hu-Friedy Co.,
      Chicago, IL, USA). All analyses were performed from six points, three from buccal aspect and
      three from the palatal aspect as mesial, middle, and distal sites for both teeth. A mean
      value of six measurements was recorded.

      Collection of Gingival Samples Gingival biopsies were obtained according to a protocol by a
      previous study [22]. Two gingival biopsies were obtained from each participant. Gingival
      sampling in periodontally healthy individuals was performed by crown lengthening procedure in
      the routine treatment protocol or before orthodontically indicated tooth extraction. In the
      periodontitis groups, biopsies were obtained from the inflamed gingival tissue with minor
      surgical periodontal procedures. All biopsies were obtained from posterior maxillary teeth
      (tooth #4, #5, #6 or #7).

      Firstly, local anesthesia was performed, and a gingival sample was dissected with a tissue
      scissor from an area with requiring either gingivectomy/crown lengthening procedures from
      patients with impaired gingival topography or before tooth extraction indicated for
      orthodontic treatment in healthy individuals. As for the periodontitis patients, a gingival
      sample was dissected using a tissue scissor during the scaling and root planning procedure
      from the gingival areas with severe gingival inflammation. One gingival sample from each
      participant was immediately placed in 10% neutral buffered formalin for 48 hours and
      underwent histological tissue processing. The other samples of each participant were
      immediately frozen at -80⁰C until the day of analysis.

      Histopathological Evaluation All histological procedures were performed by an experienced
      blinded researcher (F.G.). Gingival samples which underwent histological tissue processing
      were first washed and cleansed from formalin and were dehydrated with alcohol series. Then
      all tissues were cleared with xylene and embedded in paraffin blocks. Each block sectioned
      throughout as serial sections and three sections from each block were selected for
      Hematoxylin-eosin (H&E) staining. Fibroblast and inflammatory cell infiltration were
      evaluated from H&E stained slides under 1000x magnification via a light microscope .

      For fibroblast and inflammatory cell evaluation, the gingival area at the connective tissue
      border neighboring gingival epithelium was evaluated. A cell counting frame of 10.000 µm2
      area was selected under 1000x magnification. The total inflammatory cells (neutrophil,
      lymphocyte, eosinophil, and macrophage cells) within the frame were counted as inflammatory
      cell counting. The fibroblasts were also counted likewise. The measurements were performed
      from three different points, and the mean of these three measurements was recorded [23].

      PPAR-γ, RXR-α, and VDR Immunohistochemistry Immunohistochemistry was performed as reported in
      previous studies [23-26]. Three sections were chosen for each immunohistochemistry staining
      from each participant. Firstly, all slides were deparaffinized and dehydrated with alcohol
      series. After washing with distilled water, antigen retrieval was performed via sodium
      citrate buﬀer (pH 6.0) for two h at 70°C, and then endogenous peroxidase activity was
      suppressed with 3% hydrogen peroxide treatment. After hydrogen peroxide treatment, all slides
      were incubated with normal rabbit serum for 30 min. After normal serum incubation, primary
      antibody diluents were prepared and applied to the samples overnight at a humidified chamber
      at 4⁰C. The primary antibodies were goat polyclonal anti-PPAR-γ antibody (1:250), anti-RXR-α
      antibody (1:250), and anti-VDR antibody (1:250). After primary antibody incubation, all
      slides were washed with phosphate buffer solution (PBS) three times for five minutes (3x5)
      and biotinylated immunoglobulin G was applied for 30 min. Again a wash of 3x5 PBS, all
      samples were exposed to a streptavidin-horseradish peroxidase-conjugated reagent for another
      30 min. After washed with 3x5 PBS, all sections were treated with AEC chromogen to visualize
      staining. Counterstaining was performed with Meyer's hematoxylin, and sections were mounted.
      After allowing dry for two days, samples were examined under 400x magnification using light
      microscopy [23, 24].

      Immunohistochemical semi-quantitative H SCORE Analysis Immunohistochemistry evaluation was
      performed on three different areas in each section, and three sections were evaluated for
      each patient. Nine measurements were performed for each participant. All cells were marked
      according to their staining as no staining '0', low staining '1', mild staining '2', and
      dense staining '3'. Staining scores were converted to a numeric value as 'H score' through a
      formula (∑Pi(i+l)) which is a frequently used value for immunohistochemistry [23, 24]. In
      this formula, i shows the staining intensity score and Pi: indicates the percentage of the
      stained cells.

      RT-PCR Analysis The primers to be used for the genes were shown in table 1. The RNA isolation
      and RT-PCR analysis performed according to a previous study [27]. Briefly, for RT-PCR
      analysis, first gingival tissues were homogenized and subjected to sonication to reveal RNA
      in the tissues and tissue supernatants were obtained. Then the supernatants were submitted to
      the total mRNA isolation step. The tissues treated with Qiazole, then allowed to incubate in
      the shaker for 5 minutes. At the end of 5 minutes, chloroform was added to the medium
      containing the cells and incubation continued for 3 minutes at room temperature and then the
      samples were centrifuged. At the end of the centrifuge, the supernatant at the top of the
      tube transferred to another tube. Afterward, isopropanol was added to the supernatant and
      incubated for 10 minutes at room temperature, and the centrifugation step was repeated. At
      the end of the centrifugation, the pellet deposited at the bottom was washed with 75%
      ethanol, remaining supernatant was centrifuged again and suspended in pure, sterile water
      without RNase. mRNAs were determined by spectrophotometric method. Then using the qRT-PCR
      kit, one µg mRNA were transformed into single-step cDNA using specific primers targeting the
      gene of interest, later the genes were identified with the help of qRT-PCR (Verso, Thermo
      Fisher Scientific, Massachusetts, USA). The results were normalized to the β-actin control
      gene.

      Statistical Analysis The power of the study was over 85% based on a previous study with a
      similar study design [25]. IBM SPSS software was used for statistical analysis. One Sample
      K-S test was used to test the normality. For the fibroblast cell counts, inflammatory cell
      counts, and CAL, One Way ANOVA followed by Tukey test used. For other parameters, Mann
      Whitney U and Kruskal Wallis tests were used. Data were presented as mean ± SD or percentage
      as appropriate. p < 0.05 were considered statistically significant.
    

Intervention name(s): Gingival biopsy
Intervention type(s): Procedure
Intervention description(s): Two gingival biopsies from each participant were collected.
Observational model: Case-Only
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        Forty-two participants, 14 participants in each group, were enrolled in the study. The
        study groups were as follows.

        Healthy controls (C, mean age 41.05±1.80, seven men, seven women) Periodontitis patients
        with stage 3 grade B, (P, mean age 42.35±1.92, seven men, seven women) Patients with
        diabetes and periodontitis with stage 3 grade C, (DP, mean age 41.09±1.28, seven men, seven
        women)
      
Keywords: Diabetes Mellitus; NF-κB; PPAR-γ; Periodontitis; RXR; VDR