Sample collection
The current study included 12 patients with periodontitis (patient group, 7 males and 5 females) and 10 patients without periodontitis but with orthodontic demands (control group, 5 males, 5 females). Patients with more than 3 mm periodontal pocket depth were diagnosed as periodontal disease. Patients who had received any treatment or combined with other diseases were excluded from this study. Gingival crevicular fluid (GCF) samples and gingival tissues were obtained from patients (n=22) of Changsha Stomatological Hospital. GCF and gingival tissue were isolated and collected as described before [21, 22]. Consent forms were obtained before surgery. This study has been approved by the Ethics Committee of Changsha Stomatological Hospital.
Cell culture and transfection
PDLSCs were separated from gingival tissues of patients with orthodontic demands as described before [23, 24]. Cells were cultivated in α-minimal essential medium (α-MEM; Gibco BRL, USA) and supplemented with 10% fetal bovine serum (Gibco BRL), 100 units/ml penicillin (Invitrogen Life Technology, USA), 100 mg/ml streptomycin (Invitrogen), and 0.292 mg/ml l-glutamine (Invitrogen) at 37°C with 5% CO2. To induce oxidative stress, PDLSCs were treated with H2O2 (100 μM) for 24 h before each experiment. To evaluate the effect of Klotho on PDLSCs, cells were treated with Klotho (final concentration: 10 ng/mL for 48 h, and cells treated with the same concentration of DMSO served as controls. Short-interfering RNA (si-RNA)-targeted UCP2 (si-UCP2) was purchased from GenePharma (Shanghai, China) and transfected into cells using lipofectamine 2000 (Invitrogen).
ELISA
The level of Klotho in GCF samples (or controls) and PDLSCs, as well as the levels of ROS, malondialdehyde (MDA), SOD, and glutathione peroxidase (GSH-PX) in PDLSCs, were assessed using an ELISA kit (R&D Systems, Inc.) following the manufacturer's instructions.
Western blot assay
Protein samples from cultured PDLSCs or gingival tissues were acquired using the Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Beijing, China) and isolated on 10% SDS-PAGE. After moving onto polyvinylidene fluoride (PVDF membranes, 5% non-fat milk was added. Later, samples were treated with primary antibodies for Klotho (ab181373, Abcam, Cambridge, USA), UCP2 (ab97931), Bcl-2 (ab32124), Bax (ab32503), cleaved caspase-3 (ab2302), and GAPDH (ab8245, Abcam) overnight. Subsequently, secondary antibodies were added to the culture in darkness for 1 h. Finally, proteins were evaluated using a chemiluminescence detection system.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
According to the protocol of TUNEL assay, apoptosis of PDLSCs was detected using the In Situ Cell Death Detection kit (Roche). Apoptosis was evaluated by counting the positive cells as well as total number of cells in five random fields. Cell nuclei were positive if they were labelled with FITC (green), and DAPI staining indicated the cell nuclei under a florescence microscope. Positively stained cells were counted using image‐Pro Plus 6.0 software.
Statistical analysis
All experiments were performed at least three times (N ≥ 3). To analyze the statistical difference, the P-values between any two groups were calculated by one-way analysis of variance (ANOVA) using Graphpad 6.0 statistical software (GraphPad Software Inc., San Diego, CA, USA). The results of multiple experiments are presented as mean ± standard deviation. The result was considered statistically significant when P < 0.05.