Patients with chronic periodontitis (n=30) treated at the Department of Stomatology, Tianjin Nankai Hospital (Tianjin, China) from June 2017 to December 2017 were included in the chronic periodontitis group (18 males and 12 females). The age ranged from 21–67 years and the average age was 43.7±19.4 years. The inclusion criteria were as follows: i) Having been diagnosed of chronic periodontitis according to the standards formulated by the committee of periodontal disease research; ii) having no oral or regional inflammation, including amygdalitis and pharyngitis; iii) having no immune disease, infectious diseases or other chronic diseases; and iv) having not taken in any antibiotics or immunosuppressants in the last three months. Patients selected for physical examination in the same hospital during the same period were the control group (n=30; 14 males and 16 females). The age range was 24–62 years, and the average age was 45.2±17.7 years in the control group. Exclusion criteria were as follows: i) Having chronic diseases; ii) having been administered with antibiotics; iii) having oral inflammation; and iv) being pregnant. Written informed consent was obtained from subjects, and the study was approved by the Ethics Committee of Tianjin Nankai Hospital.

For patients with chronic periodontitis included in this study, peripheral venous blood was collected in the early morning following fasting. The specific procedure was as follows: A disposable serum separator hose produced by BD Biosciences (San Jose, CA, USA) was used to collect ~3 ml peripheral venous blood; following serum separation, the samples were centrifuged at 4°C, 1,000 × g for 10 min. The upper layer was collected and stored in a −80°C refrigerator prior to analysis.

Samples of saliva from patients and control subjects were collected in the early morning with a cotton swab. Subjects were required to have an empty stomach in the 2 h prior to saliva collection; during this time, subjects could not drink water, alcohol or beverages, eat, smoke, exercise or have severe emotional fluctuations. Subjects were in a sitting position, leaning slightly forward and the cotton swab was dipped in acid to stimulate oral saliva secretion during collection. Saliva was collected in 10 ml RNase-free tubes after 1.5 min and the upper layer liquid was collected immediately after centrifuging at 4°C and 1,000 × g for 15 min. The collected saliva supernatant was frozen in a −80°C refrigerator prior to use.

A detailed record of all periodontal clinical indicators was obtained for all patients, including plaque index (PLI), gingival index (GI), periodontal probing depth (PD) and loss of attachment (AL). PLI=the total amount of dental plaque/the total number of teeth. GI: The gingiva around each tooth was examined with a blunt periodontal probe and was divided into 4 tooth surfaces; the average score was obtained and the total score was the average score of all the analyzed teeth. PD: The depth of the gum pocket or periodontal pocket measured with a special periodontal probe. AL: Using the periodontal probe, 20–25 g of force was extended into the gingival crevicular groove; the attachment of connective tissue decreased; the depth of the exploration was >3 mm and the enamel bone boundary was detected. The degree of periodontitis was measured according to probing depth, gingival index, loss of attachment and X-ray films showing alveolar bone resorption.

Periodontal ligament tissue of the patients and control subjects were used in the present study. A third of the periodontal ligament tissue of the root of bicuspids removed due to the orthodontics was cut into small pieces of 1 mm³ and transplanted into culture bottles with a bottom area of 25 cm² containing 10% thermal non-animalized fetal bovine serum. Primary culture of tissue was performed in Dulbecco's modified Eagle's medium (DMEM; 100 U/ml, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and streptomycin (100 µg/ml) under 55% humidity and an atmosphere of 5% CO₂ at 37°C. When hPDLFs were fully confluent, they were passaged at a ratio of 1:3. Following the third passage, the cell culture medium was changed to a mineralization induction medium (phenol red DMEM, 10% heat non-immobilized fetal bovine serum (Dalian Meilunbio Biology Technology Co., Ltd., Dalian, China), 10⁻⁸ mol dexamethasone, 10 mmol β-glycerophosphate sodium, 50 µg/ml ascorbic acid) to differentiate hPDLF cells. When passaged to the fourth generation, cells (1.5×10⁴/cm² density) were seeded into a 6-well plate. At 90% confluence, the serum-free mineralization-inducing medium was replaced by mineralization induction medium and hPDLFs were further incubated for 12 h to prepare for subsequent experiments. The total mRNA of hPDLF cells was extracted by the Trizol reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Human IL-18 Quantikine ELISA kit (cat. no. DL180), Human MMP1 Quantikine ELISA kit (cat. no. DMP100), MMP2 Quantikine ELISA kit (cat. no. MMP200), MMP3 Quantikine ELISA kit (cat. no. SMP300) and MMP9 Quantikine ELISA kit (cat. no. DMP900) were used to detect IL-18 and IL-1 in serum and saliva supernatants, and the content of MMP1, MMP2, MMP3, MMP9 in hPDLFs, respectively (all from R&D Systems China Co., Ltd., Shanghai, China). The experimental operations were performed in accordance with the instructions of the kit manufacturer.

An ABI StepOne Plus Real-Time PCR System was used to perform RT-qPCR for MMP1, MMP2, MMP3, MMP9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 using RNA obtained from hPDLFs, in strict accordance with the instructions of the Takara SYBR Premix Ex TaqPCR Reaction kit (Takara Biotechnology Co., Ltd., Dalian, China). The primers used for qPCR were as follows: MMP1, forward 5′-TCTGGGGAAAACCTTTCGACT-3′ reverse, 5′-CACCAACGTATTCAAAAGCACAA-3′; MMP2, forward 5′-TGACTTTCTTGGATCGGGTCG-3′, reverse, 5′-AAGCACCACAGATGACTG-3′; MMP3, forward 5′-AGTCTTCCAATCCTACTGTTGCT-3′, reverse 5′-TCCCCGTCACCTCCAATCC-3′; MMP9, forward 5′-TGTACCGCTATGGTTACACTCG-3′, reverse, 5′-GGCAGGGACAGTTGCTTCT-3′; TIMP-1, forward 5′-CTTCTGCAATTCCGACCTCGT-3′, reverse, 5′-ACGCTGGTATAAGGTGGTCTG-3′; TIMP-2, forward 5′-AAGCGGTCAGTGAGAAGGAAG-3′, reverse, 5′-GGGGCCGTGTAGATAAACTCTAT-3′; GAPDH, forward 5′-TGTGGGCATCAATGGATTTGG-3′, reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′; and IL-18, forward 5′-CAAGGCTGGTCCATGCTCC-3′ and reverse, 5′-TGCTATCACTTCCTTTCTGTTGC-3′.

The PCR mixture (20 µl) comprised 10 µl SYBR Premix Ex Taq II, 0.5 µl ROX Reference Dye II, 2 µl cDNA templates, 0.5 µl upstream primers, 0.5 µl downstream primers, RNAase Free dH₂O 6.5 µl. The thermocycling conditions were: 95°C, 15 min; 95°C, 15 sec; 60°C, 60 sec, 35 cycles. The 2^−ΔΔCq method (21) was used to quantify expression.

hPDLFs were pretreated with pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB; 100 nM) for 24 h, and then stimulated with hPDLF with IL-18 (10 ng/ml) for 24 h. In the control group, the PDTC was replaced with a solvent (dimethyl sulfoxide, 0.1%). Radioimmunoprecipitation assay buffer (P0013B, Beyotime Institute of Biotechnology, Haimen, China) was used to extract total protein from the cells. Protein concentration was determined using a bicinchoninic acid assay. Denatured protein (50 µg/well) was then loaded into 10% SDS-polyacrylamide gels, followed by separation. Constant voltage (80 V) was applied until the samples formed a line, then the voltage was adjusted to 120 V and electrophoresis continued. Following electrophoresis, the protein was transferred to a polyvinylidene difluoride membrane on ice at 0.25 mA for 2 h. Ponceau staining for 10 min was used to detect successful transfer. Following washing with TBS-Tween buffer, the membrane was incubated for 2 h at room temperature in 5% non-fat dry milk powder on a shaker; then, primary antibody was added (P65, 8242S, 1:2,000, Cell Signaling Technology, Inc., Danvers, MA, USA; p-P65: 3031S, 1:2,000, Cell Signaling Technology, Inc.; MMP1: ab137332, 1:2,000, Abcam, Cambridge, UK; MMP2: ab37150,1:2,000, Abcam; MMP3: ab52915, 1:2,000, Abcam; MMP9: ab73734, 1:2,000, Abcam; GAPDH: ab8245, 1:5,000, Abcam) and incubated overnight at 4°C. After washing the membrane, goat anti-rabbit IgG-horseradish peroxidase secondary antibody (ab6721, 1:2,000, Abcam) was added and incubated for 1 h at room temperature. Western blots were exposed by enhanced chemiluminescence (P0018, Beyotime Institute of Biotechnology) and band gray values were analyzed using ImageJ software version 1.43 (National Institutes of Health, Bethesda, MD, USA). The results were expressed as the experimental gray value/GAPDH band optical density value, and the internal reference was GAPDH.

According the manufacturer's instructions of the CCK-8 kit (Invitrogen; Thermo Fisher Scientific, Inc.), cells were inoculated in a 96-well (3,000 cells, 100 µl) plate. The hPDLFs were stimulated with IL-18 (10 ng/ml for 24 h at 37°C) when 50% confluence was attained. In the control group, hPDLFs were stimulated with PBS for 24 h. After 24 h, CCK 8 reagent was added in the medium, which accounted for 10% of the total volume The plate was then incubated for 4 h at 37°C and 5% CO₂ incubator; 100 µl liquid was removed from each well and inserted into a 96-well enzyme-labeled plate, a chemiluminescent analyzer to determinate the absorbance values.

The results are presented as the mean ± standard deviation. Statistical analysis was performed using a Student's t-test. The correlation between variables was analyzed using linear regression analysis. All statistical analyses were performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

In this study, serum IL-18 protein levels in 30 healthy volunteers and 30 patients with chronic periodontitis were detected by ELISA. The serum IL-18 protein expression level in patients with chronic periodontitis was significantly higher than that in healthy volunteers (Fig. 1). This result suggests that there may be an association between IL-18 protein expression and chronic periodontitis.

Saliva specimens were collected from 30 patients with chronic periodontitis and IL-18 expression was detected in the saliva using ELISA (Table I). Additionally, clinical indicators of periodontitis patients were assessed and recorded, including PLI, GI, PD and AL. By performing linear regression analysis of the expression levels of IL-18 in saliva with these clinical indicators, it was determined that the expression levels of IL-18 in saliva were significantly positively correlated with each periodontal clinical index (Table II). This indicated that IL-18 expression is closely associated with periodontal destruction.

hPDLFs were stimulated with IL-18 (10 ng/ml) and the proliferation of cells was detected using CCK-8. After 24 h, the optical density value at 480 nm was detected and there was no significant difference between IL-18-stimulated cells and control cells (Fig. 2); thus, IL-18 had no effect on the viability of hPDLFs.

Following stimulation of hPDLFs with IL-18 (10 ng/ml) for 24 h, the cells were collected and the mRNA expression of MMPs in the IL-18-treated group and control group was detected by RT-qPCR. The mRNA expression levels of MMP1, MMP2, MMP3 and MMP9 were significantly increased in the IL-18-treated group compared with the controls (Fig. 3); however the mRNA expression levels of TIMP-1 and TIMP-2 did not change in the IL-18-treated group compared with the control group (Fig. 4). This indicated that IL-18 can promote the mRNA expression of MMP1, MMP2, MMP3 and MMP9 in hPDLFs, whereas it has no effect on TIMP-1 and TIMP-2.

To further investigate the effect of IL-18 on the protein expression levels of MMP1, MMP2, MMP3 and MMP9, ELISA was used to detect MMP protein levels in IL-18-stimulated (10 ng/ml) hPDLFs. MMP1, MMP2, MMP3 and MMP9 protein levels were significantly higher in the IL-18-treated group compared with the control group (Fig. 5). This result indicated that IL-18 promotes the secretion of MMP1, MMP2, MMP3 and MMP9 proteins by hPDLFs.

Previous studies have demonstrated that NF-κB can promote the transcription of MMPs, and the activation of NF-κB can increase the expression of MMP1, MMP2, MMP3 and MMP9 (22). Therefore, this study examined whether IL-18 can promote the phosphorylation of NF-κB p65 protein to regulate the expression of MMPs. hPDLFs were pretreated with pyrrolidine PDTC, an inhibitor of NF-κB, and then stimulated with hPDLF with IL-18 (10 ng/ml). After 12 h, western blot analysis was performed to detect NF-κB. The phosphorylation NF-κB p65 protein was significantly increased in the IL-18-treated group, and the expression levels of MMP1, MMP2, MMP3 and MMP9 protein were also significantly increased compared with the control. However, the phosphorylation of NF-κB p65 was significantly decreased in the IL-18 + PDTC-treated group, and the protein expression levels of MMP1, MMP2, MMP3 and MMP9 were also suppressed compared with IL-18 treatment (Fig. 6). These results suggested that IL-18 induces the expression of MMP1, MMP2, MMP3 and MMP9 by activating the NF-κB signaling pathway.