1.1 General Information
Between July 2015 and September 2016, we randomly selected 27 patients (14 males and 13 females) with periodontitis (CP), 17 patients (7 males and 10 females) with gingivitis (cg) and 20 patients (8 males and 12 females) with no periodontal disease (PH). Subjects were 24-60 years old with an average age of (33.24 ± 6.93) years. Periodontitis and gingivitis were diagnosed according to the diagnostic criteria of the Periodontal Disease Association
The inclusion criteria were: at least 20 teeth in the mouth, 4-6 teeth with severe periodontitis in all periodontal patients; no periodontal treatment in the preceding 6 months; no smoking history in the preceding 6 months; no history of head and neck radiotherapy; no antibiotics, phenytoin sodium, cyclosporine, calcium channel blockers, oral contraceptives, atropine and other drugs within the month; no serious systemic diseases and serious infections in other areas. Pregnant or lactating women were excluded. The study protocol was approved by the Ethics Committee of Linyi Central Hospital. All study participants provided written informed consent.
1.2 Experimental methods
1.2.1 Determination of clinical indicators
The researchers who received professional training in periodontal examination and passed the standard conformance test (Kappa=0.73) used the Williams probe to detect periodontal clinical indicators in all subjects, including the gingival index (GI), detection of bleeding on probing (BOP), depth of detection (PD), and clinical attachment level (CAL). Among them, PD and CAL were detected at 6 sites on the buccal (lip) side and at the distal, middle and proximal sites of the lingual side.
1.2.2 Collection of peripheral blood samples
Two mL of peripheral venous blood of all subjects was extracted and placed in an anticoagulation tube containing K2EDTA. Peripheral blood samples were collected by centrifugation (4000 r/min, 6 min) at 4°C. The supernatant was placed in a sterile Eppendorf tube and stored at -80 ° C until use.
1.2.3 Extraction of total RNA from peripheral blood samples and RT-PCR determination
One ml of Trizol was added in the serum and repeatedly pipetted, and the cell lysate was transferred to a 1.5 ml Eppendorf tube, left for 5 min, and fully lysed. Two hundred μL of chloroform was added per 1 ml of Trizol, mixed for 15 s, and left at room temperature for 3 min. After centrifugation at 4o C 12000 g for 15 min, the upper aqueous phase was aspirated and transferred to another new Eppendorf tube. Then, 0.5 ml of isopropanol was added per 1 ml of Trizol, mix, and left at room temperature for 10 min. After centrifugation at 42000g of 4oC for 10 min, the supernatant was discarded, and RNA was precipitated on the bottom of the tube. One ml of 75% ethanol was added per 1 ml of Trizol and mix vigorously. After centrifugation at 4o C at 7500 g for 5 min, the supernatant was discarded. RNA was precipitated at room temperature for 5-10 min, dissolved in 30 μL of EPC treated water, and stored at -80oC for use. RNA purity and concentration were measured by spectrophotometry at A260 and A260/280. Total RNA was extracted using a total RNA rapid extraction kit and reverse transcribed with Super M-MLV reverse transcriptase using miRNA-specific reverse transcription primers (Table 1). RT-PCR was performed in a real-time quantitative system. The amplification conditions are shown in Table 2, the primer design is shown in Table 3, and the reaction system is shown in Table 4.
1.2.4 Western blotting assays
The expression of TLR-8 and MMP-9 in cells was detected by Western blotting assays. The protein was resolved by a 4%-10% polyacrylamide gel electrophoresis and the gel was transferred to a PVDF membrane using SDS polyacrylamide gel electrophoresis. After blocking with 5% non-fat dry milk for 1 h at room temperature, the membrane was first incubated with 1:500 diluted primary antibody overnight, and after wash with TBS-T buffer, the membrane was incubated with secondary antibody for 1 h. Following rinse with TBS-T solution, enhanced chemiluminescence was carried out. The PVDF membrane was placed in a developing solution in a dark room and exposed to an X-ray film for exposure and development. Protein densitometry was performed using Image 1.6 software.
1.2.5 Preparation of leukocyte suspension
After extracting 3 mL of venous blood from all subjects, we used heparin as an anticoagulant to precipitate red blood cells, and white blood cells were taken. After wash, 1×106 / mL white blood cell suspension was prepared with RPMI1640 medium containing 10% bovine serum.
1.2.6 Determination of neutrophil chemotaxis
Using agarose glass plate method: agarose solution was prepared with 1640 solution, and after addition of 10% inactivated AB serum, the glass plate was poured ,. Each group consisted of three circular holes arranged in a straight line with a diameter and a pitch of 2.5 mm. Five μL of a white cell suspension (1×106/mL) was added in triplicates to the mesopores and Escherichia coli was added to the side wells for 24 hours, and 5 μL of the filtrate was used as the chemokine, and the other side was treated with 5 μL of the 1640 solution. The agarose glass plate was placed in a 37 ° C incubator, incubated with saturated humidity and 5% CO2 for 4 h, removed, fixed with methanol and formaldehyde. After agarose gel electrophoresis, the gel was stained with Wright, and measured under an optical microscope. The distance a cell moved from the edge of the hole to the hole on either side was measured.
1.2.7 Determination of phagocytic function of neutrophils
Staphylococcus aureus was grown on agar slants for 24 h, colonies were washed with sterile isotonic saline, washed twice with PBS, and the cells were suspended at 5×107 /ml by specific concentration method. Three drops of heparinized blood were taken on the concave slide, and three drops of Staphylococcus aureus suspension were added and after thorough mixing, they were placed in a sealed wet box. After incubation at 37 ° C for 30 min, the box was shaken once every 10 min; 1 drop was taken with a capillary suction tube, pushed onto a slide on a glass slide, fixed in methanol, stained with Giemsa, and counted by light microscopy.
1.3 Data processing
Data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison between groups. P < 0.05 was considered to have a significant statistical difference.