1. Ethics statement
This study, including the method of obtaining consent, was approved by the Clinical Ethics Committee of at the School of Stomatology, Tongji University, in accordance with government-issued guidelines and institution policies (Approval number 2018012). Written informed consent was obtained from all the participants involved in this study. All subjects were over 18 years of age, were assessed for the presence/history of periodontal disease. Smokers, pregnant women, and nursing women were excluded from the study. Probing depth, clinical attachment loss, bleeding on probing, and physiological bone loss were assessed. The periodontitis group consisted of subjects with severe periodontitis, but without other serious systemic conditions. The healthy group consisted of volunteers in general good health who showed no signs of clinical attachment loss, no physiological bone loss with probe depth ≤ 3mm, instances of bleeding on probing were < 10%, and there was no visible gingival inflammation [30]. A gingival biopsy was collected from each subject (4 per group).
2. Mice
Specific pathogen-free male mice were used in this study. Mlkl-deficient (Mlkl−/−) mice shared a common genetic C57BL/6 background with wild-type (WT) mice. Animal experiments were conducted in accordance with the guidelines of the Clinical Ethics Committee at the School of Stomatology, Tongji University (Approval number 2018011).
3. Cell culture
WT and Mlkl−/− mice, aged 6–8 weeks, were sacrificed by cervical dislocation. The mice were sanitized by immersing them in 75% alcohol for 2 min. To harvest and culture bone marrow-derived macrophages (BMDMs), the intact femur and tibia were removed and placed in phosphate-buffered saline solution. Both ends of the bone were then removed and the bone marrow cavity was rinsed with Dulbecco's modified Eagle's medium (DMEM) until the cavity turned white. Cells were harvested at room temperature, centrifuged, and resuspended in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 20 ng/mL macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ, USA). L929 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.
4. Western blots
Gingival tissues of healthy individuals and patients with periodontitis were obtained. Frozen gingival tissues were homogenized rapidly in liquid nitrogen and lysed on ice in a lysis buffer comprised of 50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulf, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin, supplemented with 1% protease inhibitor cocktail (Bimake, Houston, TX, USA). After 30 min, the lysates were centrifuged at 12,000 g for 15 min at 4°C. The protein concentration was determined using the bicinchoninic acid method (Beyotime, Shanghai, China). Twenty micrograms of total protein were separated on an 8% sodium dodecyl sulfate polyacrylamide gel via electrophoresis, then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After complete protein transfer, the membranes were blocked with 5% milk powder solution for 2 h at room temperature and incubated overnight at 4°C with the following rabbit monoclonal anti-RIPK3 (Abcam, Shanghai, China), anti-MLKL (Proteintech, Rosemont, IL, USA), anti-phospho-MLKL (p-MLKL) (Abcam), or anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA). Immunodetection was performed using the Odyssey CLx (LI-COR, Lincoln, NE, USA), and the blots were quantified using ImageJ. Treated L929 cells similarly subjected to western blotting.
5. Cell viability assay
Cells were seeded into 96-well plates and treated with 1 µg/mL Porphyromonas gingivalis lipopolysaccharide (LPS-Pg; InvivoGen, San Diego, CA, USA), 20 µM of the pan-caspase inhibitor, zVAD (Promega, Madison, WI, USA) or 30 µM Nec-1 (Cambridge Bioscience, Cambridge, UK) for 24 h. Cell survival was determined using the CellTiter-Glo luminescent cell viability assay (Promega). Luminescence was read using an Infinite M200 microplate reader (Tecan, Zürich, Switzerland).
6. RNA extraction, reverse transcription and the quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIzol (Life Technologies, Carlsbad, CA, USA). The quantity of RNA was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham MA, USA). One microgram of RNA was used for reverse transcription using the RT reagent kit (Takara, Kusatsu, Japan). The qRT-PCR was performed using SYBR Green PCR Master Mix in a LightCycler 480® (Roche, Basel, Switzerland). Fold change in mRNA levels was calculated by the Eq. 2−ΔΔCt method [31]. Primer sequences are shown in the Table.
7. Ligature-induced experimental periodontitis
The experimental periodontitis model was induced in WT and Mlkl−/− mice at eight weeks of age (ten per group). A 5 − 0 silk ligature was tied around the maxillary right second molar, while the contralateral unligated tooth served as the baseline control. Alveolar bone loss (ABL) was analyzed 7 days after ligature placement. For this, the distance between the cementoenamel junction and the alveolar bone crest (CEJ–ABC distance) was measured on both the buccal and palatal sides. Further, the maxillae of the mice were subjected to micro-computed tomography (CT) and histological analyses. Mice in which the ligatures were lost were excluded from the analyses.
8. Bone loss measurements
For micro-CT analysis, the maxillae were fixed in 4% paraformaldehyde until analysis. To calculate the ABL, the CEJ–ABC distance on the control tooth was subtracted from that on the ligated tooth.
9. Histological analyse
For histological analyses, the maxillae were fixed in 4% paraformaldehyde, decalcified in 10% EDTA solution for 3 weeks, and then embedded in paraffin. Sections were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP). TRAP-positive multinucleated (> 3 nuclei) cells were counted as osteoclasts.
10. Statistical analyse
Statistical significance between the groups was determined using the two-tailed Student’s t-test or One-way ANOVA. All analyses were performed using Prism 7.0 software (GraphPad, La Jolla, CA, USA). P < 0.05 was considered significant.