Animal models
Six-week-old Sprague-Dawley rats were obtained from the Experimental Animal Experiment Center of Chongqing Medical University. Then, the animals were then randomly allocated into seven groups as follows: (1) Control group, (2) Light group: the optimal orthodontic MF (50g), (3) Heavy group: the heavy orthodontic MF (120g), (4) Yoda1-Light group: the optimal orthodontic MF (50g) + Yoda1, (5) GsMTx4-Heavy group: the heavy orthodontic MF (120g) orthodontic MF + GsMTx4, (6) C-176-Light group: the optimal orthodontic MF (50g) + C176 and (7) ADU-S100-Heavy group: the heavy orthodontic MF (120g) orthodontic MF + ADU-S100.
Wildtype C57/B6 male mice (6 to 8 weeks) were obtained from the Experimental Animal Center of Chongqing Medical University and STING null (sting−/−) mice (6 to 8 weeks) were purchased from Shanghai Biomodel Organism Science & Technology Development Co., Ktd. Then, the animals were then randomly divided into five groups: (1) WT-Control group: the wildtype mice, (2) WT-Light group: the application of light orthodontic MF on wildtype mice (10g), (3) WT-Heavy group: the heavy orthodontic MF on wildtype mice (30g), (4) sting−/−-Control group: the light orthodontic MF on sting−/− mice (10g), (5) sting−/−-Light group: the light orthodontic MF on sting−/− mice (10g), and (6) sting−/−-Heavy group: the heavy orthodontic MF on sing−/− mice (30g). All animal experiments were performed following the National Institutes of Health Guidelines for the Use of Laboratory Animals. The institutional Animal Care and Use Committee of Chongqing Medical University approved all the study protocols (202310171728000204352).
The OTM animal model was established following the methodology outlined in a previous study 49. A nickel-titanium coil spring was fixed between the maxillary left first molar and the incisors to induce either a light or heavy MF. The appliances were promptly activated upon insertion, and their fit was assessed daily. No reactivation was conducted throughout the duration of the experiment. The animals in the heavy orthodontic MF group were administered a subcutaneous injection of Piezo1 inhibitor GsMTx4 (MedChemExpress, United States) at a dosage of 20 µl with a concentration of 3 µM or STING inhibitor C-176 (MedChemExpress, United States) at a dosage of 20 µl with a concentration of 0.5 µmol/L every other day. In contrast, the light orthodontic MF group received a subcutaneous injection of either the Piezo1 agonist Yoda 1 (MedChemExpress, United States) at a dosage of 20 µl with a concentration of 5 µM or STING agonist ADU-S100 (MedChemExpress, United States) at a dosage of 20 µl with a concentration of 5 µM every other day. Each group was killed at days 1, 3, and 7 following tooth movement. The alveolar bone blocks included the left first molar, which was harvested for subsequent analysis.
Micro-CT scanning and analysis
The samples were fixed in a 4% paraformaldehyde solution for 24 h and subsequently scanned by using a vivaCT 40 system (Scanco Medical, Switzerland). Scanning of the specimens was carried out at 70 kV and 114 mA with an integration time of 500 ms and a voxel resolution of 10 mm. The distance of OTM was measured by assessing the spacing between the cementum–enamel junction levels of the first and second left molars. In this study, a 200 mm×200 mm×600 mm cube of trabecular bone distal to the middle part of the distal buccal root of the maxillary left first molar was selected as the region of interest for analysis. The distance between the cube and the root was 100 mm. Then, parameters including the distance and bone volume/total volume (BV/TV) ratio were calculated on days 1, 3, and 7 following the OTM.
Histological analysis
Following micro-CT scanning, the left half of the maxilla from each specimen underwent decalcification in a 14% ethylenediaminetetraacetic acid (EDTA) solution at pH 7.4 for two months. Then, all the specimens were dehydrated in a series of alcohol baths and embedded in paraffin. Subsequently, the samples, which included the maxillary molars, were excised into frontal sections that were 5 mm thick in the sagittal direction.
H&E staining and TRAP staining (Servicebio, China) were performed for the histological analyses. Multinucleated cells adjacent to the tension side of the periodontal region were quantified as TRAP+ osteoclasts. Two independent investigators counted the number of TRAP+ cells. For immunohistochemistry (IHC) analysis, primary antibodies were administered and allowed to incubate at 4°C overnight. A Diaminobenzidine detection kit (ZSGB-BIO, China) was employed. Antibodies detailed information is provided in Supplementary Table 1.
Cell culture and application of static compressive MF
The PDL cells were subjected to static compressive MF using the uniform compression technique. The optimal(light) MF (2g/cm2) and heavy MF (8g/cm2) were determined based on previous literature and experiments50, 51. The groups were categorized as follows: (1) Light group: subjected to light MF for PDL cells, (2) Heavy group: subjected to heavy MF for PDL cells, (3) Yoda1-Light group: subjected to light MF for PDL cells with Yoda1 (5 µM) treatment, (4) GsMTx4-Heavy group: subjected to heavy MF for PDL cells with GsMTx4 (4 µM) treatment, (5) siITPR3-Light group: subjected to light MF for PDL cells after ITPR3 small-interfering RNA treatment, (6) siITPR3-Heavy group: subjected to heavy MF for PDL cells after ITPR3 small-interfering RNA treatment, (7) C-176-Light group: subjected to light MF for PDL cells with C-176 (20 µM) treatment, (8) ADU-S100-Heavy group: subjected to heavy MF for PDL cells with ADU-S100 (10 µM ) treatment.
Analysis of proteomics using Tandem Mass Tag (TMT) labeling and protein phosphorylation modifications
Tandem Mass Tag (TMT) labeling and protein phosphorylation modifications analysis were conducted from Novogene Co., Ltd. Statistical analysis of protein quantification results was conducted using a T-test. Proteins with significant quantitative variances between the experimental and control groups (p < 0.05, | log2FC |>1.5) were categorized as differentially expressed proteins (DEPs).
Senescence-associated β-galactosidase staining
Senescence-associated β-galactosidase (SA-β-Gal) activity was detected using Senescence β-Galactosidase Staining Kit (Beyotime, China), according to the manufacturer's instructions.
Cell TRAP staining
After applying compressive MF, PDL cells were fixed with 4% paraformaldehyde. Tartrate-resistant acid phosphatase (TRAP) staining (Servicebio, China) was performed for the PDL cells according to the instructions.
Flow cytometry analysis of cell cycle and apoptosis
Centrifuge the compressed PDL cells to collect the cell precipitates. Add 400uL of ethidium bromide (PI, 50µg/mL) and 100ul of RNase A (100 µg/mL), then incubate the mixture at 4 ℃ in the dark for 30 minutes. Detected through conventional methods employing flow cytometry, the outcomes were assessed utilizing the cell cycle fitting software ModFit.
Cell apoptosis following the application of compressive MF was assessed utilizing the Annexin V-FITC Apoptosis Staining/Detection kit (Abcam, United Kingdom).
Calcium oscillation analysis
The calcium oscillation ([Ca2+]i ) activity was assessed in PDL cells that were pre-treated with Fluo8-AM (10 mM for 15 min at 37°C), following the methodology outlined in a previous study50. Fluorescence images of cells were captured using a Leica TCS SP8 confocal microscope. The scanning rate for 8-bit images sized 1024×1024 pixels was 1.12 seconds per scan. Time-lapse images collected from a single Z plane were recorded at 3-s intervals.
Mitochondrial calcium analysis
After 1 hour MF stimulation, the mitochondrial Ca2+ ([Ca2+]m) levels were quantified in PDL cells that were pre-treated with Rhod-2 AM (5 µM) for 15 min at 37°C) PDL cells, as previously described52. Fluorescence images of cells were acquired with a Leica TCS SP8 confocal microscope. The fluorescence activity in the cells prior to the application of MF was documented by Li J et al25.
Mitochondrial membrane potential, cytoplasmic ROS, and mito-SOX analysis
The JC1-mitochondrial membrane potential assay kit (Beyotime, China) was utilized following the guidelines provided by the manufacturer. Ratio F(aggregate)/ F(monomer) was subsequently assessed. Reactive Oxygen Species Assay Kit (Beyotime, China) was used to detect cytoplasmic ROS according to the manufacturer's recommendations. Detection of fluorescence for cytoplasmic ROS was excitation wavelength of 488nm and emission wavelength of 525nm. The MitoSOX-Red Assay Kit (Thermofisher, United States) facilitated the detection of mitochondrial ROS according to the manufacturer's recommendations.
Transmission electron microscopy and Energy Dispersive X-Ray Spectroscopy analysis
After MF treatment, the cultured cells were successively fixed with 2.5% glutaraldehyde for 1 h and 2% osmium tetraoxide for 2 h. After washes with double distilled water, the cell samples were stained with 0.5% uranyl acetate for 12 h, dehydrated, polymerized, and sectioned into 70–90 nm ultrathin sections. Images were observed and captured using a Tecnai G2 TWIN TEM. The Energy Dispersive X-Ray Spectroscopy (EDX) was established as previously described53. Data on oxygen (O), carbon (C), lead (Pb), and calcium (Ca) were collected from the TEM–EDX analysis. C, O, and Pb were used as controls.
Immunofluorescence analysis
After the treatment, the cells were fixed in a 4% paraformaldehyde solution for 15 min. Cells were blocked for 2h at 4℃. Primary antibodies were diluted in a blocking solution and incubated for 18 h at 4℃. The cells were washed with PBS containing 0.1% Triton X-100, and subsequently exposed to secondary antibodies that were diluted in a blocking solution. This incubation took place at 22°C for one hour under dark conditions. Following multiple washes, coverslips were affixed to a microscope slide with an antifade reagent. Antibody details can be found in Supplementary Table 1. The immunofluorescence analysis of mitochondrial DNA was conducted following the previously described protocol54.
Western blot analysis
Frozen cells were lysed by RIPA for 15 minutes, followed by centrifugation to isolate the proteins. Proteins were separated by 8–12% SDS-PAGE, followed by transfer, blocking, washing, and incubation with specific primary and secondary antibodies. Comprehensive details can be found in Supplementary Table 1. The reagents used for western blotting are presented in Supplementary Table 1. Image lab was used to detect and visualize the protein bands, and alpha-Tubulin was utilized for normalization purposes.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Cellular RNA was extracted using TRIzol reagent (Invitrogen, United States), and RNA reverse ranscription to cDNA was performed. General PCR was performed, followed by digital imsenescence of agar gelatin electrophoresis to visualize the DNA abundance. RT-qPCR was conducted to quantitatively analyze the DNA content. Expression levels were normalized against that of ACTB. The set of deltaCq replicates for the control and tested samples, normalized against the geometric means of the reference genes, was used for statistical testing and estimation of the P values. The primers used for qRT-PCR are presented in Supplementary Table 3.
Measurement of cytosolic mtDNA
After induction of TBHP for the indicated time and dose, cytosolic mtDNA was analyzed by general PCR or RT-qPCR43, 46, 55. Half of the cells were lysed using a mild lysis buffer, while the remaining half was lysed by a strong lysis buffer. Cellular cytoplasmic lysed with 0.1% NP-40 for 20 min on ice, followed by centrifugation at 14,000×g for 20 min at 4°C. Cytosolic mtDNA from the supernatant cytosolic fraction and total mtDNA from the total lysis were isolated using a TIANamp Genomic DNA Kit. General PCR, followed by agar gelatin electrophoresis and RT-qPCR, as detailed previously, were employed for DNA analysis, with normalization by the mtDNA in the total lysate.
siRNA transfection
For the inhibition of STING, Lipofectamine 2000 (Invitrogen, United States) was employed to transfect NP cells with 100 nM ITPR3 small-interfering RNA (si-ITPR3, GenePharma, China) or the control for 72 hours, followed by immediate exposure to 100 µM TBHP. The siRNA sequences are provided in Supplementary Table 2.
Enzyme-linked immunosorbent assay
Cytokine and chemokine release from astrocytes were quantified using commercially available duo-set ELISA kits (R&D Systems, United States) for human IL-1β (DLB50) and IL-6 (D6050B). The ELISA analysis was conducted following the ELISA kits protocol.
THP-1 cell-derived macrophage Transwell assay
Supernatants were collected from treated PDL cells. THP-1 cells were added into the upper chamber at 1 × 105 cells/well with phorbol myristate acetate (PMA, 100 ng/mL; MedChemExpress, United States). The supernatant was then added to the lower chamber. Cells were incubated for 24-hr. Cells stained by crystal violet on the bottom surface were considered migrated cells. For crystal violet assay, cells were washed with PBS, then fixed for 15 min in 3.7% formaldehyde and stained with crystal violet.
THP-1 cell-derived osteoclastogenesis
THP-1 cells were seeded at a concentration of 1×105 cells per well in 24-well plates and treated with PMA (100 ng/mL) for 48 h. Subsequently, the culture medium was changed to PDL cells-derived supernatants supplemented with M-CSF (20 ng/mL, MedChemExpress, United States) and RANKL (20 ng/mL, MedChemExpress, United States). Supernatants were replaced, and cellular morphology was monitored at 2-day intervals over 12 days. TRAP staining was subsequently conducted.
Statistical analysis
All data were expressed as the means ± standard deviations (SD). The comparisons between the two groups were performed using Student’s t-test. One-way analysis of variance (ANOVA) in combination with post hoc Dunnett’s test was used for multiple comparisons. GraphPad Prism 8.0 (San Diego, USA) software was used for all statistical analyses, and statistical significance was considered at p < 0.05. All experiments were performed at least in triplicate and repeated 3 times.