A NiTi spiral tension spring (0.12 mm) was purchased from Emondi Materials Technology Co., Ltd. The orthodontic ligation wire (0.2 mm) and dynamometer were purchased from Hangzhou Bioer Co., Ltd. The antibodies used in the present study were purchased from Abcam and were as follows: Beclin-1 (cat. no. ab232461; 1:1,500), LC3-II (cat. no. ab51520; 1:1,500), P62 (cat. no. ab56416; 1:1,000), Bcl-2 (cat. no. ab185002; 1:1,000), Bax (cat. no. ab32503; 1:1,500) and caspase-3 (cat. no. ab197202; 1:800). Tartrate-resistant acid phosphatase (TRAP) staining regents were obtained from Beijing Solarbio Technology Co., Ltd.

In total, 40 male Sprague Dawley rats (6-8-week-old; weight, 247±33 g) were purchased from the experimental animal center of Southwest Medical University and the animal study was approved by the Ethics Committee of Southwest Medical University (license no: SCXK-2019-17; Luzhou, China). The rats were housed under a 12-h light/dark cycle with free access to food and water at the temperature of 23-25˚C and 40-50% humidity. The rats were randomly divided into 4 groups, with 10 rats in each group. Rats from the different groups were treated with orthodontic pressure for 0 or 1 h, as well as 1 or 7 days. The animals were initially anesthetized by intraperitoneal injection with xylazine (10 mg/kg) and ketamine (100 mg/kg) as described previously (17). To check the successful induction of anesthesia, the palpebral reflex was tested by touching the medial canthus or the inner corner of the animals' eyes. No palpebral reflex indicated the successful induction of anesthesia. A nitinol tension spring was placed between the rat incisor and the right upper first molar. The two incisors were used as anchorage and a force of 0.392 N was applied to move the maxillary first molar (Fig. 1A). The force application device for each rat was monitored daily. The animals from the various groups were euthanized at the end of the testing time points (0, 1 h, 1 and 7 days). Inhalation of carbon dioxide (used at a 30% flow displacement rate) was used for animal euthanasia, which was performed in October 2019. Absence of respiration, heartbeat and the corneal/palpebral reflex indicated the death of animal. The right first molar and its surrounding tissues were dissected and subsequently used for other experiments. The relevant experiments involving the right first molar were subsequently performed (Fig. 1B).

The prepared tissues were fixed in 4% paraformaldehyde solution for 48 h at room temperature. The tissues were buffered in PBS solution for 24 h at room temperature and transferred into 14% EDTA decalcification solution (pH: 7.3-7.5) for decalcification at room temperature. The decalcification solution was replaced daily. Following 6 weeks of decalcification, the tissues were embedded using paraffin wax and cut into 4-µm thick slices.

An initial deparaffinization step was conducted as follows: Slides were incubated in xylene twice (5 min each time), followed by washes in a descending ethanol gradient. The slides were then washed using deionized water twice (5 min each time). In total, 3% hydrogen peroxide (25 min) was used to remove endogenous peroxidase activity at room temperature. Following washing with PBS (three times, 5 min/time), goat serum (cat. no. ab7481; Abcam) was applied for blocking (20 min) at room temperature. The aforementioned primary antibodies were incubated with the tissues overnight at 4˚C. Following washing with PBS (three times), the tissues were incubated with the horseradish peroxidase-conjugated secondary antibodies (cat. no. ab97051, 1:2,000; cat. no. ab205719, 1:2,000; Abcam) for 4 h at room temperature. Finally, DAB reagents were used, and slides were counterstained with hematoxylin for 20 sec at room temperature. The relative protein expression was analyzed using Image Pro-Plus 6.0 (Media Cybernetics, Inc.) with a light microscope (magnifications, x200 and 400; BX51; Olympus Corporation). Briefly, the background of images was adjusted firstly. Five fields per slide were taken for further analysis. Regions of interest were selected and the intensity of DAB staining was calculated.

The tissues were prepared as aforementioned. Following deparaffinization, the tissues were stained with hematoxylin for 6 min at room temperature. After washing with running tap water for 5 min, 1% acid alcohol (30 sec) was used for differentiation. After washing with running tap water again for 1 min, 0.2% ammonia water (30 sec) was used for bluing. After washing with running tap water for 5 min, slides were rinsed in 95% alcohol for five times. Finally, the tissues were stained with eosin for 20 sec at room temperature, mounted and observed using light microscopy (magnifications, x200 and 400; BX51; Olympus Corporation). PDLCs were counted manually from five different fields of view per slide.

The TRAP solution was made according to the instructions provided by the manufacturer. Following deparaffinization as aforementioned, the tissues were cultured with TRAP solution for 1 h at 37˚C and washed with deionized water three times (5 min/each time). Finally, the tissues were counterstained with hematoxylin for 3 min at room temperature, dehydrated and mounted. Tissues were observed using microscopy (magnifications, x200 and 400; BX51, Olympus Corporation). Osteoclasts were counted manually from five different fields of view per slide.

The mCT system (Rigaku-mCT) was used to measure the tooth movement as previously described (18). After euthanizing at the end of testing time points (0, 1 h, 1, and 7 days) before dissection, the mCT system was used to detect the distance between the mesial aspect of the second molar and the enamel on the most distal aspect of the first molar.

The tissue samples surrounding first molar were homogenized and then lysed using Protein Lysis Buffer (Promega Corporation). Protein concentration was measured using bicinchoninic acid protein assay method (Nanjing Jiancheng Bioengineering Institute). The same amount of protein (30 µg) was separated using 10% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (EMD Millipore). Following blocking with 5% non-fat milk prepared with TBS-Tween 20 (0.1%) at room temperature for 2 h, the membranes were incubated with the primary antibodies aforementioned overnight at 4˚C. After washing, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies (Goat anti-rabbit IgG; cat. no. ab97051; 1:2,000; Goat anti-mouse IgG, cat. no. ab205719, 1:2,000; Abcam) aforementioned for 2 h at room temperature. SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.) was used as visualization reagent. ImageJ software 1.53 version (National Institutes of Health) was used to analyze the protein band intensity.

Total RNA was isolated using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the extracted RNA was reverse-transcribed into cDNA using SuperScript™ II Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.). The full temperature protocol for the reverse transcription was listed as follows: Primer annealing (65˚C, 5 min), DNA polymerization (45˚C, 50 min), enzyme deactivation (85˚C, 5 min). RT-qPCR was conducted using SYBR Premix Ex Taq™ II kit (Takara Bio, Inc.). The primer sequences used are as follows: GAPDH forward, 5'-ATGGGGAAGGTGAAGGTCG-3' and reverse, 5'-TCGGGGTCATTGATGGCAACAATA-3'; Bax forward, 5'-AGACAGGGGCCTTTTTGCTAC-3' and reverse, 5'-AATTCGCCGGAGACACTCG-3'; Bcl-2 forward, 5'-GTGAACTGGGGGAGGATTGT-3' and reverse, 5'-GGAGAAATCAAACAGAGGCCG-3'; caspase-3 forward, 5'-CTCGCTCTGGTACGGATGTG-3' and reverse, 5'-TCCCATAAATGACCCCTTCATCA-3'; Beclin-1 forward, 5'-ATGGAGGGGTCTAAGGCGTC-3' and reverse, 5'-TGGGCTGTGGTAAGTAATGGA-3'; LC3-II forward, 5'-GACCGCTGTAAGGAGGTGC-3' and reverse, 5'-AGAAGCCGAAGGTTTCTTGGG-3'; and P62 forward, 5'-GAGGCACCCCGAAACATGG-3' and reverse, 5'-ACTTATAGCGAGTTCCCACCA-3'. GAPDH gene was used as internal control gene. The thermocycling conditions were set as follows: Initial denaturation (98˚C, 2 min); 35 cycles for denaturation (94˚C, 30 sec), annealing (55˚C, 40 sec) and extension (72˚C, 60 sec); Final extension (72˚C, 5 min). The data were analyzed using the 2^-∆∆Cq method (19).

The data were analyzed using SPSS 25.0 (IBM Corp.). One-way ANOVAs followed by post-hoc Tukey's test were used to analyze the significant differences between the various groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the dynamic changes of autophagy and apoptosis during PDL reconstruction following OTM induction in rats, initially an OTM model was established (Fig. 1A). The levels of specific key autophagy and apoptotic proteins were measured at different time points post OTM (Fig. 1B). Moreover, the tooth movement was measured post-OTM induction and the data indicated that after 1 or 7 days of OTM, there was significant tooth movement compared with that in group 0 h (Fig. 1C and D).

The PDLCs were observed using H&E staining. PDLCs and PDL fibers in the control group were arranged in a regular fashion. With the extended treatment times, the PDL gap in the area where the pressure was exerted in the experimental groups was gradually narrowed and the local PDL fibers exhibited a wrinkled-like deformation. The PDLC arrangement was no longer regular, the blood vessels were compressed and the diameter of the tube was reduced (Fig. 2A). The number of PDLCs was slightly increased following 1 h of orthodontic force and significantly decreased following 1 and 7 days of treatment (Fig. 2B).

The morphology and quantity of osteoclasts were investigated using TRAP staining. The alveolar bone, in which osteoclasts were predominantly located, demonstrated apparent resorption lacuna (Fig. 2C). The number of osteoclasts in the pressure area was increased significantly following 1 h of orthodontic force application and reached its maximum value on day 7 (Fig. 2D).

Beclin-1, LC3-II and P62 were mainly expressed in the cytoplasm of PDLCs (Fig. 3A-C). The expression levels of Beclin-1 and LC3-II in the experimental groups reached their peak value following 1 h of treatment compared with that noted in the 0 h group (Fig. 3D). Subsequently, the expression levels of Beclin-1 and LC3-II decreased to the lowest levels on the 7th day (Fig. 3D). The changes in the expression levels of p62 in PDLCs were opposite to those noted for Beclin-1 and LC3-II. The expression levels of p62 in each experimental group were significantly lower than those noted in the 0 h group (Fig. 3D). At 1 h post-OTM treatment, the expression levels of P62 were at their lowest. Similar findings were observed by measuring the mRNA and protein expression levels of Beclin-1, LC3-II and p62. The expression levels of Beclin-1 and LC3-II were significantly increased following 1 h and 1 day of treatment (Fig. 3E and F). After 7 days, the levels of Beclin-1 and LC3-II reduced to baseline levels, possibly due to adaptation to continuous stress. However, the expression levels of p62 were inhibited following 1 h of treatment and were gradually increased following 1 day of treatment (Fig. 3E and F). After 7 days, the level of p62 returned to baseline levels, possibly due to adaptation to continuous stress.

The expression levels of Bcl-2 significantly increased after induction of OTM (1 h) and reached a peak at the 1 day time-point of OTM treatment (Fig. 4A and D). Subsequently, the expression levels of Bcl-2 were decreased following 7 days of OTM induction. However, a significant increase in the expression levels of Bax was observed only on the 7th day post-OTM (Fig. 4B and D). In addition, the expression levels of caspase-3 fluctuated during the time course. The expression of caspase-3 was significantly increased at the 1-h and 7-day timepoints following OTM induction (Fig. 4C and D). A slight increase was noted following 1 day of OTM induction (Fig. 4C and D). The protein and mRNA expression levels of Bcl-2, Bax and caspase-3 were also investigated using western blotting and RT-qPCR assays, respectively (Fig. 4E and F). Bcl-2 mRNA and protein levels, although raised, showed a reduction at 7 days, which was no longer significant for the protein levels compared with those at 0 h. Caspase-3 expression also stopped showing a significant raise at the 1 day compared with that at 0 h, reducing from the 1 h time-point. However, this level was increased again at day 7. The mRNA levels of Bax were significantly decreased and increased following 1 h and 7 days of OTM induction, respectively (Fig. 4E and F). Meanwhile, the protein level of Bax was significantly increased after 7 days.