Animal experimental design
180 male 8-week-old Wistar rats (weight, 220±20 g) were purchased from the Laboratory Animal and medicine Center of Shandong University (Jinan, China) and were raised in the same laboratory. The animals were housed in cages in a temperature-controlled room at 24°C under a 12:12-h light-dark cycle and received food and water without restriction. The animals were adjusted to laboratory conditions for three days without any manual intervention. Then, the Wistar rats were adapted to the animal model for environmental circadian rhythm disruption for 30 min per day for four successive days before the start of the experiment.
The rats were then divided randomly into three groups (60 in each group): the control (CON) group, the circadian rhythm disturbance (CRD) group, and the circadian rhythm recovery (REC) group. The three groups were divided equally into three subgroups (n=20 each) according to the different time points at which the rats were sacrificed (4, 6, and 8 weeks). The CRD and REC rats were placed on small platforms during the procedure at the same time, as described in the subsequent section of this article. At the scheduled time, the rats in the CRD group were sacrificed, while the rats in the CRD group were allowed to stay in cages for one week to adjust their circadian rhythm back to normal. Rats in the CON group were housed in cages in the same room.
TMJ tissue collection
All rats were sacrificed after 4, 6, and 8 weeks according to their subgroups with an overdose of pentobarbital sodium, and their bilateral TMJs were excised rapidly. The TMJs were dissected, and 5 rats’ bilateral TMJs were selected randomly from each group for hematoxylin and eosin (HE) staining and immunohistochemistry (IHC). These isolated TMJs were fixed in 10% buffered paraformaldehyde. Additionally, the remaining 15 rat joints were selected to determine the BMAL-1, ERK, P-ERK, MMPs, ADAMTS and COL2 expression by Western blotting and real-time quantitative polymerase chain reaction (PCR). These TMJ specimens were dissected and flash frozen with liquid nitrogen. Two pieces of mandibular condylar cartilage from each rat were treated as one sample to guarantee that enough protein and mRNA was available for the analysis.
Animal model for environmental CRD
The modified multiple platform method (MMPM) was selected to induce CRD in this study[18]. As shown in Figure 1, the rats were placed inside a tiled glass water tank made of organic glass (145.0 cm long × 44.0 cm wide × 45.0 cm high) containing 28 narrow circular platforms (6.5 cm in diameter). The narrow platforms were set 12 cm apart from each other so that the rats could only stand on them. During the experiment, the two tanks were filled with water until there was approximately 1 cm from the surface of the water to the platform. Then, 20 rats were placed in each tank so that they could move around freely by jumping from one platform to another among the 28 platforms. Finally, the water tank was covered with iron mesh, and food and water were placed on the iron mesh. When the rats reached the paradoxical phase of sleep, they were awakened when their faces touched the water as a result of muscle atonia. Thus, CRD was achieved by depriving the rats of paradoxical sleep. The rats were placed on the platforms for 18 h per day (14:00—8:00+1 day) under controlled room (24 ±2°C) and water (18 ± 2°C) temperatures. After each 18-h circadian rhythm disruption, the animals could sleep in their individual home cages for 6 h (beginning at 8:00). The water in the tank was changed daily throughout the CRD period.
Histological staining and immunohistochemistry
Following sacrifice, condyles and articular disks from TMJs were dissected aseptically and fixed in 4% buffered paraformaldehyde for 24 h, decalcified with 10% EDTA at 4°C for 4 weeks and then embedded in paraffin. The samples were cut into 5-μm-thick sagittal sections and stained with HE. The sections were then evaluated for degenerative changes by three blinded graders according to a modified scoring system for mouse articular cartilage, with a score of zero representing unaltered cartilage and six representing severe OA. The scores for each section from three blinded graders were averaged, resulting in total scores between 0 and 6 (Table 1) [19]. Immunohistochemical staining was carried out using the standard streptavidin-peroxidase (S-P) method. For IHC, after 3% hydrogen peroxide treatment and antigen retrieval, all tissue sections were blocked at room temperature with 5% bovine serum albumin (BSA) for 30 min and then incubated with primary antibodies overnight at 4°C. A secondary antibody, biotinylated anti-rabbit IgG, was applied for 30 min at room temperature (Vector Laboratories). The percent of positively stained cells per field (positive cell %) was determined for an average of 3 fields in each slide. The immunohistochemistry results were analyzed by image capture under a microscope at 400× magnification and quantification with Image-Pro® Plus software[20].
RNA extraction and analysis by real-time quantitative PCR
For RNA extraction, TMJ tissues were harvested and cut into small pieces (< 2 × 2 mm2). The pieces were frozen immediately in liquid nitrogen and then ground into powder. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. For RT-PCR, we used a SYBR Two Step RT-qPCR Kit (Takaba, USA, Code No. RR037A) following the manufacturer’s instructions. The cycles were as follows: initial denaturation at 95°C for 30 s, followed by 40 amplification cycles of 95°C for 5 s and 60°C for 30 s. The resulting values were quantified using the comparative cycle threshold method, and the samples were normalized to GAPDH. Quantification of the relative expression levels was determined by the 2-ΔΔCT method. The primers for measuring mRNA are described in Supplement Table 1.
Chondrocyte isolation and culture
Three-week-old Wistar rats were sacrificed with an overdose of pentobarbital sodium, and their bilateral TMJs were excised rapidly. First-passage chondrocytes were used for the subsequent experiments. The chondrocytes were cultured in DMEM containing 10% fetal bovine serum. Once reaching 80% confluence, the chondrocytes were cultured and plated onto 6-well plates for further study. After 3 days of culture when the 6-well plates reached 80% confluence, the chondrocytes were transfected with an siRNA and plasmid against BMAL-1-11 under IL-6 simulation.
Small interfering RNA (siRNA) and plasmid targeting BMAL-1 for transfection under IL-6 simulation
siRNA targeting BMAL-1 and a BMAL-1 overexpression plasmid were purchased from RiboBio Biotechnology Inc. First-passage chondrocytes were transfected with siRNA or plasmid with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After transfection, the cells were seeded on a 6-well plate and cultured for 8 h. To induce catabolic stress, cells were cultured with 10 ng/μl IL-6 for another 12 h after transfection.
Protein sample preparation and Western blotting
Cell and tissue lysates were prepared using modified RIPA buffer. Total protein concentrations were determined by a bicinchoninic acid (BCA) protein assay (Beyotime, catalog P0012). Protein solutions (approximately 15 μl) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, catalog IPVH00010). The PVDF membranes with the proteins were blocked in 5% non-fat milk at room temperature for 2 h. Then, the membranes were incubated with primary antibodies, including anti-BMAL-1 (1:1000, Abcam, catalog ab231793), anti-Erk1/2 (1:1000, Cell Signaling, catalog #4695), anti-phospho-ERK1/2 (1:1000, Cell Signaling, catalog #4370), anti-ADAMTS5 (1:1000, Bioss, catalog bs3573R), anti-MMP3 (1:1000, Servicebio, catalog GB11131), anti-MMP13 (1:1000, Abcam, catalog ab39012), anti-COL2α1 (1:1000, Sigma, catalog SAB4500366), and anti-β-actin (1:2000, Servicebio, catalog GB11001). All primary antibodies were diluted with 5% TBST buffer (with 0.1% Tween 20) and incubated overnight at 4°C. After washing with TBST buffer (with 0.1% Tween 20) three times, the membranes were incubated for 2 h with secondary horseradish peroxidase-conjugated anti-mouse (1:10,000, Servicebio, catalog GB23301) and anti-rabbit (1:10,000, Servicebio, catalog GB23303) antibodies. After washing with TBST, the target proteins were detected on the membranes with an ECL detection system (Millipore, catalog MA01821). Western band images were captured digitally, and the intensity of the bands (pixels/band) was determined in arbitrary optical density units using ImageJ densitometry analysis software.
Intra-articular adenovirus injection
Adenoviruses expressing mouse Ad-NC and Ad-bmal1 plasmids were purchased from RiboBio Biotechnology, Inc. Rat TMJ articular chondrocyte cells were cultured for 3 days to reach 80% confluence and then infected with adenovirus for 2 h. For IA adenovirus injection, Ad-NC-plasmid and Ad-bmal1-plasmid (1×109 PFUs in a total volume of 10 μl) were injected into the TMJs of mice once per week for 4, 6 and 8 weeks in the CON, CRD and REC groups.
Alcian blue staining
Alcian blue (Solarbio, catalog G2542, PH2.5) was used in this experiment. Primary cultured cells were plated at a density of 8-9×104 per well in six-well plates. At the end of the indicated culture period, the cells were washed with ice-cold PBS three times, fixed at room temperature with 37% buffered formalin for 5 min, and rinsed with 0.1 N HCl. The cells were then stained at room temperature with Alcian blue for 30 min. After washing with PBS buffer, the staining results were captured by imaging under a microscope with 200× magnification and quantified with Image-Pro® Plus software.
Statistical analysis
Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc.). In the case of data with a normal distribution and/or equal variances, significant differences between two and more than three groups were determined by a two-tailed Student’s t test and one-way ANOVA followed by Bonferroni’s post hoc comparison test, respectively. P values lower than 0.05 were considered to be statistically significant. The results are presented as the mean SEM from at least three independent experiments.