Experiments were conducted with the ethical protocol approved by the Animal Ethics Committee at Chongqing Medical University (AECCMU-2020-004). All the methods were in accordance with the approved guidelines.
Animals and TMJ inflammation induction
Adult female Sprague Dawley rats aged 10-12weekes and weighing 350-400g were used as control to CRP knock out (CRP-/-) rats of the same age and weight. The CRP-/- rats were offered by professor Zhigang Yang from Chongqing medical school. DNA sequencing results of these rats showed that with several basic groups deficiency in exons, CRP protein expression decreased in hepatic tissue together with decline in serum CPR level (Supplemental Fig.1). Animals were housed in Experimental Animal Center of Stomatological Hospital of Chongqing Medical University, maintained in a barrier room at 25 °C with 40% humidity of 12-hour light/dark cycle, with free access to food and water available ad libitum.
Inflammation was induced by 50 μl CFA (Sigma, MO, USA) injection into the upper compartment of bilateral TMJs [29]. And rats without inflammation induction were received saline injection of the same volume. Thirty-two rats were included and divided into 4 groups. Control group was the SD rats with saline injection, and CFA group with CFA injection. CRP-/- rats injected with saline were classified as CRP group, while the ones with CFA injection were into CRP+CFA group.
Tissue harvest and disc weight measurement
The heads were hemisected after sacrificed by carbon dioxide inhalation 7 days after inflammation induction. And one side of TMJ capsules opened, exposed naked condyle and separated disc tissue. The contralateral heads were not disarticulated and fixed in situ in 4% paraformaldehyde (PFA, Affymetrix, USA) at 4°C for slides section. Separated disc were clean up carefully and weighed with excess water removed. Separated disc and synovia from the same rat were then gathered together for PCR analysis.
Paraffin section preparation and staining
The PFA-fixed hemi-heads were decalcified with 14% EDTA (pH 7.5) for up to 2 months. After gradient-decalcification, paraffin was used for embedding samples with sagittal section. Five µm thick sections were obtained before stained with hematoxylin and eosin (H&E), safranin O and fast green (Solarbio, China) to observe histological change in cartilage using standard methods. And tartrate resistant phosphatase (TRAP) staining (Solarbio, China) was performed for osteoclasts activity estimation. Sections from equivalent regions of the TMJ were compared between animals.
Histological analysis
Slides with H&E staining were used for histological analysis by 2 blinded examiners separately. Disc thickness measurement was done in anterior, intermediate and posterior band in 6 randomly selected sections every joint (N=6/group) referred to Wang's study [7]. And the extended lines of intermediate zone in cartilage were gauged for the thickness of total cartilage, fibrocartilage layers and hypertrophic chondrocytes layers with safranin O and fast green staining slides.
For inflammation score of synovial membrane, scores were semiquantitatively evaluated with following parameters: one scale for inflammatory infiltration: no infiltration = 0; discrete infiltration =1; moderate infiltration = 2; intense infiltration = 3. Another scale for synovial membrane thickness: no thickening = 0; discrete thickening =1; moderate thickening = 2; intense thickening = 3 [8]. And the total number of mononucleated cells in synovial membrane of 100*100 μm square was counted for infiltrated inflammation cells measurement (N=6/group, every joint with 6 randomly selected sections).
Immunohistochemistry
Immunohistochemical staining was performed with sections been de-paraffinized, Rehydrated. Slides were incubated with 0.3% hydrogen peroxide for 20 minutes (min), before processing with serum to reduce unspecific binding. The slides were then incubated overnight with anti-CRP 1:200 (abcam, USA), anti-IL-1β 1:100 (Bioss, China), anti-IL-10 1:150 (Bioss, China), anti-TNF-α 1:100 (Bioss, China), anti-CD68 1:100 (abcam, USA), anti-Inducible Nitric Oxide Synthase (iNOS) 1:100 (Affinity, USA), anti-receptor activator of NF-κB (RANKL) 1:100 (Bioss, China). Then slides were washed and incubated with goat anti-mouse second antibody for 30 min (Zhongshan Biotechnology, China), visualized by 3,3-diaminobenzidine tetrahydrochloride (DAB) substrate (Zhongshan Biotechnology, China) and counterstained with hematoxylin.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from disc and synovia with Trizol reagent (Invitrogen, USA) following the instructions. RNA qualification, reverse transcription and polymerase chain reaction were performed as previously described in detail [30]. The sequences of the commercially synthesized primers are listed in Supplemental Table.
Enzyme linked immunosorbent assay (ELISA)
After centrifuging the blood of rats, the serum was acquired and diluted 4000 times with double distilled water. Diluted sample was added into each well of enzyme label plate, incubated at 25 ℃ for 30 min before washed for 5 times. Then 100 μl of substrate was added into the wells, and the color was developed in dark at 25 ℃ for 10 min. After that 100 μl of termination solution was applied in each well. The optical density at 405 nm was measured with 650 nm wavelength correction.
Western-Blot analysis
Protein samples from hepatic tissue were extracted using RIPA lysis buffer (Thermo Fisher Scientific, USA). The total protein was separated and transferred to a polyvinylidene fluoride membrane. The membranes were blocked with 5% BSA in PBS for 60 min at room temperature and then were probed with anti-CRP (1:1000; Abcam, USA) or anti-β-actin (1:5000; Zhengneng, China) at 4°C overnight. Then followed by the addition of goat anti‐rabbit IgG H&L secondary antibodies (1:500; Abcam, USA) for 60 min, immunoreactive proteins were detected with a chemiluminescence kit (Millipore, USA) by enhanced chemiluminescence (Amersham Pharmacia Biotech, USA) reaction.
Statistical analysis
SPSS 23.0 software (SPSS Inc., USA) was used for data analysis. Data were expressed as mean + standard deviation (SD). The normality of data distribution was tested by Shapiroee-Wilk test, and Levene's test was used to assess homogeneity of variance. Statistical comparisons were performed using independent t-test (two groups comparison) or One-way ANOVA analysis (beyond two groups comparison) followed by multiple comparisons using the Tukey's test.