Experimental animals
A total of 50 healthy adult male Wistar rats (150 g-200 g) were selected and fed under 12-h dark and 12-h light conditions with free access to food and water. This study was approved and supervised by the animal ethics committee of TongJi Hospital in Wuhan, Tongji Medical College, Huazhong University of Science and Technology. Significant efforts were made in order to minimize both the number of animals and their suffering. All procedures were strictly conducted in accordance with the codes of International Association for the Study of Pain.
Model establishment and grouping
After the rats were anesthetized by intraperitoneal injection of 10% urethane (1 g/kg), a volume of 50 µL complete Freund’s adjuvant (CFA) (Sigma, St Louis, MO, USA) was injected into ankle articular cavity. After 4-8 h, the local inflammatory response and the pain-induced sensitization of the injection joint were observed. Rats injected with sterile normal saline (NS) were regarded as the sham group. The 85 rats were grouped as follows: sham (10 rats), MA (10 rats), MA + NS (10 rats), MA + DEX (40 rats), MA + SB203580 (5 rats), MA + Anisomycin (5 rats), and MA + Anisomycin/DEX groups (5 rats). According to the DEX concentration, the MA + DEX group was further divided into 4 groups (2.5, 5, 10, 20 µg/kg) and normal saline (NS) group, with 10 rats in each group. DEX was injected intraperitoneally on the third day after MA modeling. The changes of pain sensitivity in rats were detected 15, 30, 45, 60, 90, 120, and 150 min after administration. After induction of CFA, SB203580 (Sigma) and Anisomycin (Sigma) (5 mg/kg, dissolved in 5 mg/mL DMSO) were intraperitoneally injected for three days, and the control rats were injected with the same dose of DMSO as those used in dissolving the SB203580 and Anisomycin, with 5 rats in each group. The changes of pain sensitivity were detected 3 days later. According to the experimental groups, 5 rats in each group were killed on the 1st or 10th day after CFA injection, and then the following experiments were conducted.
Measurement of ankle circumference
The circumference of affected ankle in rats was measured to judge the degree of local swelling of ankle arthritis. In quiet state, the rats were fixed to the self-made wooden rack. The ankle was gently surrounded by ordinary inelastic cotton thread to measure its circumference.
Thermal hyperalgesia
The hot plate was maintained at 55°C ± 0.5°C and the rats were placed on it to carry on the thermal hyperalgesia. The pain threshold was determined by the paw withdrawal latency (PWL). The maximum cutoff time of each animal was 15 s. Three experiments were carried out at an interval of 5-10 min per claw. The average latency of paw withdrawal of each claw was calculated, and the average value of affected claw was subtracted from the other claw, and the obtained value represented hyperalgesia of affected claw.
Paw withdrawal threshold (PWT)
The threshold of mechanical paw withdrawal of rat soles was determined by von Frey filaments. Different weights of filaments were used to act on the central part of the soles of rats to induce claw contraction. The filaments causing claw contraction was used to represent the PWT. The maximum cutoff time of each animal was 15 s.
Hematoxylin and eosin (HE) staining
After modeling and deep anesthesia [intraperitoneal injection of 10% urethane (1 g/kg)], the rats were killed by cervical dislocation and the two-sided ankle was mutilated for conducting histopathological examination. After removing the skin of ankle, the ankle was decalcified in the decalcanizer for 12 h, and then the tissue block was embedded with paraffin and then sectioned. After that, the sections were used to perform HE staining, and the changes of synovial structure, cartilage and degree of bone damage after inflammation were observed under microscope.
Safranin-O/Fast Green staining
After mutilation, the ankle was decalcified in the 20% (v/v) ethylene diamine tetraacetic acid (EDTA) solution (pH 7.2) and then dissected in sagittal plane. The ankle was processed in the Tissue-Tek VIP 1000 tissue processor (Miles, Inc., Elkhart, IN) and embedded in a single Paraplast X-tra (Thermo Fisher Scientific Inc. (Waltham, Mass). The slices were cut into 6-µm sections and installed on the slide for performing Safranin-O/Fast Green staining to evaluate the severity of cartilage injury.
Immunohistochemistry
The tissue sections were dewaxed to hydration, reacted in 92-98°C 10 g/L citric acid-sodium citrate solution (500 mL) for 15 min, incubated with hydrogen peroxide (0.882 mol/L) at room temperature for 10 min, and washed with PBS for 3 times. Then, the sections were sealed by sealing fluid containing 100 mL/L bovine serum, incubated at room temperature for 1 h, added with primary antibodies TNF-α (1:100, ab6671, Abcam, Cambridge, MA, USA) and IL-1β (1:100, ab9722, Abcam) for incubation at 4°C overnight, followed by PBS washing for 3 times and incubation with secondary antibody IgG (1:2000, ab205718, Abcam) at 37°C for 30 min. Subsequently, the sections were developed by diaminobenzidine (DAB), washed by distilled water, re-stained, dehydrated, cleaned, sealed by neutral gum, and observed under microscope. The image was analyzed by Image-Pro Plus 6.0 software.
Enzyme-linked immunosorbent assay (ELISA)
The rats were anesthetized by inhalation of ether, and then the blood sample was taken from orbit (blood sample was collected on the 1st and 10th day, respectively). After proper treatment, the concentrations of TNF-α (RTA00) and IL-1β (RLB00) in serum were measured by ELISA kit (R&D Systems, Abingdon, United Kingdom). The rat serum was added into the ELISA plates and incubated with the coating buffer at 37℃ for 2 h and then with the 10% calf serum at 4℃ overnight. Then, the blood samples were cultured with the primary antibody at 37℃ for 2 h and the secondary antibody for 1 h, The termination solution was added after the chromatogenic reaction. The optical density of each well was measured at 450 nm.
Western blot analysis
The protein of ankle joint tissues of rats was extracted and the concentration of protein was determined according to the instructions of BCA kit (Thermo Scientific Pierce; Rockford, IL). The extracted protein was added with sample loading buffer and boiled at 95°C for 10 min. Protein sample was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% [w/v]) with the voltage change from 80 v to 120 v (wet transfer, voltage of 100 mv, 45-70 min). The polyvinylidene fluoride (PVDF) membrane was sealed by 5% BSA at room temperature for 1 h and incubated with the primary antibodies p38 (1:1000, ab31828, Abcam), Pp38 (1:1000, ab47363, Abcam), β-actin (1:5000, ab32572, Abcam) and ASK1 (1:1000, ab45178, Abcam) at 4°C overnight. After TBST washing for 3 times, the membrane was incubated with the secondary antibody IgG (1:2000, ab205718, Abcam) at room temperature for 1 h, followed by TBST washing. After that, the membrane was developed by enhanced chemiluminescence (ECL) method and imaged by Bio-OAdGelEZ imager (Bio-OAd, California, USA). The target band was analyzed by Image J software (National Institutes of Health, Bethesda, Maryland, USA). The relative expression of protein was shown as the gray value of target protein/the gray value of β-actin, with β-actin as the internal reference.
Statistical analysis
All data analyses were conducted using SPSS 21.0 software (IBM Corp. Armonk, NY, USA). All the data were consistent with normal distribution by Kolmogorov-SmiRnov test, and expressed as mean ± standard deviation. Comparison of data between two groups was conducted by t-test, while comparison among multiple groups was conducted by one-way analysis of variance (ANOVA). After the ANOVA analysis, comparisons between two data were conducted by Tukey’s-multi-comparisons test. P is a two-sided test, values of P < 0.05 were considered statistically significant.