2.1 Animals and the PTOA Model
Eight-week-old male Sprague-Dawley rats (n = 24, 220–250 g) were purchased from the Animal Center of Laboratory Animal Center of Shandong University. This animal experiment was approved by the Ethics Committee on Animal Experimentation of Qingdao Municipal Hospital. Rats were housed under controlled conditions (25 ± 2°C, 70% humidity and 12-hlight-dark periods) and fed a standard diet. As previously reported, anterior cruciate ligament transection (ACLT) was performed on the right knee to cause joint instability, thereby inducing PTOA. The rats were randomized into the following four groups (n = 6/group): a sham group, an ACLT group, an ACLT + vector, and an ACLT + pcDNA-NUMB. Briefly, rats were anesthetized using 3% sodium pentobarbital via intraperitoneal injection. After sufficient disinfection, the medial skin of the patellar ligament was incised and the joint capsule was opened to dislocate the patellar bone. Flexing the knee joint exposed the anterior cruciate ligament and cut the anterior cruciate ligament. After rinsing with sterile saline, the wound was closed layer by layer and disinfected, and penicillin was injected intramuscularly to prevent infection. After 6 weeks, the rats were anesthetized with 3% sodium pentobarbital and sacrificed. The cartilage tissue was immediately taken out and embedded in paraffin for subsequent experiments. In sham control mice, a skin and capsule incision was instead conducted.
Safranin O/Fast Green staining
Cartilage tissue sections were dewaxed with 4% xylene and dehydrated with gradient alcohol. For Safranin O staining, tissues were stained with Safranin O solution (ScyTek, Logan, UT, USA). All staining was performed according to the manufacturer’s instruction, and the morphological changes were observed with an optical microscope (BX51; Olympus Corp., Tokyo, Japan).
Rat cartilage degeneration was graded for Safranin O/Fast Green-stained knee sections using the Osteoarthritis Research Society International (OARSI) scoring system. A subjective score of 0–6 was applied as previously described. Grading was performed by three blinded observers. The three grades for each section were then averaged, and the data for mice in each group were collated. Higher scores were indicative of more serious cartilage damage.
Hematoxylin-eosin (HE) staining
The cartilage tissues were taken from each rat in each group, fixed in 10% formalin, and then embedded in paraffin, and sequential serial sections were obtained. Afterward, the sections were stained with hematoxylin–eosin (HE) staining. Images were obtained using an optical microscope (BX51; Olympus Corp., Tokyo, Japan).
Human primary chondrocytes were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), streptomycin (100 mg/ml) and penicillin (100 U/ml) at 37˚C in a humidified atmosphere containing 5% CO2. Only cells within the fifth passage were used for the subsequent experiments.
Chondrocytes were cultured in 96-well plates at a density of 104 cells /well and induced with various doses of IL-1β (1, 5, or 10 ng/ml) as a cell inflammatory model, and the normal chondrocytes (NC) were used as control.
Short-hairpin NUMB (sh-NUMB) plasmid, short-hairpin BTRC (sh-BTRC) plasmid, and their respective nontargeting sequence (sh-NC) as well as NUMB and BTRC overexpression plasmid (pcDNA-NUMB and pcDNA-BTRC) and their respective nontargeting sequence (vector) were synthesized by GenePharma (Shanghai, China). Chondrocytes were grown to 70%-80% confluency in 24-well plates and then transfected with Lipofectamine 3,000 reagents (Invitrogen, CA, USA) according to the manufacturer's instructions. Following transfection for 48 h, chondrocytes were collected for subsequent experiments.
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide (MTT) assay
Cell viability was tested via MTT assay analysis. After the indicated transfection or treatment, 1 × 104 chondrocytes were added into 96-well plates, and then incubated for 72 h, followed by incubating with 0.5 mg/mL MTT (Beyotime, Shanghai, China) for 4 h. Next, the optical density (OD) value was examined via a microplate reader (Molecular Devices, San Jose, CA, USA) with a wavelength of 570 nm.
After transfection, TUNEL staining was used to detect apoptosis in chondrocytes in the presence of IL-1β (10 ng/mL). Briefly, chondrocytes seeded on glass coverslips after fixation with acetone/methanol (vol/vol) for 5 min at − 20°C. Then chondrocytes were incubated with equilibration buffer for 10 s before adding TdT enzyme for 1 h at 37°C. Subsequently, chondrocytes were incubated with reaction mixture for 60 min and cell nuclei were stained with DAPI. Immunofluorescent images/slide were observed by using fluorescence microscope(Nikon, Tokyo, Japan). The results are presented as the apoptosis indices, which was quantified as (TUNEL-positive cells)/(total cells)×100%.
Cartilage tissues and chondrocytes were collected and lysed with RIPA buffer containing protease inhibitors (Beyotime, Jiangsu, China). Equal amounts of proteins were separated with 10% SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). After blocked in 5% nonfat milk, membranes were incubated with primary antibodies against NUMB (1:1,000), BTRC (1:1,000), p-p65 (1:500), p65 (1:1,000), IκBα (1:1,000), p-IκBα (1:1,000), and GAPDH (1:1,000) at 4°C for at least 12 h, followed by incubation with HRP-conjugated secondary antibody (1:5,000) for another 2 h at room temperature. Immunoblots were visualized by an enhanced chemiluminescence detection kit (ECL kit; Millipore, Billerica, MA) under a chemiluminescence imaging analysis system (Amersham Imager 600, GE, CT, USA). Relative integrated density values were calculated using Image J software.
Chondrocytes were inoculated on polylysine-coated cover glass for 24 h, fixed with 4% paraformaldehyde for 20 min, and permeated with 0.2%Triton X-100 for 10 min. Then the cells were incubated with primary antibody (NF-κB p65) at 4°C overnight. After incubation with Alexa Fluor® 594 anti-mouse antibody (Molecular Probes, USA) for 1 h at room temperature, cells were counter-stained with DAPI (Invitrogen). Images were observed by Olympus fluorescence microscope (Nikon, Tokyo, Japan).
Enzyme-linked immunosorbent assay (ELISA)
The levels of TNF-α and IL-6 in serum and in samples of supernatants from cultured chondrocytes were measured with commercial ELISA kits (R&D Systems, MN, USA) following the instruction of manufacturer. The absorbance of samples was detected at 450 nm. Similarly, the levels of related secretory proteins such as MMP-13 and COL2A1 were also detected by ELISA kits (Sigma, St. Louis, MO, USA).
Co-immunoprecipitation (Co-IP) assay
Chondrocytes were lysed in RIPA buffer (Beyotime, Shanghai, China). Cell supernatant was collected and incubated with anti-NUMB antibody, anti-BTRC antibody or IgG at 4°C overnight. Then the mixture was incubated with 100 µL of protein A/G agarose beads (Takara Biotechnology, Dalian, China) overnight at 4℃. After incubation, the mixture was centrifuged at 3000 rpm for 5 min, and the beads were collected and washed with PBS for three times. The beads were boiled with loading buffer for 5 min and then centrifuged at 3000 rpm for 5 min to separate the proteins from the beads. The IP products were subsequently analyzed by using Western blotting.
Statistical analysis was performed with SPSS version 22.0 software. All data were presented as mean ± standard deviation (SD). Student’s t-test was performed for the comparison between two groups, and analysis of variance (ANOVA) was performed for comparison among groups. P < 0.05 was considered as statistically significant difference.