Antibodies and reagents
IL-1β was obtained from Peprotech. A740003 (a P2X7R selective antagonist) and helenalin [a nuclear factor-κB (NF-κB) signaling pathway inhibitor] were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Abcam. Antibodies against p-P65, P65 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against P2X7R, MMP13, COL2, GAPDH were obtained from abcam. Bax, Bcl-2 and cleaved caspase 3 antibody were obtained from Proteintech. Other reagents were obtained from Sigma unless noted otherwise.
Human facet joint cartilage originally were obtained from patients with FJOA who underwent surgery in our hospital. They were divided into two groups:control group(n=5) and FJOA group(n=5). Informed consent was obtained from all individual participants included in the study. Each lumbar segment was examined by computed tomography (CT). After obtaining International Ethical Principles approval and patient consent, FJ tissues were harvested and the cartilage was visually graded for degeneration from grade 0 (normal), 1-2 (early degeneration) , 3-4 (advanced degeneration) according to the Weishuapt grading system.
FJ tissues were surgically excised from the surgical patient and were fixed immediately in 4% PFA for 48 h and decalcified for 14 days. Following dehydration and embedded in paraffin wax, a portion of tissue sections were cut for hematoxylin and eosin (H&E) staining. The other were used for immunohistochemical analysis. According to the manufacturer’s protocol, the sections were incubated with anti-P2X7 antibody (1:200), anti-COL2 antibody (1:200), then incubated with enzyme-labeled goat anti-mouse/rabbit IgG secondary antibody. Tissue sections were observed with an upright microscope (Nikon, Japan). The level of cartilage degeneration was measured by the Osteoarthritis Research Society International (OARSI) scores.
Chondrocyte isolation and culture
According to Muhammad 's protocol, primary chondrocytes were extracted from the articular cartilage mentioned above for further identification, chondrocytes between passage 0 and passage 2 were used for all experiments. Stimulus include IL-1β(10ng/ml)，A740003(0, 20,50μM) , BzATP (50μM) and helenalin(1μM).
Cell viability assay
Cell viability was performed by CCK-8 kit (Dojindo,Japan). According to the protocol, chondrocytes were firsty pre seeded in 96 well plates and stimulated with different stimulants (IL-1β/A740003), then CCK-8 dye was added into each well, the absorbance was tested multifunctional microplate detector (BioTEK, USA).
After IL-1β stimulation, the amount of TNF-a and IL-6 from cell supernatants were measured according to the manufacturer's manual(R＆D systems). Each independent experiment were performed in triplicate.
Flow Cytometry was performed in consistent with manufacturer's manual (BD Biosciences, cat. no. 556421). Briefly, cells were collected after different treatments, and the chondrocytes were incubated with Annexin V-PE and 7-AAD in the dark at room temperature, then analyzed by flow cytometry(BD FACSAriaII).
Nuclear extract preparation
After resuspended in a hypotonic buffer (Beyotime, China, cat. no. P0027), primary chondrocytes were incubated at 4 °C for 30 min. They would undergo 1%Triton X-100 and vortexed vigorously for a 10 min preincubation on ice. After centrifugation for 1 min, 10 μl of nuclear extraction buffer (Beyotime, China, cat. no. P0027) was used to resuspend. The pellets were stirred for 30 min at 4 °C followed by centrifugation for 5 min. Then the supernatants were collected as the nuclear extracts for Western blotting.
Total protein of chondrocytes was extracted with the cell lysis buffer. After loaded onto the SDS-PAGE gels (10–12%), PVDF membranes were transferred . The membranes were blocked with 5% skimmed milk at room temperature for 2 h and incubated with respective first primary antibodies at 4 °C overnight. The incubated primary antibodies were as follows: anti-P2X7R antibody (anti-rabbit,1:500), anti-Bax antibody (anti-rabbit,1:200), anti-Bcl2 antibody (anti-rabbit,1:200), anti-cleaved caspase 3 antibody (anti-mouse, 1:200), anti-MMP13 antibody (anti-rabbit, 1:500), anti-COL2 antibody (anti-rabbit, 1:500), anti-P65 antibody (Anti-rabbit, 1:500), anti-P-P65 antibody (anti-rabbit, 1:500), anti-GAPDH antibody (anti-rabbit, 1:1000).The respective secondary antibody were incubated for 2h. After washing in TBST, the protein was visualized using ultra-sensitive ECL kit (Bio-Rad).
The chondrocytes were cultured (2 x10^4cells/coverslip) in 24-well plates and treated with respective stimulation:IL-1β (20 ng/ml) and(or) A740003 for 36 h.s. Then incubated with primary antibodies against MMP13(1:200), COL2(1:200), P65(1:200), followed with Alexa Fluor 488-conjugated anti-IgG second-ary antibodies and Alexa Fluor 647-conjugated anti-IgG for 2 h and stained with DAPI for 7 min. Samples were examined with an upright microscope (Nikon, Japan).
All data were analyzed using the GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA). One-way ANOVA followed by the Tukey's test were used for statistical analysis. All data were presented as P < 0.05, P < 0.01value was considered statistically significant.