The rats were provided by the Experimental Animal Center of Shandong University. All rats were sacrificed by cervical dislocation after the experiment. The processes concerning animal use were in compliance with the relevant regulations of the National Institutes of Health and approved by the Animal Care and Use Committee of Shandong Provincial Hospital affiliated with Shandong University. Great efforts were made to minimize the suffering of conscious animals.
Establishment of osteoarthritis chondrocyte model
Cartilaginous tissue was separated from the Wistar rats borned within 3 days. The cartilage tissue was then digested with 0.2% type II collagenase overnight at 37°C. The following day, the digested cells were passed through a 75μm filter and centrifuged at room temperature at 800 rpm for 4 min. Finally, the chondrocytes were incubated in culture flasks with 10% fetal bovine serum in DMEM/F12 medium containing 10% fetal bovine serum (FBS) added. Chondrocytes were treated with IL-1β (10 ng/mL) (PeproTech) for 24 h to establish the OA chondrocyte model.
Experimental groups and drug administration
The incubated cells were randomly divided into the following groups: Control group, IL-1β group, IL-1β + O3 group, Control + O3 group, IL-1β + O3 + GW9662 group and IL-1β + GW9662 group. The cells were exposed to of O3 for 30 min at a certain concentration which measured using the O3 analyzer purchased from Perma Pure Inc (model MD-050-12-f-4, Perma Pure Inc). Then cells were pretreated with the PPARγ inhibitor GW9662 (20nM/mL, MCE Technologies) for 12 h prior to O3 treatment.
Cell viability assay
The viability of cells was assessed using cell counting kit-8 assay (CCK-8 kit) (Dojindo Laboratories). Briefly, chondrocytes (6000/well) were seeded in 96-well platesand incubated at 37°C for 24 hours. After being treated with IL-1β for 24 hours, the cells were exposed to O3 at concentrations of 30, 50 or 70μg/mL for 30 min. Finally, 10μL of CCK-8 kit solution was added to each well and plates were incubated at 37°C for 2 h. Absorbance was measured at 450 nm using a microplate reader.
Western blot analysis
All proteins were extracted using a radio immunoprecipitation assay (RIPA) lysis buffer with protease and phosphatase inhibitors. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Protein samples were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk solution for 1 h at room temperature. The membranes were then probed with anti-PPARγ (sc-7273, 1:200, Santa), anti-mTOR (#2983, 1:1000; Cell Signaling Technology), p-mTOR (#5536, 1:1000; Cell Signaling Technology), anti-ULK1 (#8054, 1:1000; Cell Signaling Technology), anti-LC3II (ab48394, 1:2000, Abcam), anti-P62 (ab56416, 1:2000; Abcam), anti-Beclin-1 (ab62557, 1:1000; Abcam) and anti-β-actin (1:2000; Zhongshan Golden Bridge Biotechnology) overnight at 4°C. The following day, membranes were washed three times with TBST and incubated with goat anti-rabbit or goat anti-mouse antibody IgG (H+L)-HRP (1:5000; Wuhan Sanying) for 1 hour at room temperature. Finally, the membranes were visualized using the enhanced chemiluminescence substrate LumiGLO (Millipore, MA, USA).
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from chondrocytes using TRIzol reagent (Takara, Shiga, Japan). cDNA was amplified using a PrimeScript RT reagent kit (RR047A; Takara). mRNA expression was measured with qRT-PCR using a SYBR® GrenERTM SuperMix (Takara) with the following conditions:denature at 95℃for 30 s, anneal at 60℃for 30s, extend at 95℃for 5 s. The primer sequences were as follows: β-actin, forward, 5′-GGGAAATCGTGCGTGAC-3′ and reverse 5′-AGGCTGGAAAAGAGCCT-3′; IL-6,forward,5′ATTGTATGAACAGCGATGATGCAC-3′, and reverse 5′-CCAGGTAGAAACGGA ACTCCAGA-3′; TNF-α,forward 5′-TTCCAATGGGCTTTCGGAAC-3′ and reverse 5′-AGACATCT TCAGCAGCCTTGTGAG -3′;MMP-13, forward 5′- TGATGATGAAACCTGGACAAGCA-3′ and reverse5′- GAACGTCATCATCTGGGAGCA-3′; MMP-3, forward 5′TGATGGGCCTGGAAT GGTC-3′and reverse 5′-TTCATGAGCAGCAACCAGGAATAG-3′. β-actin was used as the internal control, and the level of gene expression was analyzed using the 2-△△Ct method.
The cells were fixed with 4% paraformaldehyde for 30 min. Before blocking with goat serum, the cells were permeabilized with 0.3% Triton X-100. The chondrocytes were then incubated with anti-LC3 (ab48394, 1:100, Abcam) and anti-P62 (ab56416, 1:100; Abcam) overnight at 4°C. The following day, cells were incubated with secondary antibody (1:200; Wuhan Sanying) at room temperature for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with anti-fade medium. The stained chondrocytes were observed using immunofluorescence microscopy.
Transmission electron microscopy (TEM)
The cells were then gathered using a cell scraper and centrifuged to form cell clumps. The cell clumps were fixed with ice-cold 3% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in osmium tetroxide, embedded in Epon epoxy resin. The samples were then cut into ultrathin sections and further stained using uranyl acetate and lead citrate. Finally, the ultrathin sections were viewed using transmission electron microscopy.
SPSS 22.0 software (IBM Corp. Armonk, NY, USA) was applied for data analysis. Measurement data were described as mean ± standard deviation (SD). The differences among multiple groups were analyzed using one-way analysis of variance (ANOVA), followed by pairwise comparisons using Tukey’s multiple comparisons test. The P value was calculated using a two-tailed test, and P < 0.05 was considered to indicate a statistically significant difference.