Il 1β

The CCK-8 assay (Dojindo Molecular Technologies, Inc.) was performed to test cell viability. Following treatment with 8 nM paclitaxel (APeXBIO Technology LLC), 20 ng/ml recombinant human IL-1β (Sangon Biotech Co., Ltd.), 0.1 µg/ml tunicamycin (Beijing Solarbio Science & Technology Co., Ltd.), paclitaxel + IL-1β, paclitaxel + tunicamycin and blank control, then 2x10³ A549 cells were seeded into 96-well plates and incubated at 37˚C with 5% CO₂ for 48 h. At 0, 12, 24 and 48 h, 10 µl CCK-8 reagent was added to each well and incubated with the cells for 2 h. Absorbance was measured at 450 nm by a microplate reader (Thermo Fisher Scientific, Inc.).

Acadesine (AICAR, A838530), quercetin (Q817161), etoricoxib (E831542), compound C (D864421) were all purchased from Shanghai MACKLIN; TNF-α (Cat no. SEKR-0009), IL-1β (Cat no. SEKR-0002), IL-6 (Cat no. SEKR-0005) content was detected by ELISA kit (Solarbio, Beijing, China). Antibodies: Iba1 Rabbit mAb (Cat no. 17198), AMPKα Antibody (Cat no. 2532), phospho-AMPKα (Thr172) (40H9) Rabbit mAb (Cat no. 2535), JNK (Cat no. 9252), p-JNK (Thr183/Tyr185) Cat no. 9251, ERK, p-ERK (Thr202/Tyr204) (Cat no. 4377), p38 MAPK Antibody (Cat no. 9212) and p-p38 (Thr180/Tyr182) (Cat no. 9212), and HRP-labeled secondary antibody were all purchased from Cell Signaling Technology.

Vectors and oligonucleotides were synthesized by Guangzhou RiboBio Co., Ltd. The following vectors and oligonucleotides were used for transfection: miR-17-5p mimic (miR-17-5p, 5'-CAAAGUGCUUACAGUGCAGGUAG-3'; 50 nM), mimic negative control (miR-NC, 5'-UCGCUUGGUGCAGGUCGGGAA-3'; 50 nM), miR-17-5p inhibitor (in-miR-17-5p, 5'-CUACCUGCACUGUAAGCACUUUG-3'; 100 nM), inhibitor control (in-miR-NC, 5'-CAGUACUUUUGUGUAGUACAA-3'; 100 nM), EZH2 overexpression vector (EZH2; 50 nM), empty overexpression vector (pcDNA; 50 nM), small interfering RNA (si-RNA) targeting EZH2 (si-EZH2, 5'-GAGGGAAAGUGUAUGAUAATT-3'; 100 nM), siRNA control (si-NC, 5'-GGGAAAGAGUAUAUAGUGATT-3'; 100 nM), miR-19b-3p mimic (miR-19b-3p, 5'-UGUGCAAAUCCAUGCAAAACUGA-3'; 50 nM) and miR-19b-3p inhibitor (in-miR-19b-3p, 5'-UCAGUUUUGCAUGGAUUUGCACA-3'; 100 nM). At 70% confluency, cells were transfected using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After transfection for 48 h at 37˚C, chondrocytes were treated with 10 ng/ml IL-1β (Beijing Solarbio Science and Technology Co., Ltd.) for 24 h at 37˚C.

A total of 36 male newborn (weight, 5–6 g) Sprague-Dawley rats, which were all reared at room temperature under 12/12 h day/night cycles, were purchased from the Experimental Animal Centre of the Kunming Medical University (Kunming, China) for chondrocyte extraction. Briefly, after rats were sacrificed by cervical dislocation without anesthesia, articular cartilage was harvested from the knee joints. The cartilage tissue was cut into 1–3 mm³ pieces followed by digestion with 2 mg/ml collagenase II (Sigma-Aldrich; Merck KGaA) for 3 h at 37°C. Finally, the digested chondrocytes were suspended in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% penicillin/streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) and 10% FBS (Zhejiang Tianhang Biotechnology Co., Ltd.) at 37°C with 5% CO₂ in a humidified incubator. The chondrocytes adhered to the plate after 2–3 days of culture, at which point the tissue pieces were discarded and the medium replaced. The cells were cultured for three passages for chondrocyte identification and use in subsequent experiments. IL-1β (10 ng/ml; Beijing Solarbio Science & Technology Co., Ltd.) was used to stimulate chondrocytes to establish the in vitro OA model as previously described (21 (link)). The present study was approved by the Ethics Committee of Qujing First People's Hospital (approval no. 19-025).

ARPE-19 cell culture medium was collected into a centrifuge tube and centrifuged at 1,000 x g for 10 min at 4˚C. Subsequently, the concentration of TNF-α (cat. no. SEKH-0047; Beijing Solarbio Science & Technology Co., Ltd.), IL-1β (cat. no. F01220) and IL-6 (cat. no. F01310; both from Shanghai Xitang Biotechnology Co., Ltd.) was measured by ELISA kits according to the manufacturer's instructions. The absorbance of each well was measured using a microplate reader at 450 nm. The concentration of nesfatin-1 was determined with a commercially available ELISA kit (cat. no. JL19919-96T; Shanghai Jianglai Biological Technology Co., Ltd.) in accordance with the manufacturer's instructions.