Tak 242

Chondrocytes were digested from joints of 2‐3‐week‐old male SD rats.20 Briefly, rats were euthanized and immersed into 75% alcohol for 5 minutes. Articular cartilage was resected, digested with 0.25% Trypsin‐EDTA for 30 minutes and collagenase II (Gibco) for 4 hours at 37°C and filtrated through the 200‐mesh strainer. For culture, chondrocytes were seeded at density of 1.8 × 10⁴ cells/cm² in high‐glucose DMEM with 1% penicillin/streptomycin (Solarbio) and 10% FBS (Gibco) in a 37°C and 5% CO₂ incubator. The phenotype at P1 was identified by typical morphology and immunostaining of type 2 collagen (COL2A1; Abcam) using Inverted Ti‐E fluorescence microscope (Nikon). For experiments, unless otherwise mentioned, chondrocytes (passage 3 to 5) were seeded at density of 3 × 10⁴ cells/cm² and incubated with amurensin H for 2 hours, then, stimulated by 10 ng/mL human IL‐1β (PeproTech) in DMEM with 2.5% FBS. Besides, BAY61‐3606 (Syk inhibitor, Sigma) or TAK‐242 (TLR4 inhibitor, Solarbio) were used to confirm TLR4 signalling.

According to the previous study (Ding et al., 2015 (link)), OB-6 cells were purchased from the Cell Bank of CAS Shanghai Institute of Biological Sciences (Shanghai, China). DEX was used to pretreat cells for 24 h, and the effect of DEX at different concentrations (0.25, 0.5, 1, 2, 5 μM) was observed using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the IC50 values were calculated. Then the cells pretreated with 1 μM DEX were treated with 0, 0.25, 0.1, 1, or 5 μM Ato for 72 h (Yan et al., 2015 (link)). In addition, at the same time of 0.1 μM Ato treatment, cells were transfected with miR-186 inhibitor or the NC (GenePharma) according to Lipfectamine^TM 2000.
According to the previous studies (Muller et al., 2017 (link); Pourgonabadi et al., 2017 (link)), miR-186 inhibitor-treated cells were transfected with 25 μM TAK242 or dimethyl sulfoxide (DMSO) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 72 h for subsequent experiments.