Tnf α

The TMJ synoviocytes were assigned to control (N), TNF‐α + CHX group (TC, induction by 10 ng/mL TNF‐α [Sino Biological] and 10 μg/mL cycloheximide [CHX, Abmole]), TNF‐α + CHX + SM164 group (TCS, induction by 10 ng/mL TNF‐α, 10 μg/mL CHX, and 10 ng/mL SM164 [Medchemexpress]; synoviocytes were challenged with 10 ng/mL SM164 for 2 h before induction with TNF‐α + CHX), TNF‐α + CHX + Nec‐1 group (TCN, induction by 10 ng/mL TNF‐α, 10 μg/mL of CHX, and 40 μM Nec‐1 [Medchemexpress]; synoviocytes were challenged with 40 μM Nec‐1 for 1 hour before induction by TNF‐α + CHX). After 6 h, the conditioned media (CM) in N, TC, and TCN groups were centrifugated (1000 g, 5 min), collected and stored (−80°C).
As described previously,
²⁵ (link) the chondrocytes were prepared from condylar fracture tissue, cultured into passage 3, then seeded into plates. Before treatment, TMJ synoviocytes‐CM (N‐CM, TC‐CM, or TCN‐CM) were diluted at 1:1 with medium. Gene expression was analysed based on samples harvested at 6, 12 or 24 h of treatment.

The 3T3-L1 cell line used in this study was purchased from American Type Culture Collection (ATCC, VA, USA). 3T3-L1 was cultured in DMEM/High Glucose (Hyclone, PA, USA) with 10% Fetal Bovine Serum (Gibco, NY, USA). 3T3-L1 cells were seeded in 24-well plates (4 × 10⁵/cm²). After 24 h, we treated the 3T3-L1 with TNFα (MedChemExpress, Monmouth Junction, USA) at the concentration of 1 ng/mL for 5 days to induce 3T3-L1 insulin resistance. We treated 3T3-L1 cells with TNFα and DHM (1 μM, Sigma Chemical Inc., Louis, MO, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance. We treated 3T3-L1 cells with TNFα, DHM, and Compound C (the AMPK inhibitor, 5 μM, MedChemExpress, Monmouth Junction, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance through AMPK. We treated 3T3-L1 cells with TNFα, DHM, and STO-609 (the CaMKK inhibitor, 10 ng/mL, MedChemExpress, Monmouth Junction, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance through CaMKK. We treated 3T3-L1 cells with TNFα, DHM, and U73122 (1 μM, MedChemExpress, Monmouth Junction, USA) for 5 days to demonstrate DHM ameliorated inflammation-induced insulin resistance through the PLC-IP3 receptor pathway.

The C2C12 cell line used in this study was purchased from American Type Culture Collection (ATCC). C2C12 cells were cultured in DMEM/HIGH GLUCOSE (Hyclone) with 10% foetal bovine serum (Gibco). C2C12 cells were seeded in 24‐well plates (4 × 10⁵ /cm²). After 24 h, we treated the C2C12 cells with TNF‐α (MedChemExpress, Monmouth Junction, USA) at the concentration of 1 ng/ml for 7 days to induce C2C12 cell muscle atrophy. Dissolve 3.2 mg DHM into 10 ml DMSO to prepare a 1 mM DHM solution. In C2C12 cell experiments, the DHM solution was mixed into the cell culture medium at a ratio of 1/1,000. We treated C2C12 cells with TNF‐α and DHM (1 μM, purity ≥ 98%, Sigma Chemical Inc.) for 7 days to demonstrate DHM resisted inflammation‐induced muscle atrophy. We treated C2C12 cells with TNF‐α, DHM and Compound C (5 μM, MedChemExpress) for 7 days to demonstrate DHM resisted inflammation‐induced muscle atrophy through AMPK. We treated C2C12 cells with TNF‐α, DHM and STO‐609 (10 ng/ml, MedChemExpress) for 7 days to demonstrate DHM‐resisted inflammation‐induced muscle atrophy through CaMKK. We treated C2C12 cells with TNF‐α, DHM and ryanodine (100 nM, MedChemExpress) for 7 days to demonstrate DHM resisted inflammation‐induced muscle atrophy through the ryanodine receptor.