Raw264.7 cells

RAW264.7 cells and C2C12 cells were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; 25 mM glucose; Gibco, USA) containing 10% FBS, 1% penicillin/streptomycin, and with 20 ng/mL RANKL at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. On the third or fifth day, the cells were cultured with RANKL-free and glucose-free medium for 12h. Then, the medium was collected for indirect co-culturing with C2C12 cells. The medium from RAW264.7 cells without RANKL induction was also collected as control.
C2C12 myoblasts were cultured in DMEM supplemented with 10% FBS (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. On the second day, the medium was changed to DMEM with 2% horse serum (Gibco, USA) to induce differentiation for an additional 4 days. The medium was replaced every two days.
The differentiated C2C12 myoblasts (10⁴ cells/well) were cultured with the conditioned mediums (CMs) containing 40% medium collected from RAW264.7 cells (non-RANKL-treated, CCM; RANKL-treated for 3 days and 5 days, CM3 and CM5) and 60 mM of glucose (n = 6) for 24 h in a 24-well plate.

Mouse mononuclear macrophage RAW264.7 cells were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing fetal bovine serum and 10% penicillin-streptomycin in a CO2 incubator at 37°C. RAW264.7 cells in logarithmic growth phase were inoculated into a 96-well plate for 24 h before treating with different concentrations (0, 50, 100, 200, 400, 600, 800, 1600, and 3200 μg/mL) of TDXD solution. Cell proliferation was determined by CCK-8 assay (Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the instruction manual. Using a Benchmark microplate reader (Bio-Rad, Hercules, CA, USA), the absorbance value of each well was measured at 450 nm. Subsequently, in further experiments, TDXD concentrations of 400, 600, and 800 μg/mL were used for low-, medium-, and high-dose groups, respectively.

MTT assay was used to assess the cell viability of tiamulin and the target compounds on the viability of RAW 264.7 macrophages. RAW 264.7 cells were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). DMEM supplemented with 10% FBS containing RAW 264.7 cells at a density of 1.0 × 10⁵ cells per well were added into 96-well plates and incubated at 5% CO₂ and 37 °C for 24 h. Finally, the absorbance at 490 nm was recorded using a microplate spectrophotometer (BIO-TEK Instrument Inc, USA). The method was performed as described in reference³² (link).

Polyethylene glycol (PEG) coated ferrous oxide magnetic nanoparticles (USPIO) with carboxyl groups on the surface, provided by Dongna Biotech. Co. (Nanjing, China). 1-Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sulfo-N-hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). Mouse anti-rabbit IL-6 monoclonal antibody and a nonspecific IgG antibody were purchased from Biosynthesis Biotech. Co. (Beijing, China). Borate-buffered saline (BBS) and phosphate-buffered saline (PBS) were obtained from Leagene Biotech. Co. (Beijing, China). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Co. (Carlsbad, USA). Lipopolysaccharide (LPS) was acquired from Sigma Co. (St Louis, USA). RAW264.7 cells and human umbilical vein endothelial cells (HUVEC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).