Dexamethasone (dex)

Murine osteoblast precursor cell line MC3T3-E1 was obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). Cells were maintained in α-MEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), 50 µM ascorbate, 1% penicillin-streptomycin, and 10 mM β-glycerophosphate in a humidified atmosphere with 5% CO₂ at 37 °C. Cells at the third or fourth passage were used in the experiments. For dexamethasone treatment, cells were cultured in α-MEM supplemented with 1% FBS for 24 h and then stimulated with different concentrations of dexamethasone (0, 10⁻⁸, 10⁻⁷, 10⁻⁶, and 10⁻⁵ M; Macklin, Shanghai, China) (Belka, Nickel & Kurth, 2019 (link)).

Sheep preadipocytes were isolated from the tail fat of a 70-day-old fetus in Hu sheep as described by Cai et al. [17 ]. The cells were seeded in 6-well plates overnight and cultured in DEME/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 2% penicillin/streptomycin at 37 °C in a humidified 5% CO₂ incubator. The following day, cells were cultured in a new fresh medium containing the PDGF-D-overexpression lentiviral vector (Genechem, Shanghai, China) for 12 h. Preadipocytes were then cultured in new differentiation medium containing 10% FBS, 1 μM dexamethasone (Macklin, Shanghai, China), 0.5 mM isobutylmethylxanthine (Macklin), and 10 mg/mL insulin (Macklin) for 2 days, followed by 10 mg/mL insulin alone for 2 days. Virus without PDGF-D overexpression served as negative control. The date that cells were cultured with differentiation medium was set as the first day (1 d).

The dried over-ground portion of C. japonicum, collected in Hubei province (batch No. 201027), was purchased from Anhui Guanghe Medical Technology Co., Ltd. (Bozhou, China) and authenticated by Prof. Zhong Li from Guangdong Pharmaceutical University.
Acetonitrile, methanol and ethanol were provided by Merck Chemicals (Darmstadt, Germany). Fluorescein sodium salt (FL), methylglyoxal (MGO), fructose, bovine serum albumin (BSA), 2,2-azobis (2-methyl-propionamidine) dihydrochloride (AAPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-car-boxylic acid (Trolox^®) were supplied by Macklin (Shanghai, China). Macroporous resins (D101, HPD-100, HPD-500, HPD-600, HP-20, DM130, NKA-9, and AB-8) were obtained from Beijing Inluck science and technology Co. Ltd. (Beijing, China). LPS from Escherichia coli O55:B5 was supplied by Sigma (L2880; Sigma, Saint Louis, MO, USA). Dexamethasone was supplied by Macklin (Shanghai, China).

HTMCs were purchased from BNCC (338506, Shanghai, China), which were authenticated by STR profiling. We confirmed that no mycoplasma contamination was detected by regular examinations. Cells were cultured in Dulbecco’s Modified EagIe’s Medium: F-12 (DMEM/F12) (Cytiva, HyClone Laboratories, Logan, UT, USA) containing 10% foetal bovine serum (BI, USA) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Gibco, Life Technologies Corporation, NY, USA) at 37°C and 5% CO₂. To identify the characteristics of HTMCs, cells were treated with 500 nM DEX (Shanghai Macklin Biochemical Co., Ltd, China) for 7 days, and the expression of myocilin was evaluated by WB (He et al., 2019 (link)). For drug testing, tBHP (Damas-beta, China) and MET (Sigma-Aldrich, St. Louis, USA) were dissolved in DMEM/F12. HTMCs were pre-treated with tBHP solution for 1 hr to induce oxidative damage, followed by 24-hr incubation in normal culture medium containing MET at certain concentrations.