Dexamethasone d4902

Dexamethasone (D4902) and LPS (L2880, from E. coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-acetylcysteine (A9165-25G) was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Chloral hydrate (R24095) was acquired from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Wright–Giemsa Stain solution was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The other reagents used were of analytical grade.

The rats were habituated to the various experimental procedures daily for two weeks before the experiment began. The eighteen animals were randomly assigned to three groups: control (CON; n = 5), DEXA placebo (DEX; n = 6), and DEXA supplemented with vitamin D₃ (DEX + VD; n = 7). The experiment lasted for 28 days. Throughout this time, the CON group was treated intraperitoneally with saline, and the two other groups were treated with dexamethasone at 2 mg/kg/day (dexamethasone D4902, DEXA, Sigma-Aldrich, St Paul, MN, USA) in the same manner. The DEX + VD group was orally supplemented with vitamin D₃ (Juvit D3, PPF HASCO-LEK. SA., Wrocław, Poland) at 600 IU/kg/day, and the DEX group was orally supplemented with vegetable oil as a placebo. Blood was collected on Day 1 and Day 28 of the experiment from the tail vein under isoflurane anesthesia. On Day 29 of the experiment, all animals were sacrificed using decapitation. The brains were collected, and the hippocampus was dissected, weighed, frozen in liquid nitrogen, and stored at −80 °C for further analysis.

HEK-293, C3H10T1/2, and C2C12 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The above cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), which contained 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cell culturing dishes, flasks, 6-well plates, and 24-well plates were incubated at 37 °C with 5% CO₂. DMEM, FBS, and trypsin were purchased from Gibco (Chinese branch). BMP9 (sc-514211), ALP (sc-271431), RUNX2 (sc-12488), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-32233) antibodies, retinoic acid receptor beta (RARβ) specific inhibitor Le135 (sc-204053), and ATRA (sc-200898) were bought from Santa Cruz Biotechnology (Chinese branch). Osteopontin (OPN, ab8448) and RARβ (ab53161) antibodies were purchased from Abcam (Chinese branch). Serpina3n (AF4709) antibody was sourced from R&D systems (Chinese branch). Dexamethasone (D4902) was obtained from Sigma-Aldrich (Chinese branch). Dylight 594 conjugated secondary antibody (A23410) was purchased from Abbkine (Chinese branch). Alkaline Phosphatase Assay Kit (C3206) and Alizarin Red S (ST1078) were bought from Beyotime (Shanghai, China).

Dulbecco’s modified Eagle’s medium, calf serum, fetal bovine serum (FBS), penicillin–streptomycin, and phosphate-buffered saline were obtained from Gibco BRL (Grand Island, NY, USA). Antibodies against-β-actin (sc-47778), type 2 deiodinase (DIO2; sc-98716), and uncoupling protein 2 (UCP2; sc-6526), and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against voltage-dependent anion channel (VDAC; 4661s) was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against UCP1 (ab23841) and total oxidative phosphorylation (OXPHOS) complex (ab110413) were purchased from Abcam (Cambridge, MA, USA). Antibody against total myosin heavy chain was purchased from Developmental Studies Hybridoma Bank (Iowa city, IA, USA). 3-Isobutyl-1-methylxanthine (IBMX, l7018), withaferin A (WFA; W4394), withanolide A (WNA; W2145), and dexamethasone (D4902) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Radioimmunoprecipitation assay buffer (89900) and protease- and phosphatase-inhibitor cocktails (78440) were purchased from Thermo Scientific-Pierce (Rockford, IL, USA).