Bacterial lipopolysaccharides lps

Undenatured ethanol, chloroform and glacial acetic acid were purchased from HmbG chemicals, Hamburg, Germany. Iodine potassium iodide, ferric chloride, sodium hydroxide, sulphuric acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), β-mercaptoethanol in PBS, sullforhodamine B (SRB), phorbol 12-myristate 13-acetate (PMA), trichloroacetic acid (TCA), Trizma base, bacterial lipopolysaccharides (LPS), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA. RPMI 1640 (Gibco), 1X antibiotic (Gibco), and foetal bovine serum (FBS) were purchased from Thermo Fischer Scientific, USA.

Cell culture materials (DMEM/F-12, L-glutamine, Hanks’ balanced salt solution, 0.05% trypsin, and antibiotic/antimycotic) were purchased from Thermo Fisher. Fetal bovine serum (FBS) was obtained from Atlas Biologicals. Benzyl cinnamate (cinnamein), Griess reagent, bacterial lipopolysaccharides (LPS) and polyinosinic-polycytidilic acid (poly IC) were procured from Sigma-Aldrich (St. Louis, MO).

Synovial tissues were obtained from 10 OA patients (4 females and 6 males; median age was 73.3 [77.5–69.5] years old) and 10 OA-DB patients (2 females and 8 males; median age was 74.6 [83.4–65.8] years old) who underwent joint replacement surgery and gave informed consent. This study was reviewed and approved by the Local Ethics Committee. Samples were subsequently embedded in paraffin for obtaining 4 µm-thick histological section. For in vitro experiments, we used the immortalized cell line of monocytes TPH-1 that was maintained in Roswell Park Memorial Institute Medium (RPMI)-1640 (ThermoFisher, Madrid, Spain) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin (Lonza, Basel, Switzerland). TPH-1 were differentiated into macrophages after treatment with phorbol-12-myristate-13-acetate (PMA; 500 nM) (Sigma-Aldrich, St Lois, USA) for 3 h [8 (link)]. Thereafter, macrophages were incubated in RPMI-1640 2% FBS with 1 g/l glucose (normal glucose, NG) or 4.5 g/l (high glucose, HG) and stimulated with bacterial lipopolysaccharides (LPS; 1 ug/ml) (Sigma-Aldrich, San Luis, MO, USA), a classical inductor of M1-like macrophages [14 (link), 29 ], in the presence or absence of a slow-releasing H₂S donor, GYY-4137 (500 µM) (SantaCruz Biotechnology, Heidelberg, Germany), based on previous research [46 , 66 (link)].