Heparin sodium

For isolation of peripheral blood monocytes (PBMo), blood samples from the jugular vein of healthy turtles were collected into a 50 mL centrifuge tube pre-filled with 200 µL of 1% heparin sodium (Sangon, Beijing, China). Histopaque 1083 (Sigma) was used for gradient separation according to the manufacturer’s instruction. Isolated PBMo was washed twice with RPMI-1640 (Gibco, Grand Island, NY, USA) and resuspended in complete RPMI-1640 medium supplemented with 10% heat-inactivated newborn calf serum (Gibco), 100 units/mL penicillin and 10 µg/mL streptomycin. PBMo was cultured in 24-well plates for 12 h to allow adhesion. The adherent cells were washed once with RPMI-1640, and incubated in complete RPMI-1640 medium containing tIL-1β (expressed in the baculovirus system) at the final concentration of 200 ng/mL. Lipopolysaccharide (LPS, Sigma) was used at a final concentration of 1 μg/mL as positive control and RPMI-1640 medium was used as negative control. The mRNA transcription levels of cox-2, il-18, il-1β, casp-1 and β-actin were determined by qPCR after stimulation with tIL-1β peptide or LPS or RPMI-1640 at 1, 2 and 4 h post-treatment, respectively. Each treatment consisted of three replicates.

Rutin and 4-mercaptophenylboronic acid (MPBA) were obtained from Aladdin Reagent Co. (China). Silver nitrate, sodium borohydride (NaBH₄), NH₄HCO₃, and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Heparin sodium was provided by Sangon Biotech (Shanghai) Co., Ltd. Polyvinylpyrrolidone (PVP, Mw: 10 kDa), carrageenan, and urethane were obtained from Sigma-Aldrich Co. (United States). Endothelial cell basal medium (ECM), fetal bovine serum (FBS), and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Thermo Fisher Scientific Co. (United States).