Lipopolysaccharide lps from e coli 0111 b4

Adult eels (Anguilla anguilla) of about 200g (±25g) were purchased from a commercial fishery, Valenciana de acuicultura (Puçol, Valencia). Fish were maintained in our facilities in recirculating water at 15°C under a photoperiod of 12:12 (light:dark). They were acclimated to laboratory conditions for at least 15 days before experimentation. All experiments described comply with the guidelines of the European Union Council (2010/63/EU) for the use of laboratory animals and have been approved by the Department of Environment of the Generalitat de Valencia under the reference code 2014/pesca/RGP/028. Eels were divided into 4 different tanks, 5 individuals per tank. Three different experimental challenges were carried out (1 challenge/tank), which consisted of an intraperitoneal (ip) administration of 250 μl maximum volume of either; 6 mg/kg of lipopolysaccharide (LPS) from E. coli 0111:B4 (Sigma-Aldrich), 1 mg/kg of peptidoglycan (PGN) from E. coli 0111:B4 (Invivogen) or 3 mg/kg of poly I:C (Invivogen). Control individuals were injected via the intraperitoneal route with 250 μl of saline buffer. At 12 h post-treatment eels were killed by an overdose of benzocaine (Sigma-Aldrich) and liver, spleen and head-kidney were immediately dissected under sterile conditions and frozen in liquid nitrogen.

A scanning multi-well spectrophotometer (Multiskan spectrum, Thermo Fisher Scientific, Waltham, MA, USA) was utilized to measure absorbances. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin and phosphate buffered saline (PBS) were purchased from Biowest (Nuaillé, France). Lipopolysaccharide (LPS) (from E. coli 0111:B4) and phorbol 12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The activity of transcription factors NF-κB/AP-1 was evaluated using the THP-1-XBlue^TM-MD2-CD14 cell line [obtained from Invivogen (San Diego, CA, USA)], expressing an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene, as was described previously [54 (link),59 (link)]. The complexes 1–5 were applied to the cells at the concentrations of 200 nM and 50 nM, pomiferin (at 200 nM), and a reference drug prednisone at 1 μM, respectively. Lipopolysaccharide (LPS) from E. coli 0111:B4 (Sigma-Aldrich) dissolved in PBS was used to trigger an inflammation-like reaction. The activity of the SEAP was determined by Quanti-Blue^TM reagent (Invivogen, San Diego, CA, USA) according to the manufacturer’s instructions 24 h after lipopolysaccharide stimulation. The activity of NF-κB/AP-1 was determined spectrophotometrically using the FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) at 655 nm and compared with that of the vehicle.