Interleukin 6 (il 6)

Clinical data comprising baseline demographic characteristics, medical history, vital signs, the cause of infections and other clinical findings were collected prospectively. The Sequential Organ Failure Assessment (SOFA) score was calculated upon hospital admission on the basis of age, medical history, vital signs, and laboratory results. Samples of venous blood were collected in tubes containing heparin or ethylenediamine tetra-acetic acid in patients with sepsis on days 1, 3 and 7 of ICU admission, or in healthy volunteers upon enrollment. Moreover, blood samples continued to be taken on the ward if patients were discharged alive from the ICU on days 3 or 7. Blood samples were centrifuged (2000×g) for 10 min and stored at − 80 °C for further analyses.
Enzyme-linked immunosorbent assay kits were used to measure plasma ferritin (Abcam, Cambridge, MA, USA), EPO (Abcam), sTfR (R&D Systems, Minneapolis, MN, USA), hepcidin (Uscn Life Sciences, Wuhan, China), and IL-6 (USCN Life Sciences). To make the test more specific, the ratio of sTfR (measured in nmol/L) to log ferritin (ng/mL) was calculated. Hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and red blood cell distribution width (RDW) were measured by an automatic blood cell analyzer (XN-2000; Sysmex, Kobe, Japan).

ELISA was performed using rat pulmonary artery homogenates to measure MCP-1/CCL2 (Pierce), RANTES/CCL5 (Invitrogen), IL-6, TNF-α, MIP-1/CCL4 (USCN Life Sciences), and TGF-β (R&D) as described previously [17] .

Concentrations of cytokines and oxidative modification products were determined in 10% (weight/volume) lung homogenate prepared using 0.1 M ice-cold phosphate buffer (PBS, pH 7.4) by Polytron homogenizer PT 1200 E (5-times for 25 s, 1200 rpm; Kinematica AG, Switzerland). Homogenates were then frozen 3 times and centrifuged (12,000 rpm, 15 min, 4 °C). Final supernatants were then stored at −70 °C until the analysis was performed.
Concentrations of IL-6, IL-1β, IL-10 (USCN Life Science Inc., Wuhan, China), and RAGE (MyBioSource, San Diego, CA, USA) were quantified using rabbit-specific ELISA kits according to the manufacturers’ instructions. Data were expressed in pg/mL.
Protein oxidative damage was determined using OxiSelect^TM Nitrotyrosine ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed in 3-nitrotyrosine nanomole concentration (nM, 3NT). Lipid oxidative damage expressed by the concentration of thiobarbituric acid reacting substances (TBARS) was determined by OxiSelect^TM TBARS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as malondialdehyde in micromole concentration (μM MDA).
Total antioxidant capacity (TAC) was determined using an ELISA kit according to the manufacturer’s instructions (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as micromole concentration of copper reducing equivalents (μM CRE).