Oxidative Stress and Antioxidant Enzymes in Jejunal Mucosa

The jejunal mucosa samples were weighed, homogenized with chilled sterile saline (1 : 4; weight/volume), and centrifugated at 4,000 g for 15 min at 4°C to obtain the supernatant. The activities of superoxide dismutase (SOD; #A001-1) and glutathione peroxidase (GPX; #A005-1) and the level of reduced glutathione (GSH; #A006-1) in plasma and jejunal supernatant and the malondialdehyde (MDA; #A003-1-2) concentration in jejunal supernatant were measured using the commercial kits purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, Jiangsu, China). For measurement of H₂O₂ content, jejunal mucosa and plasma samples were homogenized with acetone followed by centrifugation at 8,000 g at 4°C. Then, the H₂O₂ level was detected using the titanium sulfate method in conformity with the manufacturer's descriptions (#bc3595; Solarbio; Beijing, China). Jejunal glutathione reductase (GR) activity was measured by reduced nicotinamide adenine dinucleotide phosphate- (NADPH-) dependent reduction of oxidized glutathione method as elucidated previously [26 (link)]. Jejunal thioredoxin reductase (TRXR) activity was tested using a colorimetric kit (#KA0883; Abnova, Beijing, China). Jejunal 4-hydroxy-2-nonenoic acid (4-HNE) level was measured by an enzyme-linked immunosorbent assay in accordance with the manufacturer's instruction (#E-EL-0128c; Elabscience; Wuhan, Hubei, China).