50 mm phosphate buffer

Rat kidney homogenates prepared from the kidneys of newly killed male Wistar rats, 130–180 g in weight, were mixed with or without the suspension of extracts or compounds, which were dissolved in 10% EtOH (final concentration: 0.4%). The mixtures were then incubated with 12.5 mM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes Inc., Eugene, Oregon), which was dissolved in 100% EtOH (final concentration: 0.2%), at 37 °C for 30 min. A 50 mM phosphate buffer (Wako Pure Chemical Industries, Osaka, Japan) solution at pH 7.4 was also used. DCFH-DA is a stable compound that is hydrolyzed by intracellular esterase to create a reduced, nonfluorescent compound of 2’,7’-dichlorodihydrofluorescein (DCFH). The ROS produced by the homogenates oxidizes the DCFH to a highly fluorescent 2’,7’-dichlorofluorescein (DCF). A microplate fluorescence spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT) with excitation and emission wavelengths of 460 and 530 nm, respectively, was used to track the fluorescence intensity of the oxidized DCF [23 (link)]. As a positive regulation, Trolox has been used as an important standard oxidant.

On day 50, intestinal samples were collected during deep anesthesia by intraperitoneal injection of Nembutal (Dainippon Sumitomo, Tokyo, Japan). For HE staining, specimens of the jejunum were fixed with 4% paraformaldehyde in a 50 mM phosphate buffer (Wako, Osaka, Japan), pH 7.4, for 24 h at 4 °C and stained with HE after dehydration, embedding, and slicing. The structure and morphological changes were observed and analyzed using a microscope (Olympus, Tokyo, Japan). Villus length was determined by measuring the distance from the crypt base to the villus tip using ImageJ software (Version 1.50, National Institutes of Health, Bethesda, MD, USA). The degree of inflammation was evaluated using an intestinal inflammation scoring system based on the following parameters: (1) inflammatory cell infiltration, (2) damage to the surface epithelium, and (3) irregular villous and crypt loss [22 (link)]. We also counted eosinophil infiltration in the lamina propria of the jejunal mucosa. Five to seven animals from each experimental group were evaluated, and a minimum of 15 well-oriented villi from each section were measured and observed by microscopy.