Evaluation of histomorphology of the jejunum This assay was carried out according to the previous method described by Dong et?al. into 1% (vol/vol) glutaraldehyde answer and another (1?cm??1?cm??1?cm) sample embedded into 4% buffered formaldehyde for the morphological measurements, the rest sample storied at??80?C for further study. 2.2. Evaluation of histomorphology of the jejunum This assay was carried out according to the previous method explained by Dong et?al. (2014). Jejunum samples that fixed in 1% (vol/vol) glutaraldehyde answer were examined NS13001 using a Philips 420 transmission electron microscope (Philips, Amsterdam, The Netherlands) at 80?kV. The jejunum samples fixed in 4% buffered formaldehyde were dried using a graded series of xylene and ethanol and then embedded into paraffin for histological processing according to the method explained before (Dong et?al., 2014). Ten slides of the middle site of each sample were obtained, and the images were acquired. Villus length, villus width, and crypt depth were metered, and the villus area was calculated using the following formula: for 15?min at a heat of 4?C. The supernatant was used to measure the secreted immunoglobulin A (sIgA) concentration using an ELISA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) (Lebacq-Verheyden et?al., 1972). 2.4. Concentrations of jejunum inflammatory cytokine The concentrations of the inflammatory cytokines in the jejunum, including interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF-), were calculated by ELISA assay packages according to their manufacturer’s instructions (Elabscience Biotechnology Co. Ltd, Wuhan, China). 2.5. Concentrations of jejunum digestive enzyme The jejunum samples were homogenized with a hand-held homogenizer in 1?mL of cold PBS (pH?=?7.4, 0.01?mol/L). The homogenate was centrifuged at 500 for 10?min at a heat of 4?C, and the supernatants were collected. The activity of the digestive enzymes sucrase, maltase, lactase, amylase, lipase, and chymotrypsin were determined according to the manufacturer’s instructions of Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). 2.6. Measurement of redox status Jejunum samples were homogenized NS13001 in 0.9% sodium chloride solution on ice and then centrifuged at 3,500 for 15?min at a heat of 4?C. Both serum and jejunum supernatant answer were used to measure the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), and malondialdehyde (MDA) according to the manufacturer’s instructions of corresponding assay packages (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein content was tested with a bicinchoninic acid (BCA) protein assay kit purchased from Nanjing Jiancheng Bioengineering Institute. 2.7. Measurement of jejunum mitochondrial redox status Jejunum mitochondria was isolated using the mitochondria isolation kit (Solarbio, Beijing, China). The levels of protein content, manganese superoxide dismutase (MnSOD), GSH-Px, GSH, and -glutamylcysteine ligase (-GCL) were calculated according to the manufacturer’s instructions with the corresponding assay packages (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The ROS concentration was measured using a ROS assay kit bought from the Nanjing Jiancheng Institute of Bioengineering. Briefly, NS13001 the mitochondria were incubated with 10?mol/L of dichlorodihydrofluorescein diacetate (DCFH-DA) and 10?mmol/L of DNA stain Hoechst 33,342?at 37?C for 30?min. Then the DCFH fluorescence of the mitochondria was measured at an emission wavelength of 500?nm and an excitation wavelength of 525?nm with a fluorescence reader (SpectraMax Gemini EM; Molecular Devices, Franklin Lakes, NJ, USA). The results were expressed as the mean DCFH-DA fluorescence intensity over that of the control. The mitochondrial membrane potential (MMP) level was calculated using an MMP assay kit bought from the Nanjing Jiancheng Institute of Bioengineering. Briefly, the mitochondria were loaded with 1??JC-1 dye at 37?C for 20?min, and then analyzed, after washing, by a fluorescence microscope. The MMP was calculated as the increase in ratio of green and reddish fluorescence. The results were calculated as the ratio of the fluorescence of aggregates (reddish) to that of the monomers (green). The number of apoptotic cells and necrotic cells was tested by an Alexa Fluor 488 Annexin V/lifeless Cell Apoptosis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and carried out according to their manufacturer’s instructions. Protein carbonyls (PC) and 8-hydroxy-2-deoxyguanosine (8-OHdG) in the jejunum sample were calculated Efnb2 using their respective ELISA assay kits and carried out following the manufacturer’s instructions (Nanjing Jiancheng Institute of.