Female 6- to 8-wk-old C57BL/6 mice weighing 15-20 g were purchased from Orient Bio (Sungnam, KyungKiDo, South Korea). All animals were bred and housed in standard shoebox cages in a climate-controlled room with an ambient temperature of 23 �� 2 ��C under a 12-h light-dark cycle for 7 d. The animals were fed standard laboratory chow, allowed water ad libitum, and randomly assigned to control or Enzalutamide supplier experimental groups. The mice were fasted for 18 h before AP was induced. Experimental design AP was induced by intraperitoneal injections of supramaximal concentrations of the stable cholecystokinin analogue cerulein (50 ��g/kg) or saline, hourly for 6 h[6]. Prior to injecting the NJ4 treatment group with cerulein, NJ4 (1 ��g/kg, 5 ��g/kg, or 10 ��g/kg, n = 6) or saline (control group, n = 6) were intraperitoneally administered (1 h before the first cerulein injection).
Mice were killed 6 h after the last cerulein injection was administered. Blood samples were taken to determine serum amylase, lipase, and cytokine levels. For histological examination and scoring, the entire pancreas and lungs were rapidly removed from each mouse and fixed in formalin. To measure tissue myeloperoxidase (MPO) activity, an indicator of neutrophil sequestration, and for real-time reverse transcriptase polymerase chain reaction (RT-PCR) studies, the pancreas and lungs were stored at -80 ��C. Histological analysis The pancreases from each treatment group were examined and semi-quantitatively described in terms of necrosis, vacuolization, inflammation, and edema.
A tissue section representing a minimum of 100 fields was examined for each sample and scored on a scale of 0-3 (0 being normal and 3 being severe disease) on the basis of the number of necrotic acinar cells and the presence of interstitial edema and interstitial inflammation. Measurement of serum amylase and lipase Blood samples to determine serum amylase and lipase were obtained 6 h after inducing pancreatitis. Mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (4 mg/kg). After anesthetization, blood was withdrawn from the heart of each mouse into a syringe. Serum amylase and lipase were measured using an assay kit from BioAssay Systems (CA).
Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) for IL-1��, IL-6, and TNF-�� were carried out in duplicate in 96-well plates coated with 100 ��L aliquots of anti-mouse IL-1��, IL-6, and TNF-�� monoclonal antibodies in phosphate buffered saline (PBS) at pH 7.4 during an overnight incubation at 4 ��C. The plates were washed in PBS containing 0.05% Tween-20 and blocked with PBS containing 10% FBS GSK-3 for 2 h. After additional washes, standards and samples were added and incubated at room temperature for 2 h.