Experiment design Effect of emodin on expression of claudin 4, claudin 5 and occludin, as well as on pulmonary dye extravasation, a marker to evaluate alveolar epithelial barrier, was detected in rats with acute pancreatitis. Time course of pulmonary edema and inflammation was recorded. Rats with acute pancreatitis were randomly allocated into pancreatitis group and emodin treatment group. Rats in pancreatitis group were injected with emodin via the external jugular vein 3 h after sodium taurocholate infusion. Rats in sham operation group were injected with normal saline at the same time point and served as a control group. Lung tissue samples were obtained 6 h after emodin injection, and maintained at 80? until assay. Blood samples were obtained from the inferior cava vein by direct puncture. Lung tissue samples were fixed in 4 neutral phosphate buffered formalin and embedded in paraffin wax for histology examination. Serum amylase activity was detected to confirm the appropriate induction of pancreatitis. Measurement of serum amylase level Serum amylase level was measured by incubating serum with 4,6 ethylidene p nitrophenyl 1 Dmaltoheptoside for 2 min at 37?, with its absorbance detected once a minute for 2 min at 405 nm by high through universal microplate assay .
Lung tissue sections were stained with hematoxylin and eosin. An experienced pathologist and a pancreatic specialist assessed tissue alterations under light microscope in a blinded fashion and scored them with Sunitinib kinase inhibitor a grading system . The grading involved measurements of inflammatory infiltration, pulmonary edema and alveolar collapse, each on a scale of 0 3, giving a maximum score of 9. Measurement of pulmonary cytokine level and MPO activity TNF and IL 6 levels in lung tissue samples were measured using a sandwich enzyme linked immunospecific assay according to its manufacturer?s instructions. Absorbance was measured at 450 nm by high through universal microplate assay. Tissue homogenate was corrected with the protein concentration and expressed as per protein in lung tissue . Sequestration of neutrophils in lung tissue samples was evaluated by measuring tissue MPO activity . Briefly, lung tissue samples were homogenized with 0.
5 hexadecyltrimethylammonium bromide in 50 mmol L phosphate buffer . Homogenate was sonicated for 10 Maraviroc s, freeze thawed three times, and centrifuged at 14 000 g for 15 min. The resulted suspension was used for assay. The assay mixture contained 20 L of supernatant, 10 L of tetramethylbenzidine , and 70 L of H2O2 . MPO activity was assessed photometrically at 630 nm. The results were corrected with the protein concentration and expressed as the activity of per protein in lung tissue . Evaluation of pulmonary edema and alveolar epithelial barrier function Severity of pulmonary edema was estimated by measuring water content in lung tissue samples.