And ChemicalsHU210 [(6aR)-trans-3-(1,1-Dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl -6H-dibenzo[b,d]pyran-9-methanol], AM251 [(N-(Piperidin-1-yl)-5-(4-iodophenyl)-1- (2,4-dichlorophenyl)- 4-methyl-1H-pyrazole-3-carboxamide)], were purchased from Tocris (Tocris-Bioscience, Ellisville, MO, USA). Both chemicals were dissolved in a solvent consisted of ethanol, Tween80 and normal saline (NS), volume ratio 1:1:18, to concentration 1022M, at 37uC using an ultrasonicator, and then were further diluted in NS to 1025 M, and again to 1027M with perfusion fluid just before use under the conditions that were determined in pilot study [22]. The modified Krebs-Ringer solution was composed of 117.5 mM NaCl, 4.7 mM KCl, 2.4 mM CaCl2, 1.1 mM MgCl2, 1.1 mM NaH2PO4, 25 mM NaHCO3, 11.1 mM glucose, 0.05 of bovine serum albumin, and 4 dextran. Other agents, if sources were not mentioned above, were purchased from Sigma (34540-22-2 Shanghai, China).Amylase and Lipopolysaccharide LevelsThe assays of amylase and lipopolysaccharide (LPS) levels in the serum from AP or control rats were performed based on the manufacturer recommended procedures (Cat. No: C016 for amylase assay kit, Jiancheng Technology, Nanjing, China; and Cat. No: CE32545 for LPS assay kit, Chinese Horseshoe Crab Reagent Co. Ltd., Xiamen, China).Data AnalysisAll data are expressed as mean 6 SEM. Student’s t-test or 1113-59-3 biological activity single factor analysis of variance (ANOVA) was performed using the SPSS 13.0 software (SPSS Co. Ltd., Shanghai, China). P-values of ,0.05 were considered statistically significant.Assays for Inflammatory MediatorsThe levels of interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant 1 (CINC1/KC) in the serum of rat and in the venous effluent from the isolated rat stomach were quantified using the rat IL-6 and KC ELISA kits based 18325633 on the manufacturer recommended protocols (Cat. no: F01731D for rat interleukin 6 ELISA kits; Cat. no: F01723D for rat KC ELISA kits. H-Y Biological Co. Ltd., Shanghai, China). The optical density was determined at 490 nm for absorbance in an enzyme-linked immunoabsorbent assay instrument (Microplate Reader, Model Elx800; BioTek, Winooski, VT, USA). Each specimen was measured three times and the measurement was repeated in 6 rat samples of each group. The data in the venous effluent from the isolated rat stomach was presented with the difference of IL-6 or KC level between the perfusion and the effluent, being considered as the release of IL-6 and KC from the rat stomach.Results Results from Experiment In VivoPathological changes in the pancreas of AP rats. Under light microscopy, it was evident that after treatment with sodium taurocholate, rats developed severe acute pancreatitis with obvious edema, vacuolization and serious necroses in the acinar cells of the pancreatic 1527786 tissues. And the histological scores in AP rats were much higher than those of the control rats (Fig. 1). Combined with the increased level of amylase activity in the serum of AP rats, theGastrin and Somatostatin Levels in Animal SpecimensGastrin and somatostatin levels in the animal serum and in the isolated stomach venous effluent were measured using commercially-available gastrin and somatostatin radioimmunoassay Kits (Gastrin Kit: Cat. No. G01PJB, North Institute of Biologic Technology, Beijing, China. Somatostatin Kit: Cat. No. S111013, Second Military Medical University, Shanghai, China). Measurement procedures were based on the manufacturers’ recommendations as des.And ChemicalsHU210 [(6aR)-trans-3-(1,1-Dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl -6H-dibenzo[b,d]pyran-9-methanol], AM251 [(N-(Piperidin-1-yl)-5-(4-iodophenyl)-1- (2,4-dichlorophenyl)- 4-methyl-1H-pyrazole-3-carboxamide)], were purchased from Tocris (Tocris-Bioscience, Ellisville, MO, USA). Both chemicals were dissolved in a solvent consisted of ethanol, Tween80 and normal saline (NS), volume ratio 1:1:18, to concentration 1022M, at 37uC using an ultrasonicator, and then were further diluted in NS to 1025 M, and again to 1027M with perfusion fluid just before use under the conditions that were determined in pilot study [22]. The modified Krebs-Ringer solution was composed of 117.5 mM NaCl, 4.7 mM KCl, 2.4 mM CaCl2, 1.1 mM MgCl2, 1.1 mM NaH2PO4, 25 mM NaHCO3, 11.1 mM glucose, 0.05 of bovine serum albumin, and 4 dextran. Other agents, if sources were not mentioned above, were purchased from Sigma (Shanghai, China).Amylase and Lipopolysaccharide LevelsThe assays of amylase and lipopolysaccharide (LPS) levels in the serum from AP or control rats were performed based on the manufacturer recommended procedures (Cat. No: C016 for amylase assay kit, Jiancheng Technology, Nanjing, China; and Cat. No: CE32545 for LPS assay kit, Chinese Horseshoe Crab Reagent Co. Ltd., Xiamen, China).Data AnalysisAll data are expressed as mean 6 SEM. Student’s t-test or single factor analysis of variance (ANOVA) was performed using the SPSS 13.0 software (SPSS Co. Ltd., Shanghai, China). P-values of ,0.05 were considered statistically significant.Assays for Inflammatory MediatorsThe levels of interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant 1 (CINC1/KC) in the serum of rat and in the venous effluent from the isolated rat stomach were quantified using the rat IL-6 and KC ELISA kits based 18325633 on the manufacturer recommended protocols (Cat. no: F01731D for rat interleukin 6 ELISA kits; Cat. no: F01723D for rat KC ELISA kits. H-Y Biological Co. Ltd., Shanghai, China). The optical density was determined at 490 nm for absorbance in an enzyme-linked immunoabsorbent assay instrument (Microplate Reader, Model Elx800; BioTek, Winooski, VT, USA). Each specimen was measured three times and the measurement was repeated in 6 rat samples of each group. The data in the venous effluent from the isolated rat stomach was presented with the difference of IL-6 or KC level between the perfusion and the effluent, being considered as the release of IL-6 and KC from the rat stomach.Results Results from Experiment In VivoPathological changes in the pancreas of AP rats. Under light microscopy, it was evident that after treatment with sodium taurocholate, rats developed severe acute pancreatitis with obvious edema, vacuolization and serious necroses in the acinar cells of the pancreatic 1527786 tissues. And the histological scores in AP rats were much higher than those of the control rats (Fig. 1). Combined with the increased level of amylase activity in the serum of AP rats, theGastrin and Somatostatin Levels in Animal SpecimensGastrin and somatostatin levels in the animal serum and in the isolated stomach venous effluent were measured using commercially-available gastrin and somatostatin radioimmunoassay Kits (Gastrin Kit: Cat. No. G01PJB, North Institute of Biologic Technology, Beijing, China. Somatostatin Kit: Cat. No. S111013, Second Military Medical University, Shanghai, China). Measurement procedures were based on the manufacturers’ recommendations as des.
