Hypercoagulable state and thrombosis are major lethal causes of ulcerate colitis (UC). The aim of the present study is to explore the change and role of protein C (PC) system in UC thrombosis. 4% dextran sulfate sodium (DSS) was used to induce the UC model, and the body weight, the length of colon, and the weight of spleen were measured after intake of DSS as drinking water for 1 week. The macroscore and microscore were examined. The quantity of macrophage in colon smooth muscle was observed by immunofluorescence, and TNF-α and IL-6 levels in plasma were evaluated by ELISA. Intravital microscopy was applied to observe colonic mucosal microvascular circulation, activities of PC and protein S (PS) were determined by immunoturbidimetry, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) expressions were detected by immunohistochemistry. In vitro, TNF-α and IL-6 levels were tested in supernatant of macrophage separated from colonic tissue. After stimulation of mouse colonic mucosa microvascular endothelial cells by TNF-α and IL-6 respectively, the activities of PC, PS, activated protein C (APC) were evaluated, and the expressions of EPCR and TM were detected by Western blotting. The results revealed that compared with control, the DSS mouse showed weight loss (P < 0.05), a shortened colon (P < 0.05), and swelled spleen (P < 0.05), accompanied by higher histological score (P < 0.05), as well as infiltration of macrophages, elevated TNF-α and IL-6 levels in plasma (P < 0.01). The intravital microscopy results revealed that compared with control, DSS mice showed significantly enhanced adhesion of leukocytes and colonic mucosal microvascular endothelial cells (P < 0.01), meanwhile, decreased activity of PC and PS in plasma (P < 0.01 or P < 0.05), and down-regulated expression of EPCR (P < 0.01). The degree of inflammation was negatively correlated with the PC activity. In vitro, TNF-α and IL-6 levels were increased in the supernatant of macrophages from DSS mice colonic tissue (P < 0.05), and after incubation of TNF-α or IL-6 with colonic mucosal microvascular endothelial cells, the APC activity was decreased (P < 0.05 or P < 0.01), and expression of EPCR was down regulated (P < 0.05). These results suggest that PC system is inhibited in UC mouse. Presumably, the mechanism may be due to the secretion of cytokines from macrophages and subsequential influence on the function of endothelia cells. Furthermore, enhancement of PC system activity may serve as a new strategy for the treatment of UC.
Animals ; Blood Coagulation Factors ; metabolism ; Colitis, Ulcerative ; chemically induced ; physiopathology ; Dextran Sulfate ; Immunohistochemistry ; Inflammation ; Interleukin-6 ; blood ; Intestinal Mucosa ; pathology ; Macrophages ; cytology ; Mice ; Protein C ; metabolism ; Receptors, Cell Surface ; metabolism ; Spleen ; pathology ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo clarify the role of mast cells and neuropeptides substance P (SP), somatostatin (SS), and vasoactive intestinal peptide (VIP) in dextran sulfate sodium (DSS)-induced colitis in rats.

METHODSExperimental colitis was induced in Sprague-Dawley rats (180-200 g, n=20) by oral ingestion of 4% (w/v) DSS in drinking water for 7 days. Control rats (n=5) drank water and were sacrificed on day 0. Mast cell number, histamine levels in whole blood and tissue, tissue levels of SP, SS and, VIP in the distal colon of the rats were measured on day 8, day 13, and day 18 of experimentation.

RESULTSOral administration of 4% DSS solution for 7 days resulted in surface epithelial loss and crypt loss in the distal colon. Mast cell count increased on day 8 (1.75±1.09/mm vs. 0.38±0.24/mm, P<0.05) and day 13 (1.55±1.01/mm vs. 0.38±0.24/mm, P<0.05) after DSS treatment. Whole blood histamine levels were increased on day 8 (266.93±35.62 ng/mL vs. 76.87±32.28 ng/mL, P<0.01) and gradually decreased by day 13 and day 18 after DSS treatment. Histamine levels in the distal colon were decreased on day 8 (1.77±0.65 ng/mg vs. 3.06±0.87 ng/mg, P<0.05) and recovered to control levels by day 13 after DSS treatment. SP level in the distal colon gradually increased and were raised significantly by day 13 (8777.14±3056.14 pg/mL vs. 4739.66±3299.81 pg/mL, P<0.05) after DSS treatment. SS and VIP levels in the distal colon were not changed.

CONCLUSIONSMast cell degranulation followed by histamine release may play an important role in the pathogenesis of colitis induced by DSS. SP may be a significant substance in the progression of inflammation and the recovery process of DSS-induced colitis.

Animals ; Colitis ; chemically induced ; pathology ; physiopathology ; Dextran Sulfate ; Histamine ; analysis ; Male ; Mast Cells ; physiology ; Neuropeptides ; physiology ; Rats ; Rats, Sprague-Dawley ; Somatostatin ; analysis ; Substance P ; analysis ; Vasoactive Intestinal Peptide ; analysis

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4% acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.
Acetic Acid/toxicity ; Animals ; Colitis/chemically induced/*pathology/*physiopathology ; Colon/drug effects/enzymology/*innervation/pathology ; Indicators and Reagents/toxicity ; Male ; Muscle Contraction/drug effects ; Muscle, Smooth/drug effects/metabolism ; Myenteric Plexus/pathology ; NADPH Dehydrogenase/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Neuromuscular Junction/drug effects/*metabolism ; Nitrergic Neurons/drug effects/*metabolism ; Nitric Oxide/*metabolism ; Peroxidase/metabolism ; Potassium Chloride/pharmacology ; Rats ; Rats, Sprague-Dawley