| Ulcerative colitis (UC), a form of inflammatory bowel disease (IBD), is characterized by contiguous chronic non-specific inflammation of the colonic and rectal lamina propria. Clinical manifestations include abdominal pain, diarrhea accompanied by mucus or liquor puris or blood. It has been considered in the past that UC was more common in Europe while more rare in the Asian-African. Since 1978, epidemiological data have shown that the incidence and prevalence of UC in China displayed a remarkable increase and its etiology and pathogenesis were intricate. Increasing researches disclosed that the abnormality of immune response involved in the pathogenesis of UC. Lymphocytes or antigen-presenting cells have already been a main focus in recent studies, while little is reported on the pathogenic role of neutrophils in UC. Neutrophil elastase (NE), the product of activated neutrophils, involving in tissue injury, has been studied in acute lung injury, emphysema, arthritis and other diseases, while its role in pathogenesis of UC is still unclear. This experiment was designed to observe the effect of NE inhibitor on acute experimental colitis in BALB/c mice and to explore the role of neutrophil elastase in the pathogenesis of UC.Aims:1. To investigate the expression of neutrophil elastase (NE) in plasma and the colon with ulcerative colitis in mice.2. To investigate the effect of neutrophil elastase inhibitor in the prevention and treatment of ulcerative colitis in mice.Methods:1. A total of 52 healthy male BALB/c mice were randomly divided into four groups as follows:①normal control group (N group, n=12);②experimental group (UC group, n=12);③experimental group +low-dose ONO-5046 intervention group (UC+L group,n=14);④experimental group +high-dose ONO-5046 intervention group (UC+H group,n=14). The ulcerative colitis model was induced in mice by providing 5% DSS dissolved in sterile distilled water ad-libitum for 6 days in experimental groups; mice in N group were provided with sterile distilled water ad-libitum for 9 days; mice in UC group were treated with PBS 0.2 ml intraperitoneally twice a day for nine days from day 0 on, and then on day 1 provided with 5% DSS for 6 days followed by sterile distilled wate for 2 days; mice in UC+L group and UC+H group were treated with ONO-5046 25mg/kg and 50mg/kg respectively, which was dissolved in 0.2 ml PBS, intraperitoneally twice a day for nine days, and other interventions were in accordance with UC group. Mice of each group were sacrificed on day 5 and day 9 respectively.2. Colitis was evaluated by the body weight, fecal trait, fecal blood, disease activity index and histological score.3. Samples from colon and plasma in all mice were obtained after intervention. The concentration and the amount of NE were examined by enzyme-linked immunosorbent assay (ELISA) in both plasma and culture supernatant of colonic mucosa. The expression of NE in colon was analyzed by Western blotting.Results:1. On day 7 (treated with DSS for 6 days), compared to N group, a remarkable decrease in body weight was observed in UC group (P<0.01), and scores of the fecal trait, fecal blood and disease activity index were significantly increased (P<0.001).2. Body weight: compared to N group, a significant decrease in body weight was observed in other groups (P<0.05) on day 5 (treated with DSS for 4 days) and no significant difference was found between UC+H and N group (P>0.05) on day 9 (treated without DSS for 2 days).Compared with UC group, weight in UC+L group was little increased (P>0.05), and that in UC+H group, which was increased in comparison with UC+L group (P<0.05), was remarkably increased (P<0.05).3. Fecal trait scores: compared to N group, a significant increase in fecal trait scores was observed in other groups (P<0.05) on day 5 and a remarkable increase was found (P<0.01) on day 9.Compared with UC group, the scores of UC+L group were little dropped (P>0.05), while those in UC+H group, which were lower than UC+L group (P<0.05), were largely dropped (P<0.05).4. Fecal blood scores: no significant differences were viewed among four groups on day 5 (P>0.05);an obvious elevation was verified in UC group compared with N group on day 9 (P<0.001);when comparing with UC group, scores in UC+L group were slightly dropped (P>0.05),however ,those in UC+H group, despite higher than N group (P<0.05), were conspicuously dropped (P<0.01); scores in UC+H group were lower than UC+L group (P<0.05).5. Disease activity index: compared to N group, a remarkable increase in scores of disease activity index was observed in other groups (P<0.01) on day 5 and a significant increase was found (P<0.05) on day 9.Compared with UC group, the scores of UC+L group were little dropped (P>0.05), while those in UC+H group, which were lower than UC+L group (P<0.05), were largely dropped (P<0.05).6. Histological scores in colon: the scores in UC group were raised on either day 5 or day 9 compared to N group (P<0.01); furthermore, the scores on day 9 were higher than those on day 5 (P<0.05). Scores in UC+L group were dropped slightly (P>0.05), while scores in UC+H group, lower than UC+L group (P<0.01), were dropped dramatically (P<0.01). Scores in both UC+L and UC+H group were still higher than N group (P<0.01).7. Concentration of NE in plasma: compared to N group, the NE concentration in UC group was increased on either day 5 or day 9 (P<0.01); moreover, the concentration on day 9 was dramatically increased compared with that on day 5 (P<0.05). In comparison with UC group, UC+L group was decreased little (P>0.05), while UC+H group, lower than UC+L group (P<0.01), was decreased significantly (P<0.01), and they both were significantly increased compared to N group on day 5 (P<0.05), while UC+H group had no distinguished difference from N group on day 9 (P>0.05).8. Amount of NE in culture supernatant of colonic mucosa: on either day 5 or day 9, the NE amount was markedly raised in UC group comparing with N group (P<0.01), and the amount was higher on day 9 than that on day 5 (P<0.01). UC+L group was dropped slightly (P>0.05), while UC+H group, lower than UC+L group (P<0.01), was declined remarkably (P<0.01) when compared to UC group; they were both higher than N group on day 5 or day 9 (P<0.01).9. Expression of NE protein in the distal colon: on either day 5 or day 9, the NE expression level was markedly raised in UC group comparing with N group (P<0.01), and the expression level was higher on day 9 than that on day 5(P<0.05). UC+L group was dropped slightly (P>0.05), while UC+H group, lower than UC+L group (P<0.01), was declined remarkably (P<0.01) when compared to UC group; they were both higher than N group on day 5 or day 9 (P<0.01).10. Correlationship between concentration of NE in plasma and expression of NE protein in the distal colon with disease activity index and histological scores in colon:on either day 5 or day 9, the concentration of NE in plasma and the expression of NE in colon tissues were remarkably positive correlation with DAI and histological scores (P<0.05).Conclusions:1. The fact that murine body weight was decreased dramatically and the scores of fecal trait or fecal blood were increased remarkably on day 7 (treated with DSS for 6 days), resembling the characteristics of UC in human, indicated the establishment of UC model in mice.2. The expressions of NE in both plasma and colon, which was well correlated with DAI and histological scores, increased evidently in model group, which revealed NE may involve in the pathogenesis of UC. 3. Interference with NE inhibitor in mice could prevent from weight loss and fecal blood, meanwhile ameliorate histological injury, displaying a dose-dependent effect, which demonstrated that utilization of NE inhibitor might be a novel preventative and therapeutic approach in ulcerative colitis. |
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