| The aim of this paper is to investigate the prevention and cure mechanisms of Anemonin on diarrhea in mice induced with PRV and E.coli mixed infection. Methods: Mice were randomly assigned into four groups: the prevention group, the treatment group, the self-healing group and the control group. Each mice was injected intraperitoneally with 0.9 billion E.coli O101 and inoculated with 0.1mL(1×104.5TCID50)PRV to induce diarrhea in all groups except the control group. The mice of prevention group were inoculated with anemonin (1mg/ml) twice a day 7 days before the infection and last till the end of the test. The mice of treatment group were inoculated with 0.2ml anemonin (1mg/ml) twice a day post infection. The mice of self-healing group were treated with physiological saline as the substitute twice a day post infection. At the same time, the mice of control group were also treated with physiological saline as the substitute. The clinical symptoms were observed everyday. 3 mice of each group were randomly selected for dissection on the every days post infection, and the pathologic and ultra structural pathologic changes were observed by the conventional slicing and electron microscopy methods respectively, the expression of mucosal repair gene were analysised by the Immunohistochemistry and PCR methods.The results as follow:Clinical symptoms: 6h post infection the mice appeared depression, fatigue, chill, diarrhea and poor appetite. The symptoms of self-healing mice were most severe.The mice of prevention group had milder clinical symptoms than that of treatment group. 120h post infection the symptoms were decreased gradually. The mice of control group showed normally.Pathologic changes: The mice of prevention group, treatment group,and self-healing group appeared hemorrhage in intestine severely, especially the duodenum, and intestinal tract was thinner than the normal. The mice of control group showed normally. 96h post infection, the symptoms of prevention group and treatment group were decreased. The symptoms of self-healing group were gradually decreased until 120h post infection.Histopathological changes: the intestinal villus of the infected mice was acutely injuried. The intestinal villus displaied atrophy and dehydration; the villus tips appeared vacuolization and swelling, but the bases constricted. 96h post infection the symptoms of prevention group and treatment group were gradually decreased. The symptoms of self-healing group were gradually decreased until 120h post infection. Control group mice had the normal intestinal organizational structure.Ultrastructural Changes: under SEM, the villous were injuries, dehydration and atrophy. The microvillus were loss, sparse and clutter, the numbers of goblet cells were increased. 120h post infection, the morphosis of villus was gradually restored. The ultrastructural changes of treatment group were lower than that of self-healing group, and the prevention group was the lowest one, it showed dehydration and the injury lighter. 96h post infection, the injury were gradually restored. The mice in control group had the normal intestinal organizational structure.Immunohistochemistry results showed that, positive staining of PCNA were almost concentrate upon the nucleus of intestinal gland cells in the crypts and a small quantity positive staining, and also be finded in intestinal mucosa. The mainly positive staining of EGFR was in the membrane of epithelial cells and cytoplasm near the membrane, and the crypt also had a small quantity positive staining. The positive staining of TGFαand TGFβ1 were in the intracytoplasm of intestinal lymphocyte, macrophages and some epithelial cells. The PCNA, EGFR, TGFαand TGFβ1 of self-healing group, treatment group and prevention group were all higher than that of control group, and the prevention group was the highest one. in 96h post infection the PCNA, EGFR, TGFαand TGFβ1of prevention group was significantly higher than that of control group(P<0.05). The treatment group was lower than that of prevention group and the self-healing was the lowest one. Self-healing group, treatment group and prevention group had no differences with control group 96h post the infection(P>0.05).The result of RT-PCR showed that the TGFβ1 and EGFR of self-healing group, treatment group and prevention group were all higher than that of control group, and the treatment group was higher than that of self-healing group, the prevention group was the highest one. Self-healing group, treatment group and prevention group had no differences with control group at the later period of the infection (P>0.05). In 96h infection, the TGFβ1 and EGFR of self-healing, treatment group and prevention group were all significantly higher than that of control group (P<0.01). The treatment group and prevention group were no significant difference but they were all higher than that of the self-healing group (P<0.05).Conclusion:According to clinical symptoms, pathologic changes, histopathology changes and ultrastructural changes of the mice in each group, Anemonin had significantly effects on therapeutic action and prevention effect on mice diarrhea infected by PRV and E.coli, and the prevention group was significantly higher than that of treatment group.Immunohistochemistry results showed that the PCNA, EGFR, TGFαand TGFβ1 of self-healing group, treatment group and prevention group were all higher than that of control group, treatment group and prevention group were all higher than that of the self-healing group. The results showed that anemonin can promote the expression of PCNA, EGFR, TGFαand TGFβ1.The result of RT-PCR showed that the EGFR and TGFβ1 of self-healing group, treatment group and prevention group were significantly higher than that of control group, treatment group and prevention group were all higher than that of the self-healing group. The results showed that anemonin can promote the expression of EGFR and TGFβ1.To sum up the results suggest that Anemonin can significantly preventive mice diarrhea infected by PRV and E.coli, the mechanism of the Anemonin for control diarrhea maybe has some relationships to regulation the expression of mucosal repair gene PCNA, EGFR, TGFαandTGFβ1 . |
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