| Objective:To study effect of montmorillonite powder, mesalazine and mesalazine combined montmorillonite powder on intestinal epithelial cell apoptosis and proliferation of rats with UC induced by TNBS, and explore recovery mechanism of damaged intestinal mucosa after two medications or combination therapy treat rats with experimental UC.Methods:82male SD rats were randomly divided into5groups including normal group, model group, montmorillonite powder group, mesalazine group and mesalazine combined montmorillonite powder group.18rats per group, normal group is10rats. In addition to rats in the normal group, rats in other groups were used to establish model of experimental UC by TNBS/ethanol method. Two rats were randomly selected and killed at24hours after modeling, and the pathological changes of the colon were observed. And eight rats were randomly selected and injected99TcBN nucleus norfloxacin.The creation of the model were specifically imaged by SPECT in order to confirm the presence of intestinal inflammation. After the success of modeling,according to body surface area, the daily dose of saline, mesalazine, montmorillonite powder, mesalazine were given to rats by gastro-tube. Half of rats were killed at5th day and at12day,resoectively, after the treatment, and colon specimens of rats were collected and kept.TUNEL method was used to assay intestinal cell apoptosis rate, Immunohistochemistry was used to detect VEGF, PCNA-positive cells expressing rates, using The quantitative PCR was used to measured expression of VEGF and PCNA in intestinal tissue.Results:1.Make model successfully:The rats at24hours after making model had diarrhea with bloody stools and weight loss. Colon tissue stained with HE by light microscopic observation has mucosal ischemia and necrosis, big ulceration area, submucosal edema, accompanied by neutrophil infiltration, muscle also scattered neutrophil. At the same time, glands structural is damaged and goblet cells decreased-The presence of intestinal inflammation are imaged and confirmed by SPECT.2. Results of apoptosis in intestinal tissue:Compared with the normal group, intestinal epithelial cell apoptosis rate in model group and in three intervention groups was significantly in increase at5th day and12th day after treatment (P=0.000); Compared with model group, intestinal epithelial cell apoptosis rate in three intervention groups was significantly lower at5th day after treatment (P=0.000); Among three intervention groups,the apoptosis rate in mesalazine combined montmorillonite group was more significantly lower than in montmorillonite powder group and mesalamine group (P=0.000).But montmorillonite powder group compared with mesalazine group at5th day after treatment (P=0.347). The apoptosis rate in three intervention groups was more significantly lower than in the model group at12th day after treatment (P=0.000); Among the three intervention groups, the apoptosis rate in mesalazine combined montmorillonite group was significantly lower than that in montmorillonite powder and mesalazine group, but the apoptosis rate in mesalazine group was significantly lower than in montmorillonite powder group at12th day after treatment (P=0.000); Among three intervention groups, the apoptosis rate at12th day was more significantly lower than at5th day (P=0.000).3. Results of intestinal tissue PCNA and VEGF expression by immunohistochemical detection:①PCNA:Compared with the normal group, intestinal tissue PCNA positive expression rate in model group and in three intervention groups was significantly lower at5th day and12th day after treatment (P=0.000);The intestinal tissue PCNA positive expression rate in mesalazine combined montmorillonite powder group compared with normal group at5th day and12th day after treatment (5th day P=0.222,12th days P=0.133); The intestinal tissue PCNA positive expression rate in montmorillonite powder group and mesalamine group compared with the normal group at5th day and12th day after treatment (P<0.05); The intestinal tissue PCNA positive expression rate in three intervention groups was significantly higher than in the model group at5th day after treatment (P=0.000); Among three intervention groups, the differences were not statistically significant at5th day after treatment (P>0.05); The intestinal tissue PCNA positive expression rate in three intervention groups was significantly higher than in the model group at12th day after treatment (P=0.000); Among three intervention groups, the differences were not statistically significant at12th day after treatment (P>0.05); The PCNA positive expression rate in montmorillonite powder group at12th day after treatment were in increase than at5th day (P=0.044); The PCNA positive expression rate in mesalazine group and mesalazine combined montmorillonite powder group was not statistically significant at5th and12th day after treatment (P>0.05).￢EGF:Compared with the normal group, intestinal tissue VEGF positive expression rate in model group was significantly higher at5th day and12th day after intervention (P=0.000); The intestinal tissue VEGF positive expression rate in mesalazine combined montmorillonite powder group compared with normal group at5th day and12th day after intervention (5th day P=0.739,12th day P=0.746); The intestinal tissue VEGF positive expression rate in montmorillonite powder group and mesalamine group compared with the normal group at5th day and12th day after intervention (P<0.05); The intestinal tissue VEGF positive expression rate in the three intervention groups was significantly lower than in the model group at5th day after intervention (P=0.000); Among the three intervention groups, the intestinal tissue VEGF positive expression rate in mesalazine combined montmorillonite powder group was significantly lower than in mesalazine group, ones in mesalazine group was significantly lower than in montmorillonite powder group at5th day after intervention (P=0.000); The intestinal tissue VEGF positive expression rate in the three intervention groups was significantly lower than in the model group at12th day after intervention (P<0.05); Among the three intervention groups, the intestinal tissue VEGF positive expression rate in mesalazine combined montmorillonite powder group was significantly lower than in the mesalazine group, ones in mesalazine group was significantly lower than in montmorillonite powder group at12th day after intervention (P <0.05); The intestinal tissue VEGF positive expression rate at12th day after montmorillonite powder group was significantly lower than at5th day (P=0.006), The intestinal tissue VEGF positive expression rate at5th day after mesalazine combined montmorillonite powder group and mesalazine group compared with ones at12th day (P>0.05).4. Results of intestinal tissue PCNA and VEGF expression by fluorescent quantitative PCR detection:①PCNA:The intestinal tissue PCNA expression in model group was significantly lower than in normal group(P=0.047); Normal group compared with montmorillonite powder group, mesalazine group and mesalazine combined montmorillonite powder group(P>0.05); Three intervention groups were compared with the model group(P>0.05); The comparison among the three intervention groups was not statistically significant (P>0.05).②VEGF:Model group compared with the normal group, intestinal tissue VEGF expression significantly increased (P=0.002); Normal group compared with montmorillonite powder group, mesalazine group and mesalazine combined montmorillonite powder group (P>0.05); Three intervention groups were compared with the model group(P>0.05); The comparison among the three intervention groups was not statistically significant (P=0.556); montmorillonite powder group and mesalazine group compared with mesalazine combined montmorillonite powder group (P<0.05).Conclusion:1. The pathological changes in the colonitis induced by TNBS/ethanol method are confirmed and rat with experimental UC model are established successfully by light microscopic observation and SPECT.2.Experimental results show that intestinal epithelial cell apoptosis involved in the pathogenesis of UC, and montmorillonite powder combined with mesalazine therapy can inhibit apoptosis and promote cell proliferation, and montmorillonite powder could help mesalazine reduce intestinal and vascular permeability, and both mixed effects is better than single mesalazine.3.In this experiment, the effect of single drug or combination therapy at12th day were better than at5th day. |
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