| Aims: The aims of this research are to treat mice infected with candida albicans by combining somatostatin with traditional Chinese medicine Qingyitang;observe the Hematoxylin-eosin staining in the intestinal structure,colony counting value(CFU);detect the apoptotic cell and of intestinal structure and the expression of ERK/MAPK signal protein with FCM(flow cytometry)and western blot;compare the treatment effect of using separate and combined application of somatostatin and Qingyitang;as well as to study and probe into the possibility mechanism in protecting the intestinal mucosal barrier.Methods: 110 Kunming female mice of SPF(specific pathogen free)level were selected.Weight: 18-20 g,Age: about 4 weeks.20 mice were randomly selected as blank control group through randomization(N),and 80 mice were taken as model group infected by candida albicans.The mice weight of each group had no significant difference after grouping.Two weeks later,6 mice were randomly selected from the blank control group and model group.CFU colony counting value and HE were performed on their intestinal structures.Observation was conducted under the optical microscopy.It was confirmed that the intestinal model infected by candida albicans was successfully made.The rest 63 mice(11 died during the modeling period)of model group were randomly divided into model group and experiment group(somatostatin group,Qingyitang group,drug combination group),and drugs were adopted for intervention treatment.Equivalent drugs were used to intervene the mice for 7 days,and observation was conducted on mice's general situation;for the mice of intervention treatment and blank control group,cervical dislocation was adopted for execution.The terminal ileum,ileocecal junction and right-half colon of the mice were taken out in sterilized way,and HE microscopic observation was conducted to calculate the candida albicans colony of the intestinal contents,detect the D-lactate content,ZO-1 protein expression and distribution with immunofluorescence test,and to detect the expression of protein in ERK/MAPK signal pathway with WB.All experiment data were expressed in the form of mean ± standard deviation((x|-)±s).Analysis was conducted according to the SPSS18.0 statistic software.Results1.Observation on the general situation of mice: mice of the control group were lively.They ate and drank freely with increased weight,smooth and bright hair;for the mice in the model group,however,the intake and weight declined significantly with mussy hair,declined reaction sensitivity,mental haziness.They lost their appetite and became lazy,sleepiness and thin.Open surgery was conducted on the mice of model group and control group,discovering that the color of mice intestinal structure of the control group was ruddy without abnormality.The intestine peristalsis was normal;the color of the mice intestinal structure of the control group was dark with hyperemia in pipe wall but without intestine peristalsis.2.HE staining condition of model group infected by candida albicans: when the model group was completed,HE staining was conducted on the mice's intestinal structure of model group and control group.The results showed that the intestinal mucosas of mice of control group were complete without inflammatory cell and edema.The mucous epithelium structure was normal,and the glands were in regular arrangement and clear structure without abnormality;for the model group,however,the intestinal mucosa structures were damaged.Multiple lesions can be seen on the surface.Most glands structures were incomplete,and little residues can be seen in some lesions or the structures have been totally damaged.3.In terms of intestinal model infected by candida albicans,the colony counting value was calculated when the model has been built and when the experiment has finished.Open surgery was conducted to acquire intestinal structure and detect CFUs.The results showed that the CFUs of intestinal structure of mice model group were significantly higher than that of the control group no matter when the group has been set up or after the experiment.4.The treatment effect of somatostatin and/or Qingyitang on the mice of intestinal model infected by candida albicans4.1 CFUs detection results: when the intestinal mode infected by candida albicans was set up,somatostatin and/or Qingyitang was adopted for treatment.When detecting the CFUs of mice's intestinal structure of each group,the result shows that the CFUs of the mice's intestinal structure of the model group was significantly higher than the control group(p<0.05).Compared to the model group,however,the CFUs of somatostatin and or Qingyitang treatment group was on a declining curve.The drugs combination group had the most significant effect(p<0.01).4.2 Blood D-lactic acid detection results: The blood D lactic acid level can indirectly reflect the damage condition of intestinal mucosa.The blood D lactic acid content of the mice of the five groups were 0.082±0.021,10.14±0.813,4.099±1.016,5.128±1.020,and 2.672±0.743(ng/ml)respectively.When the model infected by candida albicans was set up,the D lactic acid content has increased rapidly,was significantly higher than the control group(p<0.05).while in somatostatin and/or Qingyitang group,however,the D lactic acid began to decline,was significantly lower than the control group(p<0.05),but was still higher than the control group.4.3 Detection of ZO-1 distribution,the tight junction protein of intestinal epithelium:Fluorescene detection was conducted to the distribution of tight junction protein of intestinal epithelium ZO-1 and discovered that the ZO-1 protein of mice of control group was in complete annular distribution with high density and clear cell compartment;after treating the mice with somatostatin and/or Qingyitang,improvement was made in ZO-1distribution and shape with increased density.More specifically,the ZO-1 protein distribution improvement of the somatostatin and Qingyitang combined treatment group was the most significant.4.4 Apoptosis detection results: The detection of the mice's intestinal mucosal tissues of five groups with Flow Cytometry showed that compared with the control group,the cell apoptosis in the mice's intestinal mucosa tissues of the five groups increased significantly(p<0.05).After treating them with somatostatin and/or Qingyitang,the apoptosis rate has declined at various degrees.More specially,the combined treatment group has declined significantly,reaching to the control group.5.The influence of somatostatin and/or Qingyitang treatment on the ERK/MAPK channel of intestinal structure model infected by candida albicans: for the changes of mice of each Western blot group in ERK/MAPK pathway p-ERK 1/2,MEK,Ras protein,the results showed that the expression of ERK/MAPK pathway protein ERKl/2,p ERKl/2,MEK and Ras in model group increased significantly.After treated by somatostatin and/or Qingyitang,the expression of each protein declined at various degrees.The most significant decline was shown in the combined treatment group,and was of significant statistical difference.Conclusion: 1.Successfully duplicate the model infected by candida albicans;2.the combined application of somatostatin and Qingyitang can reduce the colonization quantity of candida albicans in the mice's intestinal,lower apoptosis and lactic acid content,increase ZO-1 expression and plays a role in protecting the intestinal mucosal barrier.3.The protecting effect of combined application of somatostatin and Qingyitang on intestinal mucosal barrier is probably related to restricting ERK/MAPK signal pathway. |
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