| Objectiv : To observe the effects of Pingwei capsule on closed protein Occludin,Claudin-4,ZO-1 and the changes of fecal microbial community richness and diversity in rats with liver depression and spleen deficiency diarrhea irritable bowel syndrome.To provide experimental basis for clinical application of Pingwei capsule in the treatment of irritable bowel syndrome.Methods:One week after adaptive feeding of 48 healthy Wistar males,divided into 10 normal groups and 38 modules.21 days after model establishment,3 rats each and normal group were randomly selected for HE staining to observe colon histopathological morphology,and observe relevant behavioral testing of recorded rats to verify whether the model was successful.After making the mold,Rats were randomly grouped again,model group(model),pilveronium bromide group(PWBJ),low dose group(PWⅠ),medium dose group(PWⅡ),high dose group(PWⅢ),Seven in each group,Treatment,For 14 days,Twice a day,At least 8h.interval.Behavioral experiments were conducted and plasma,colonic tissue and fecal samples were collected after the treatment.The general condition and weight gain of rats were observed and recorded.The content D-Lactic acid in rat plasma was detected by ELISA method,the expression of ZO-1、Occludin、Claudin-4m RNA in rat colon tissue was detected by real-time fluorescence quantitative technique,and the protein content in rat colon tissue was detected by immunohistochemical SP method.Changes of microbial community richness and diversity in rat feces were analyzed by 16 s RNA gene sequencing.Result:(1)General situation: compared with the contrl,model rats were thin,lazy and prone to piling up,dry and yellow hair,irritable and irritable mood,sharp whirring after irritation,strong aggression,loose stools,perianal and tail wrapped in thin stool black gray and dirty and smelly;compared with the model,the spirit and activity of the rats in each group improved significantly after treatment,stool formation and diarrhea improved significantly.(2)Weight change: compared with contrl,the weight of model rats decreased significantly(P<0.001);compared with model,there was no significant difference in body weight(P>0.05).After treatment,the weight of PWBJ 、 PWⅠ 、 PWⅡ groups increased significantly(P<0.05).There was no significant difference in body weight between PWBJ、PWⅡ、PWⅢ three groups(P>0.05).(3)The fecal water content of rats increased significantly(P<0.05,P<0.01)compared with contrl,and decreased significantly(P<0.01)after treatment.(4)Open field experiment: compared with contrl,the total distance,the number of upright and the number of crossing the central field of model rats were significantly reduced(P<0.001,P<0.01),and the total distance,the number of upright and the number of crossing the central field of each group were significantly increased(P<0.001,P<0.01,P<0.05)after treatment.(5)Sugar water bias rate: compared with contrl,the sugar water bias rate of model rats decreased significantly(P<0.001,P<0.05),compared with model,the sugar water bias rate increased significantly(P<0.05)after treatment,the difference was statistically significant.(6)Plasma D-LA content: compared with the contrl,the plasma D-LA content of model rats increased(P<0.05),compared with the treatment group,the plasma D-LA content expression decreased(P<0.01,P<0.05),the difference was statistically significant.(7)Morphological changes of colonic tissue: the colonic mucosa of rats were observed under light microscope.The epithelial structure and cell arrangement of each layer of mucosa were intact,no obvious hyperemia and edema were found,and the manifestations of each group were similar.model,a small amount of inflammatory cell infiltration was seen in the colon tissue of rats.(8)RT-q PCR test results:Compared with contrl,Significant decrease in Occludin m RNA,ZO-1m RNA levels in colon tissues in model rats(P<0.001),Significant increase in Claudin-4m RNA levels(P<0.01),In contrast to the model,Significant increase in Occludin m RNA,ZO-1m RNA levels in colon tissue in rats after treatment(P<0.001,P<0.05),Claudin-4m RNA levels decreased significantly(P<0.01),Differences are statistical significant.(9)The results of immunohistochemical SP showed that the expression of Occludin 、 ZO-1 protein in colon decreased significantly(P<0.05,P<0.001)expression of Claudin-4 increased significantly(P<0.05,P<0.001).The expression of Claudin-4 decreased(P<0.05,P<0.001).The difference was statistically significant.(10)Changes in intestinal microbial diversity: compared with contrl,There was a marked increase in otus in model,And after treatment,the otus of each group decreased significantly.The microbial community structure of intestinal flora in 6 groups was mainly composed of phylum firmicutes,phylum bacteroidetes and phylum deforma,The relative abundance of model phylum increased,The relative abundance decreased after treatment;The relative abundance of the intestinal flora of PWBJ group was higher than that of model;group The genus is mainly composed of Lactobacillus,Enterococcus,Polycobacterium,The relative abundance of probiotic Lactobacillus decreased significantly in model,Compared with model,the relative abundance of Lactobacillus in different dosage groups of Pingwei capsule was significantly higherConclusion:(1)4% acetic acid enema,restraint stress and tail-clamping stimulation can successfully establish a IBS-D rat model of liver depression and spleen deficiency.(2)Pingwei Capsule can effectively restore the body weight of the model rats,relieve the diarrhea degree of the rats,improve the changes of anxiety and depression-like behavior,and play the role of regulating emotional disorders.(3)Pingwei capsule can reduce the D-LA content in rat plasma,up-regulate the level of ZO-1、Occludin and down-regulate the level of Claudin-4,indicating that the pathogenesis of IBS-D is related to the change of intestinal mucosal barrier function,that is,the level of ZO-1、Occludin、Claudin-4.Pingwei capsule may play a therapeutic role by improving intestinal mucosal barrier function and intestinal flora. |
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