| Aims:Diarrhea irritable bowel syndrome(IBS-D)is a common clinical functional bowel disease.The high incidence of this disease poses great challenges to the quality of life and health care resources of patients.The pathophysiological mechanism of IBSD is complex.At present,its pathogenesis is unclear.IBS-D patients often have obvious gastrointestinal motility abnormalities,showing mild or severe diarrhea with abdominal pain and other symptoms.It was found that the destruction of intestinal barrier function and changes of intestinal microecology were the important factors of its pathogenesis.The purpose of this study was to explore the changes of intestinal microecology composition and tight junction proteins related to intestinal barrier in IBS-D rats,so as to provide experimental data and theoretical basis for the pathogenesis of IBS-D.Methods: Wistar male rats were randomly divided into control and model groups.Gastrointestinal electrodes needed to be implanted in antrum,duodenum and colon of the two groups.In addition,gastrointestinal motility was measured by gastric emptying,intestinal propulsion and colonic beading experiment;The model evaluation included abdominal withdrawal reflex score(AWR)test,HE staining,water content detection of rat feces;D-lactate content in plasma was detected by ELISA;Anxiety and depression degree of IBS-D rats was evaluated by open field test and sugar water preference test;Intestinal microecology composition was detected by 16SrRNA;Serine protease activity in feces was detected by azocasein method;molecular biology was used to detect the expression of aquaporin(AQP3,AQP8),occludin,claudin-4,protein activated receptor 2(PAR2)and extracellular regulated protein kinases(ERK),and the localization of AQP3 and claudin-4 was analyzed.Results: Compared with the control group,the IBS-D rats showed mild depression,disordered hair,slow or slightly reduced weight growth,loose stool,and the perianal area was polluted by stool.With the increase of modeling days,these changes are more significant.In the model group,AWR score and fecal water content were significantly increased(P < 0.001)and HE staining showed that the colon mucosa was intact without inflammatory cell infiltration.The results of gastrointestinal motility test showed that in the model group,the migrating motor complex was disordered,the colonic myoelectric activity was increased(P < 0.05),the gastric emptying was slowed down,the intestinal propulsion was accelerated(P < 0.05),and colonic beading latency time was shortened(P < 0.05).The above changes show that IBS-D model is successful.The content of D-lactate in the plasma of the model group was significantly increased by ELISA(P < 0.05).The detection results of the intestinal flora showed that at the level of phylum,compared with the control group,IBS-D rats showed decreased Bacteroidetes and increased Actinobacteria.The genus level results showed that the Lactobacillus in the model group was significantly decreased than that in the control group(P <0.05).Unidentified_Ruminococcaceae,Intestinimonas and Enterococcus increased.The activity of serine protease in faeces of model group was significantly increased(P < 0.05).qPCR showed that the expression of AQP3 and AQP8 mRNA in colon tissue of the model group was significantly decreased(P < 0.01),the expression of occludin and ZO-1 mRNA was also decreased,but the expression of claudin-4 mRNA was significantly increased.Western blot showed that the expression of AQP3,AQP8,occludin and claudin-4 was consistent with that of qPCR.The expression of PAR2 and ERK was significantly increased(P < 0.05).Conclusion: 1.IBS-D rats show an obvious gastrointestinal motility disorder,the inhibition of gastric motility,and the increase electrical activity of small intestine and colon.Specifically,gastric emptying is slowed down,intestinal propulsion is accelerated,colonic beading latency time is shortened.2.In IBS-D rats,the expression of AQP3 and AQP8,occludin and ZO-1 decreased,and the expression of claudin-4 increased,which results in the abnormal function of colon barrier,and even diarrhea.3.The intestinal flora of IBS-D rats is imbalanced,which shows that some beneficial bacteria decreased and the pathogenic bacteria increased significantly.This change may enhance the activity of serine protease,then activate PAR2 / ERK signal pathway,make the expression of tight junction protein abnormal,and lead to the change of intestinal barrier function. |
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