Abstract

Objective To investigate the role of intestinal immune dysfunction in the pathogenesis of irritable bowel syndrome(IBS) and to study the effects of Clostridium butylicum on the regulation of intestinal immune disorders.Methods A total of 50 male 6-week-old C57BL/6 mice were randomly divided into three groups,including the experimental group (n =20),the control group (n =20) and the Clostridium butylicum group(n =10).A mouse model of constipation-predominant IBS (C-IBS) was established by perfusing sodium butyrate solution(200 μl,concentration of 500 mmol/L) into the mouse colon twice a day for three consecutive days.The mice in control group were intrarectally perfused with normal saline enema (200 μl).Two hours before the perfusion of sodium butyrate into colon,the mice in Clostridium butylicum group were given Clostridium butylicum 500 μl(viable cell concentration of 1×109 CFU/ml) by oral gavage once a day for six days.The colorectal distention test(CRD) was carried out for evaluation of clinical parameters.HE staining of intestinal tissue section was performed for histopathological assessment of colonic mucosal inflammation.Intestinal intraepithelial lymphocytes (IELs) and lamina propria mononuclear cells (LPMCs) were isolated and analyzed by flow cytometry to evaluate the correlation between IBS and intestinal immune dysfunction/abnormal activation of intestinal immune cells in mouse model of C-IBS,and to assess the regulatory effects of Clostridium butylicum on the intestinal immune disorder.Results (1) Compared with the control group,the mice in experimental group showed a significant change in physiological parameters,histological structure of colon,inflammatory cells infiltration and low-grade inflammatory state.There was a significant increase in scores of CRD and a decrease in lowest sensory threshold (t=8.926 and t=6.103,both P<0.001) ; (2) There was a decrease in the numbers of DC in IELs (t =2.878 and t =3.086,both P<0.05),but an increase in the numbers of macrophage (t=3.191,P<0.05) and the memory T cells in mice with IBS (t=3.071,P<0.05) as compared with that in control group; (3)DCs were decreased (t=2.880 and t=2.664,both P<0.05),but memory T cells were increased (t =3.732 and 2.682,P<0.01 and P<0.05) in the LPMCs of mice in experimental group; (4)There was no significant difference in the physiological index between the mice in control group and the Clostridium butylicum group.Levels of memory T cells,macrophages and DCs in the IELs were close to the normal level (6 d,t =1.103,0.0213,0.418,all P>0.05),and levels of macrophages and DCs in the LPMCs of mice in the Clostridium butylicum group were also similar to that in the control group (6 d,t =0.782,0.347,both P>0.05) ; (5) Compared with the mice in experimental group,the level of memory T cells in LPMCs of mice treated with Clostridium butylicum was dramatically declined (6 d,t=2.346,P=0.0470,P<0.05),however,which was still higher than that of mice in control group (6 d,t =2.233,P =0.0476,P<0.05).The intestinal immune function was restored to normal level with Clostridium butylicum intervention.Conclusion The pathophysiologic mechanism of IBS might be closely related to the abnormal activation of intestinal immune cellsand disordered functional state in the intestinal mucosa.Clostridium butylicum could regulate the intestinal immune homeostasis and restore the physiological function of gastrointestinal tract. Key words: C-IBS; Visceral hypersensitivity; Immune system dysfunction; Low-grade inflammatory state; Clostridium butylicum

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