ABSTRACT
Objective To explore the role and mechanism of protein disulfide isomerase A3 (PDIA3) in abnormal colonic mucosa immune response of irritable bowel syndrome (IBS) rats with visceral hypersensitivity.Methods A total of 48 SD rats were divided into blank control group (n=12),empty virus group (IBS-1) (n=12),PDIA3 silencing group (IBS-2) (n=12) and model control group(IBS-3) (n=12).The expression of CD103 in ileocecus colonic tissues was detected by immunohistochemistry.Dendritic cells (D C) of mesenteric lymph node were isolated by flow cytometry.CD4+/CD8+ T cells of spleen were separated by immune-magnetic beads sorting technique.DC and CD4+/CD8+ T cells were co-cultured.The proliferation ability of lymphocytes promoted by DC was measured by methyl thiazolyl tetrazolium (MTT).The secretion levels of interlcukin (IL) 4 and IL-9 of CD4+/CD8+ T cells stimulated by DC were determined by enzyme linked immunosorbent assay (ELISA) method.Independent sample t-test was performed for statistical analysis.Results The cell number of CD103 positive DC of rats colon of blank control group,IBS-3 group was 6.25±1.14 and 10.83± 1.03(t=10.07,P＜0.05);that of IBS-2 group was 7.42 ± 0.90,and compared with that of IBS-3 group,the difference was statistically significant (t=-9.25,P ＜ 0.05).The MTT value of CD4+ T cells proliferation stimulated by DC of blank control group and IBm3 group was 0.54±0.01 and 0.60±0.01 (t=3.373,P＜0.05);that of IBS-2 group was 0.53±0.01,and compared with that of IBS-3 group,the difference was statistically significant (t =-3.139,P ＜ 0.05).The MTT value of CD8+ T cells proliferation stimulated by DC was 0.52±0.01 and 0.59±0.00(t=3.539,P＜0.01);that of IBS-2 group was 0.54±0.01,and compared with that of IBS-3 group,the difference was statistically significant (t=-3.183,P＜0.05).The level of IL-4 secreted by CD4+T cells promoted by DC of blank control group and IBS-3 group was 10.24±0.09 and 16.61±1.00 (t=3.222,P＜0.05);that of IBS-2 group was 11.75±0.54,and compared with that of IBS-3 group,the difference was statistically significant (t=-3.539,P＜0.01).The level of IL-9 secreted by CD4+T cells stimulated by DC of blank control group and IBS-3 group was 15.86±10.19 and 43.51±11.32 (t=4.529,P＜0.05);that of IBS-2 group was 29.05±2.09,and compared with that of IBS-3 group,the difference was statistically significant (t=-6.841,P＜0.01).The level of IL-4 secreted by CD8+T cells promoted by DC of blank control group and IBS-3 group was 7.35±0.12 and 13.91±0.57 (t=19.557,P＜0.01);that of IBS-2 group was 8.63± 0.24,and compared with that of IBS-3 group,the difference was statistically significant (t =-14.782,P＜0.01).The level of IL-9 secreted by CD8+T cells stimulated by DC of blank control group and IBS-3 group was 29.12±5.14 and 60.70±11.02 (t=4.122,P＜0.05);that of IBS-2 group was 37.17±2.65,and compared with that of IBS-3 group,the difference was statistically significant (t=-3.255,P＜ 0.05).Conclusions Protein disulfide isomerase A3 may mediate the process of DC activating T lymphocyte levels of related cytokines,cause abnormal immune response of colonic mucosa and promote the genesis of IBS visceral hypersensitivity.
ABSTRACT
Objective The aim of this study was to explore the establishment method of an animal model of irritable bowel syndrome ( IBS) and the evaluation of this animal model.Methods 30 adult SD rats were randomly divided into two groups: acetic acid irritation and bondage stress group ( n=10 ) , bondage stress group ( n =10 ) , and normal control group ( n=10 ) .The rats of the intervention group received an intra-colonic infusion of 0.4% acetic acid irritation combined with bondage stress to establish an animal model of IBS.The colonic sensitivity of the intervention group rats was assessed by stool test and colorectal distension ( CRD) test.Hydrochloric acid toluidine blue staining was used to observe the number degranulation phenomenon of mast cells in the ileocecum.Results On the 7th day, the number of soft feces was 8 and loose stool was 4 in the model group, significantly higher than that in the bondage stress group(0 and 0) (P<0.05),and normal control group (1 and 0) (P<0.05).On the 10th day, when the AWR=2, the average rectal distension volume was 1.2 mL, significantly lower than that in the bondage stress group(1.37mL) (P <0.05),also significantly lower than in the normal control group (1.49 mL) (P<0.05), and when the AWR=4, the average rectal distension volume was 1.49 mL, significantly lower than that in the bondage stress group(1.74mL) (P<0.05),and the normal control group (1.77 mL) (P<0.05).These results indicated that the visceral sensitivity of the model group was significantly higher than that in the bondage stress group and normal control group.Histological analysis showed that the rats of all groups had no obvious inflammatory changes.Conclusions Chronic bondage stress combined with intra-colonic infusion of 0.4%acetic acid irritation can be used to increase the visceral sensitivity and amount and degranulation of mast cells in the intestinal tissue in rats.This established rat model shows pathogenetic changes resembling the pathogenesis of human irritable bowel syndrome, and provides a useful animal model for further studies of the pathogenesis of this disease.